Two New 4-Hydroxy-2-pyridone Alkaloids with Antimicrobial and Cytotoxic Activities from Arthrinium sp. GZWMJZ-606 Endophytic with Houttuynia cordata Thunb

Abstract

Two new 4-hydroxy-2-pyridone alkaloids furanpydone A and B (1 and 2), along with two known compounds N-hydroxyapiosporamide (3) and apiosporamide (4) were isolated from the endophytic fungus Arthrinium sp. GZWMJZ-606 in Houttuynia cordata Thunb. Furanpydone A and B had unusual 5-(7-oxabicyclo[2.2.1]heptane)-4-hydroxy-2-pyridone skeleton. Their structures including absolute configurations were determined on the basis of spectroscopic analysis, as well as the X-ray diffraction experiment. Compound 1 showed inhibitory activity against ten cancer cell lines (MKN-45, HCT116, K562, A549, DU145, SF126, A-375, 786O, 5637, and PATU8988T) with IC₅₀ values from 4.35 to 9.72 µM. Compounds 1, 3 and 4 showed moderate inhibitory effects against four Gram-positive strains (Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus Subtilis, Clostridium perfringens) and one Gram-negative strain (Ralstonia solanacarum) with MIC values from 1.56 to 25 µM. However, compounds 1–4 showed no obvious inhibitory activity against two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two pathogenic fungi (Candida albicans and Candida glabrata) at 50 µM. These results show that compounds 1–4 are expected to be developed as lead compounds for antibacterial or anti-tumor drugs.

1. Introduction

Since ricinine [1] and ilicicolin H [2] were found in the early 1970s, a series of 4-hydroxy-2-pyridinone alkaloids with diverse structures were reported [3,4,5,6]. These alkaloids were mainly isolated from plants and fungi and had good biological activity [7]. According to the position and type of substituents, C-3 was often replaced by alkanes (e.g., septoriamycin from Septoria pistaciarum [8]) or terpenes (e.g., tenelin from Beauveria tenella and Beauveria bassiana [9]), and C-5 was often replaced by phenyl (e.g., sambutoxin from Fusarium sambucinum [10]) or cyclohexyl (e.g., torrubiellone A-B from Torrubiella sp. [11]). There were also a small number of derivatives whose C-6 was replaced by alkanes (e.g., pyridomacrolidin from Beauveria basiana [12]). These kind of compounds usually have anti-inflammatory, antibacterial, cytotoxicity, antimalarial, antiviral, insecticidal, antioxidant, anti-fibrosis, neuroprotection, inhibition of protein tyrosine kinase, and so on [13,14,15,16], which have attracted widespread attention.

In the past few years, there were some new 4-hydroxy-2-pyridones discovered from fungi, such as (+)-didymellamide B, (±)-didymellamide E, (+)-N-hydroxyapiosporamide, and didymellamides F–H which were isolated from Coniochaeta cephalothecoides [17], and arthpyrones A and B with novel oxabicyclo[3.3.1]-nonane ring which were isolated from Arthrinium arundinis ZSDS1-F3 [18]. Three new 4-hydroxy-2-pyridone alkaloids citridones E–G with antibacterial activity were isolated from the endophytic fungus Penicillium sumatrense GZWMJZ-313 in our previous studies [19]. In order to obtain more compounds of this type from endophytic fungus, Arthrinium sp. GZWMJZ-606 was isolated from Houttuynia cordata Thunb. Further chemical investigation of this fungal strain led to the isolation of two new 4-hydroxy-2-pyridone derivatives (1 and 2) which we named furaprazone A and B (Figure 1), along with the known N-hydroxyapiosporamide (3) [18,20] and apiosporamide (4) [21]. Compounds 1 and 2 were the first reported compounds with 5-(7-oxabicyclo[2.2.1]heptane)-4-hydroxy-2-pyridone skeleton. Compared with the previously reported 1,2-epoxyhexane [17,21], 2-oxobicyclo [3.3.1] nonane [18] or benzene [22], 7-oxadicyclo [2.2.1] heptane can improve some biological activities [23,24,25]. Herein, the isolation, structure elucidation, the antimicrobial and cytotoxic activity of these compounds are described.

2. Results and Discussion

Structure Elucidation

Compound 1 was obtained as a yellow crystal. The molecular formula was deduced as C₂₄H₃₁NO₇ based on the HRESIMS ion peak at m/z 468.19861 [M + Na]⁺ (calcd. for C₂₄H₃₁NO₇Na = 468.19927). Its IR (KBr) spectrum exhibited absorptions at 3434 cm⁻¹ (hydroxy), 1649 cm⁻¹ (carbonyl), and 1605/1552/1446 cm⁻¹ (aromatic heterocycle). Compound 1 had the same molecular formula with N-hydroxyapiosporamide (3) and showed a high degree of similarity in UV absorption. The NMR spectra displayed two methyls, five sp³-methylenes, eight sp³-methines, three sp²-methines, one sp³-quaternary carbon, five sp²-quaternary carbons (including two carbonyls) (

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR data of 1–4.

Position	1 b		2 a		3 b		4 b
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	31.0, CH2	0.86–0.91, m 1.95, d (11.1)	29.6, CH2	0.77–0.85, m 1.83, dd (12.2, 3.0)	31.0, CH2	0.87–0.91, m 1.91–1.94, m	31.0, CH2	0.86–0.92, m 1.90–1.95, m
2	36.6, CH2	0.99–1.11, m 1.74, overlap	35.1, CH2	0.92–1.00, m 1.67, d (12.1)	36.6, CH2	1.00–1.09, m 1.73–1.77, overlap	36.6, CH2	1.00–1.08, m 1.73–1.78, overlap
3	34.4, CH	1.48–1.53, m	32.6, CH	1.43–1.49, overlap	34.3, CH	1.49–1.52, m	34.4, CH	1.48–1.53, m
4	43.2, CH2	0.80, t (12.2) 1.72–1.80, overlap	41.4, CH2	0.72–0.78, overlap 1.69–1.72, overlap	43.1, CH2	0.80, t (12.2) 1.73–1.77, overlap	43.2, CH2	0.79, t (12.2) 1.73–1.78, overlap
5	43.2, CH	1.83, “t” like (10.8)	41.4, CH	1.75, “t” like (10.1)	43.3, CH	1.81–1.85, overlap	43.2, CH	1.80–1.86, overlap
6	131.7, CH	5.41, d (9.7)	130.4, CH	5.37, d (9.9)	131.7, CH	5.42, d (9.9)	131.7, CH	5.41, d (9.8)
7	132.6, CH	5.58–5.63, m	131.7, CH	5.56–5.59, m	132.5, CH	5.58–5.62, m	132.6, CH	5.60, ddd (9.8, 4.5, 2.7)
8	32.3, CH	2.83–2.90, m	30.6, CH	2.71–2.76, m	32.3, CH	2.82–2.86, m	32.4, CH	2.80–2.86, m
9	54.5, CH	4.42–4.47, overlap	51.8, CH	4.33, dd (11.4, 5.7)	54.5, CH	4.45, dd (11.3, 5.7)	54.2, CH	4.43, dd (11.4, 5.8)
10	37.5, CH	1.56–1.60, overlap	35.8, CH	1.41–1.49, m	37.6, CH	1.56–1.60, m	37.6, CH	1.54–1.60, m
11	22.9, CH3	0.94, d (6.5)	22.5, CH3	0.87, d (6.5)	23.0, CH3	0.94, d (6.5)	22.9, CH3	0.93, d (6.5)
12	18.4, CH3	0.82, d (7.0)	17.9, CH3	0.74, d (7.3)	18.4, CH3	0.83, d (7.3)	18.4, CH3	0.83, d (7.2)
13	211.4, C		209.6, C		211.7, C		212.0, C
14	108.2, C		106.7, C		108.6, C		108.8, C
15	159.9, C		161.8, C		159.6, C		163.9, C
16	139.5, CH	7.93, s	139.1, CH	7.33, d (6.0)	140.0, CH	8.04, s	139.9, CH	7.58, s
17	111.0, C		110.8, C		114.5, C		116.6, C
18	173.2, C		175.7, C		175.6, C		179.3, C
19	89.4, C		88.0, C		70.4, C		70.4, C
20	82.1, CH	3.87, brs	80.5, CH	3.58, dd (6.1, 1.4)	60.5, CH	3.66, d (3.7)	60.5, CH	3.64, “t” like (2.1)
21	82.9, CH	4.00, d (4.3)	81.2, CH	3.78, “t” like (4.7)	57.7, CH	3.43, “t” like (3.3)	57.6, CH	3.42, “t” like (3.3)
22	78.6, CH	4.42–4.47, overlap	76.6, CH	4.30, “t” like (5.1)	67.2, CH	4.12–4.14, m	67.2, CH	4.13, ddd (8.6, 5.7, 2.8)
23	24.1, CH2	1.68–1.73, overlap 2.20–2.27, overlap	23.0, CH2	1.45–1.54, m 2.02–2.10, overlap	25.7, CH2	1.34–1.38, m 1.81–1.85, overlap	25.8, CH2	1.32–1.38, m 1.80–1.86, overlap
24	33.1, CH2	1.60–1.65, overlap 2.20–2.27, overlap	31.8, CH2	1.45–1.49, overlap 2.01–2.08, overlap	31.8, CH2	1.70, ddd (13.9, 10.2, 2.8) 2.26, ddd (14.4, 8.4, 2.5)	31.6, CH2	1.70, ddd (13.5, 10.4, 2.5) 2.22, dd (13.3, 8.6)
20-OH				4.77, d (6.1)
21-OH				5.31, d (4.7)
-NH				11.38, brs

^a measured in DMSO-d₆, ^b measured in methanol-d₄ solvent.

), which was also similar to those of compound 3, especially for the important ¹H NMR signals, such as two methyl groups at H₃-11(δ_H 0.94) and H₃-12 (δ_H 0.82), a single special hydrogen signal at H-16 (δ_H 7.93), two olefinic protons at H-6 (δ_H 5.41) and H-7 (δ_H 5.60). The above evidence suggested that compound 1 has a similar skeleton with compound 3. The ¹H-¹H COSY correlations (Figure 2) from H₂-1 (δ_H 0.88 and 1.95) to H-10 (δ_H 1.58), H-3 (δ_H 1.50) to H₃-11, and H-8 (δ_H 2.85) to H-12 proved the existence of a decalin moiety. The relative configurations of this part were confirmed by the NOESY correlations (Figure 2) from H-10 to H₃-11/H₃-12 and H-5 (δ_H 1.83) to H-3/H-9 (δ_H 4.45), and indicated that compound 1 has the same decalin moiety as compound 3. The ¹H-¹H COSY correlations from H-20 (δ_H 3.87) to H₂-24 (δ_H 1.63 and 2.25) and the HMBC correlations (Figure 2) from H-21 (δ_H 4.00) and H₂-23 (δ_H 1.70 and 2.25) to C-19 (δ_C 89.4) confirmed the presence of an oxygenated cyclohexane moiety in compound 1. However, there was a large chemical shift difference between these two compounds at C-19/20/21/22 (δ_C 89.4, 82.1, 82.9, 78.6 for 1; 70.4, 60.5, 57.7, 67.2 for 3). Nevertheless, there is still one degree of unsaturation in the structure 1, implying an oxygen bridge in this cyclohexane moiety, but the HMBC correlations cannot be used to confirm it. The key HMBC correlations from H-9 to C-13 (δ_C 211.4), H-16 to C-15 (δ_C 159.9)/C-18 (δ_C 173.2)/C-19 indicated that the decalin and hexane moieties substituted at C-13 and C-17 of the 4-hydroxy-2-pyridinone part. The crystal of compound 1 was fortunately acquired in methanol/water (v/v, 1:1) solution. The results of the X-ray (Figure 3) analysis (Flack parameter = −0.15 (11), CCDC: 2218951) confirmed an oxygen bridge between C-19 and C-22 forming the furan ring and led to the final determination of its absolute configuration as 3R, 5S, 8R, 9R, 10R, 19S, 20S, 21S, 22S. This novel 4-hydroxy-2-pyridone was named furanpydone A.

Compound 2 was obtained as a yellow powder. The molecular formula was deduced as C₂₄H₃₁O₆N based on the HRESIMS peak at m/z 452.20319 ([M + Na]⁺, calcd. for 452.20436), which has ten degrees of unsaturation as furanpydone A (1), but one less oxygen atom than it. According to IR (KBr) spectrum data, they seem to have similar functional groups at 3445 cm⁻¹ (hydroxy), 1652 cm⁻¹ (carbonyl), and 1604/1557/1456 cm⁻¹ (aromatic heterocycle). According to 1D NMR and HSQC data, compound 2 displayed two methyl (δ_H/C 0.74/17.9, 0.87/22.5), five sp³-methylene (δ_H/C 1.50 and 2.06/23.0; 0.81 and 1.83/29.6; 1.47 and 2.04/31.8; 0.96 and 1.67/35.1; 0.75 and 1.70/41.4), eight sp³-methines (δ_H/C 2.74/30.6, 1.46/32.6, 1.45/35.8, 1.75/41.4, 4.33/51.8, 4.30/76.6, 3.58/80.5, 3.78/81.2), three sp²-methines (δ_H/C 5.37/130.4, 5.57/131.7, 7.33/139.1), one sp³-quaternary carbon (δ_C 88.0), and five sp²-quaternary carbons (δ_C 106.7, 110.8, 161.8, 175.7, 209.6), which were extremely similar to compound 1 (Table 1 and Table S1) suggested the similar structure of these two compounds. The ¹H-¹H COSY correlations (Figure 2) from H₂-1 (δ_H 0.81 and 1.83) to H-10 (δ_H 1.45), H-3 (δ_H 1.46) to H₃-11 (δ_H 0.87), H-8 (δ_H 2.74) to H₃-12 (δ_H 0.74), H-20 (δ_H 3.58) to H₂-24 (δ_H 1.47 and 2.04), the key HMBC correlations (Figure 2) from H-9 (δ_H 4.33) to C-13 (δ_C 209.6), H-16 (δ_H 7.33) to C-15 (δ_C 161.8)/18 (δ_C 175.7)/19 (δ_C 88.0), H-21 (δ_H 3.78) to C-19 further confirmed that compound 2 and 1 have the same skeleton structure. Analysis of the NMR spectral data revealed that the chemical shift of C-15 (δ_C 161.8) moved to a lower field, which was similar to compound 4, the key ¹H-¹H COSY correlation between H-16 and H-NH (δ_H 11.38) confirmed the absence of an N-hydroxy group in 2. The key NOESY correlations from H-10 to H₃-11/H₃-12, H-5 (δ_H 1.75) to H-3 (δ_H 1.47)/H-9 (δ_H 4.33), as well as the same chemical shift for C-19/20/21/22/23/24 with furanpydone A (1) suggested that these two compounds had the same relative configuration. The similarity of electronic circular dichroism (ECD) curve of compound 2 (213 (−5.83), 228 (−11.74), 265 (+5.40), 310 (+7.09), 341 (−1.63) to 1 (217 (−2.73), 242 (−3.51), 270 (+2.00), 316 (+2.75), 343 (−0.27)) (Figure 4) along with the similar optical rotation values (1: −89.7, 2: −80.0) indicated the same absolute configuration for 2 and 1. This novel 4-hydroxy-2-pyridone was named furanpydone B.

We propose a possible biosynthetic pathway for compounds 1–4. Didymellamide B was the key intermediate in the biosynthesis of these compounds [18]. The intermediates a and b were obtained by reduction from didymellamide B. Compound 2 was obtained by oxidation, hydration and cyclization reaction from a, and compound 1 was syntheszed by further oxidation. Compound 4 was obtained by two oxidation reactions from b, and compound 3 was syntheszed by further oxidation. (Figure 5).

Compounds 1–4 were tested for their antimicrobial activities against nine pathogenic microorganisms. As shown in

Table 2. Antimicrobial activity of 1–4 (MIC, µM), n = 3.

Pathogenic Bacteria	1	2	3	4	Positive Drug
E. coli ATCC 11775	>50	>50	>50	>50	0.10 *
P. aeruginosa ATCC 10145	>50	>50	>50	>50	1.56 *
S. aureus ATCC6538	12.5	>50	12.5	6.25	0.20 *
MRSA ATCC 43300	12.5	>50	25.0	6.25	0.38 *
B. subtilis ATCC 6051	>50	>50	>50	1.56	6.25 *
C. perfringens ATCC 13124	>50	>50	>50	3.13	0.047 *
R. solanacarum	>50	>50	>50	6.25	3.12 *
C. albicans ATCC10231	>50	>50	>50	>50	3.13 #
C. glabrata ATCC2001	>50	>50	>50	>50	3.13 #

* the positive drug is ciprofloxacin, ^# the positive drug is amphotericin B.

, compound 4 exhibited broad inhibitory activities against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Clostridium perfringens, and Ralstonia solanacarum with the MIC values ranging from 1.56 to 6.25 µM. Compounds 1 and 3 showed moderate selective activities against S. aureus and MRSA with the MIC values of 12.5–25.0 µM. Compounds 1–4 showed no obvious inhibitory activity against two Gram-negative bacteria (E. coli and P. aeruginosa) and two pathogenic fungi (C. albicans and C. glabrata) at 50 µM. According to the results, it seems that the compounds with ternary epoxide showed better antibacterial activity than those with furan ring, but the effect of N-OH needs more research to determine.

The antiproliferative activities against 18 cancer cell lines and one normal cell line were assayed by the CCK-8 method. Compound 1 showed significant cytotoxicity against 10 cancer cell lines, compound 3 showed activities against HCT116 and 786-O cell lines (

Table 3. Cytotoxic activity (µM, IC₅₀ ± SD), n = 3.

Cell Line	1	2	3	4	Dox
A549	6.47 ± 0.31	>10	>10	>10	0.849 ± 0.013
MKN-45	5.41 ± 0.09	>10	>10	>10	0.307 ± 0.005
HCT116	5.64 ± 0.05	>10	6.09 ± 0.02	>10	0.121 ± 0.005
K562	9.22 ± 0.93	>10	>10	>10	0.948 ± 0.058
DU145	9.01 ± 0.07	>10	>10	>10	0.189 ± 0.003
SF126	9.72 ± 0.46	>10	>10	>10	0.164 ± 0.016
A-375	7.16 ± 0.17	>10	>10	>10	0.064 ± 0.003
786-O	5.93 ± 0.13	>10	9.13 ± 0.48	>10	0.726 ± 0.028
PATU8988T	6.46 ± 0.09	>10	>10	>10	0.167 ± 0.012
5637	4.35 ± 0.08	>10	>10	>10	0.185 ± 0.002
HeLa	>10	>10	>10	>10	0.177 ± 0.006
TE-1	>10	>10	>10	>10	0.240 ± 0.030
GBC-SD	>10	>10	>10	>10	0.592 ± 0.069
MCF-7	>10	>10	>10	>10	0.966 ± 0.011
HepG2	>10	>10	>10	>10	0.619 ± 0.054
CAL-62	>10	>10	>10	>10	0.277 ± 0.019
HOS	>10	>10	>10	>10	0.090 ± 0.013
A-673	>10	>10	>10	>10	0.380 ± 0.030
L-02	7.09 ± 0.10	>10	9.70 ± 0.06	>10	0.243 ± 0.005

). The compounds with furan ring showed better antiproliferative activities than those with ternary epoxide. At the same time, nitrogen hydroxyl is the necessary group for maintaining the inhibitory activity.

3. Materials and Methods

3.1. General Experimental Procedures

The NMR spectra were recorded on Bruker Advance NEO 600 spectrometer (Bruker Corporation, Zurich, Switzerland) using TMS as an internal standard. MS analysis were carried out on Agilent 1100 instrument (Agilent Technologies, Santa Clara, CA, USA) and Thermo ultimate 3000/Q EXACTIVE FOCUS mass spectrometers (Thermo Scientific™, Waltham, MA, USA), respectively. Optical rotations were determined on Rudolph Autopol1 automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were detected on a Cary 60-UV-Vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were determined on an iCAN 9 infrared spectrophotometer (Tianjin Nengpu Technology Co., Ltd, Tianjin, China) with KBr disks. X-ray data were generated using a Bruker Smart-1000 CCD (Bruker Corporation, Billerica, MA, USA) area detector diffractometer with graphite monochromatic Cu-Kα radiation. Column chromatography was performed on silica gel (200–300 mesh; Qingdao Puke Parting Materials Co., Ltd., Qingdao, China), Sephadex LH-20 gel (Amersham Biosciences, Uppsala, Sweden). HPLC separation was performed on HITACHI Primaide with an ODS-A column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Melting point instrument (SGW X-4).

3.2. Fungal Material

The endophytic fungus Arthrinium sp. GZWMJZ-606 was isolated from the leaves of Houttuynia cordata Thunb., which was collected from Longli, Guizhou, China. The leaves were treated with 75% alcohol for 30 s, and the residual alcohol was washed with sterile water. Then 1 g of fresh leaves was grinded into a pulp and 10 mL sterile water added. The suspension (100 μL) was deposited on a rice agar plate, which was prepared from rice powder (10 g), agar (18 g), and 1 L water containing chloramphenicol (0.3%) as a bacterial inhibitor, and incubated at 28 °C for 5 days. Monoclonal was selected and streaked to purity using the same agar medium. This strain was determined as Arthrinium sp. by the phylogenetic tree (Figure S1) of the ITS sequence (GenBank No. OP810989). The strain was deposited in our laboratory of Guizhou in 20% glycerol at −80 °C.

3.3. Fermentation and Extraction

The fungal strain GZWMJZ-606 was cultured on PDA at 28 °C for 3 days and then was cut into 100 × 1000 mL Erlenmeyer flasks, each containing a solid medium prepared from 100 g rice and 110 mL distilled water. These flasks were incubated at room temperature under static conditions for 40 days. The cultures were extracted three times by EtOAc (each 500 mL) and the combined EtOAc solutions were dried in vacuo to yield the extract (480.0 g).

3.4. Isolation and Purification

The EtOAc extract (480.0 g) was fractionated into 19 fractions (Fr.1–Fr.19) by chromatography on a silica gel column using step gradient elution of petroleum ether (PE)-EtOAc (v/v, 100:1–1:1) and CH₂Cl₂-MeOH (v/v, 20:1–1:1). Fr.17 (9.4 g) was further separated into 15 subfractions (Fr.17.1–Fr.17.15) by Sephadex LH-20 (CH₂Cl₂-MeOH, v/v, 1:1). Fr.17.11 (207.6 mg) was purified by semipreparative HPLC on an ODS-A column eluting with 60% MeCN-H₂O containing 0.05% trifluoroacetic acid (TFA) to yield compound 1 (35.6 mg, t_R 11.1 min). Fr.17.14 (75.8 mg) was purified by semipreparative HPLC on an ODS-A column (60% MeCN-H₂O containing 0.05% TFA) to yield compound 2 (6.8 mg, t_R 9.3 min). Fr.17.2 (830.5 mg) was further separated into 7 subfractions (Fr.17.2.1–Fr.17.2.7). Compound 4 (12.2 mg, t_R 10.1 min) was obtained from Fr.17.2.1 (57.1 mg) by semipreparative HPLC (55% MeCN-H₂O containing 0.05% TFA). Fr.16 (1.6 g) was further separated into 6 subfractions (Fr.16.1–Fr.16.6), and Fr.16.4 (120.5 mg) was performed on a semipreparative ODS-A column (61% MeCN-H₂O containing 0.05% TFA) to yield compound 3 (38.6 mg, t_R 8.4 min).

3.5. Physical Properties and Spectral Data of 1–4

Compound 1: yellow crystal; m.p. 167.5–168.5 °C; ECD (1.12 mM, MeOH) λmax (Δε) 217 (−2.73), 242 (−3.51), 270 (+2.00), 316 (+2.75), 343 (−0.27) nm; [α${]}_{\mathrm{D}}^{22}$−89.7 (c 0.58, MeOH); UV (MeOH) λ_max (log ε) 281 (0.75), 341 (0.72) nm; IR (KBr) ν_max 3434, 2913, 2953, 1649, 1605, 1446 cm⁻¹; ¹H NMR and ¹³C NMR data see Table 1 and Table S1 and Figures S3–S10; HRESIMS m/z 468.19861 [M + Na]⁺ (Figure S2), molecular formula: C₂₄H₃₁NO₇.

X-ray crystallographic analyses of 1: C₂₄H₃₁NO₇·CH₃OH, orthorhombic, M = 477.54, a = 7.6539 (3) Å, b = 14.5093 (6) Å, c = 21.7608 (11) Å, α = 90°, β = 90°, γ = 90°, V = 2416.60 (18) ų, T = 150 K, space group P21 21 21, Z = 4, μ (Cu Kα) = 0.807 mm⁻¹, 8074 reflections measured, 4534 independent reflections (R_int = 0.019). The final R₁ values were 0.0724 (I > 2σ (I)). The final wR (F²) values were 0.1921 (I > 2σ (I)). The final R₁ values were 0.0767 (all data). The final wR(F²) values were 0.1991 (all data). The goodness of fit on F² was 1.022. Flack parameter = −0.15 (11). CCDC: 2218951.

Compound 2: yellow powder; ECD (1.17 mM, MeOH) λmax (Δε) 213 (−5.83), 228 (−11.74), 265 (+5.40), 310 (+7.09), 341 (−1.63) [α${]}_{\mathrm{D}}^{22}$−80.0 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 235 (1.05), 270 (0.50), 338 (0.74) nm; IR (KBr) ν_max 3445, 2909, 1652, 1604, 1456 cm⁻¹; ¹H NMR and ¹³C NMR data see Table 1 and Figures S12–S17; HRESIMS m/z 452.20319 [M + Na]⁺ (Figure S11), molecular formula: C₂₄H₃₁NO₆.

Compound 3: yellow solid; the molecular formula is C₂₄H₃₁NO₇ (m/z 444.1 [M − H]⁻) determined by ESIMS. [α${]}_{\mathrm{D}}^{22}$−57.4 (c 2.3, MeOH); based on ¹H NMR and ¹³C NMR data (Table 1, Figures S18 and S19) proved that compound 3 was N-hydroxyapiosporamide.

Compound 4: faint yellow solid powder; the molecular formula is C₂₄H₃₁NO₆ (m/z 452.5 [M + Na]⁺) determined by ESIMS. [α${]}_{\mathrm{D}}^{22}$−32.2 (c 0.87, MeOH); based on ¹H NMR and ¹³C NMR data (Table 1, Figures S20 and S21) proved that compound 4 was apiosporamide.

3.6. Antimicrobial Activities Assay

The isolated compounds were evaluated for antibacterial activity against pathogenic microorganisms including three Gram-negative strains (Escherichia. coli ATCC11775, Pseudomonas aeruginosa ATCC10145, Ralstonia solanacarum [26]), and four Gram-positive strains (Staphylococcus aureus ATCC6538, methicillin-resistant S. aureus ATCC43300 MRSA, Clostridium perfringens ATCC13124, and Bacillus subtilis ATCC6051), and two pathogenic fungi (Candida albicans ATCC10231 and Candida glabrata ATCC2001). The tested bacterial suspensions were incubated in Luria–Bertani (LB) medium and fungi in Mueller–Hinton agar (HMA) medium at 28 °C for 12 h and diluted to be 1 × 10⁶ CFU/mL by the same medium. Then, the DMSO solution of each compound was diluted into the corresponding concentration using the LB or MHA medium; 100 µL solution of compound was added into the first well of a 96-well plate and resulted the initial tested concentration of each compound to be 50 µmol/L (DMSO < 0.5‰ in each well) and the concentration of each compound to be 25 µmol/L (DMSO < 0.5‰ in each well) in the second well of a 96-well plate after then following this method in sequence, adding 100 µL microbial suspension into a 96-well plate. The ciprofloxacin and DMSO were used as the positive and negative controls, respectively. All experiments were repeated three times. MIC values were assessed by whether compounds can inhibit the growth of microorganisms [19].

3.7. Cytotoxic Activity Assay

Cell proliferation was measured with the CCK-8 method. By the dye of WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-etrazolium, monosodium salt) was reduced by dehydrogenase in cells to form a water-soluble tetrazolium salt product (formazan dye) with orange color. In the measurement, the amount of the formazan dye is proportional to the number of living cells. Finally, the cell viability can be estimated by recording the optical density (OD) of formazan dye at 450 nm using a microplate reader [27].

A cell suspension of 100 μL was dispensed (adherent cell viewed 5 × 10⁴/mL and suspension cell viewed 9 × 10⁴/mL) in 96-well plates. With doxorubicin hydrochloride as positive drug and DMSO as control, plates were pre-cultured for 24 h, followed by treatments with various concentrations of compound (eight concentration gradients were set for each sample for IC₅₀ determination and three multiple holes were set for each concentration, n = 3). Keep the 96-well plates at 37 °C in an incubator with 5% CO₂ for 72 h. After the aspiration of the old medium, the 10-fold diluted CCK-8 (100 μL) solution was added to each well of the plate, which was then incubated for another 3 h. An absorbance microplate reader was used to measure the absorbance at 450 nm. The optical density values (OD) of each well represented the survival/proliferation of cells. The toxicity is expressed by cell inhibition. The half inhibitory concentration (IC₅₀) was defined as the concentration causing 50% inhibition, each group of data has 8 concentration gradient responses. The IC₅₀ value is calculated by curve fitting using the software GraphPad Prism 8 (version 8.0.2, from GraphPad Software Inc., Boston, MA, USA), the experimental results are expressed in IC₅₀ ± SD [28,29].

Cell inhibition rate = (OD_Control − OD_Drug)/(OD_Control − OD_Blank) × 100%.

The tested cell lines: A549: human lung cancer cells; MKN-45: human gastric cancer cells; HCT116: human colon cancer cells; K562: human chronic myeloid leukemia cells; DU145: human prostate cancer cells; SF126: human brain tumor cells; A-375: human malignant melanoma cells; MCF-7: human breast cancer cells; 786-O: human renal clear cell adenocarcinoma cells; PATU8988T: human pancreatic cancer cells; 5637: human bladder cancer cells; HeLa: human cervical cancer cells; TE-1: human esophageal cancer cells; GBC-SD: human gallbladder cancer cells; HepG2: human hepatoma cells; CAL-62: human thyroid cancer cells; HOS: human osteosarcoma cells; A-673: human rhabdomyosarcoma cells; L-02: human normal liver cells.

4. Conclusions

Two new 4-hydroxy-2-pyridone alkaloids were isolated from an endophytic fungus Aspergillus sp. GZWMJZ-606, which was obtained from Houttuynia cordata Thunb. Compounds 1 and 2 are the first example of 4-hydroxy-2-pyridone alkaloids possessing novel 7-oxidicyclo[2.2.1]heptane part. Compound 1 exhibited broad-spectrum cytotoxicity against 10 cancer cell lines with the IC₅₀ values of 4.35–9.72 µM, and showed selective activities against S. aureus and MRSA S. aureus with MIC values of 12.5 µM. The discovery of novel 4-hydroxy-2-pyridinone alkaloids can provide a material basis for the discovery of potential drug molecules.