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

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.


Structure Elucidation
Compound 1 was obtained as a yellow crystal. The molecular formula was deduced as C 24 H 31 NO 7 based on the HRESIMS ion peak at m/z 468.19861 [M + Na] + (calcd. for C 24 H 31 NO 7 Na = 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 sp 3 -methylenes, eight sp 3 -methines, three sp 2 -methines, one sp 3 -quaternary carbon, five sp 2 -quaternary carbons (including two carbonyls) (Table 1), which was also similar to those of compound 3, especially for the important 1 H NMR signals, such as two methyl groups at H 3 -11(δ H 0.94) and H 3 -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 1 H-1 H COSY correlations ( Figure 2) 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.    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 2 was obtained as a yellow powder. The molecular formula was deduced as C 24 H 31 O 6 N 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 sp 3     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  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). 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  Compounds 1-4 were tested for their antimicrobial activities against nine pathogenic microorganisms. As shown in Table 2, 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). 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.

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.

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).

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 × 10 6 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].

Cytotoxic Activity Assay
Cell proliferation was measured with the CCK-8 method. By the dye of WST-8 (2-(2methoxy-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 × 10 4 /mL and suspension cell viewed 9 × 10 4 /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 IC 50 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% CO 2 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 (IC 50 ) was defined as the concentration causing 50% inhibition, each group of data has 8 concentration gradient responses. The IC 50 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 IC 50 ± SD [28,29].

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.