Penifuranone A: A Novel Alkaloid from the Mangrove Endophytic Fungus Penicillium crustosum SCNU-F0006

One previously undescribed alkaloid, named penifuranone A (1), and three known compounds (2–4) were isolated from the mangrove endophytic fungus Penicillium crustosum SCNU-F0006. The structure of the new alkaloid (1) was elucidated based on extensive spectroscopic data analysis and single-crystal X-ray diffraction analysis. Four natural isolates and one new synthetic derivative of penifuranone A, compound 1a, were screened for their antimicrobial, antioxidant, and anti-inflammatory activities. Bioassays revealed that penifuranone A (1) exhibited strong anti-inflammatory activity in vitro by inhibiting nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells with an IC50 value of 42.2 μM. The docking study revealed that compound 1 exhibited an ideal fit within the active site of the murine inducible nitric oxide synthase (iNOS), establishing characteristic hydrogen bonds.


Introduction
Natural products remain an important source of lead compounds and innovative medicines.From 1981 to 2019, 49.5% of all drugs approved by FDA were derived from natural products or their derivatives [1].Marine microorganisms are considered to be an important source of natural products with marvelous structures and numerous biological activities [2].Mangrove forests are salt-tolerant woody plants that form coastal intertidal and fragile ecosystems at the intersection between land and water [3].The intricate root system of mangroves shelters abundant microorganisms [4].Among the mangrove microorganisms, mangrove fungi are extremely interesting and considered as an important resource of valuable natural products with chemical structures and biological diversity [5,6].Some of the key secondary metabolites derived from mangrove endophytic fungi include alkaloids [7,8], terpenoids [9], polyketides [10,11], peptides [12,13], etc. Alkaloids constitute one of the largest categories among these secondary metabolites and have a wide range of biological activities, such as anticancer [14,15], anti-neurodegenerative [16], anti-diabetic [17], anti-inflammatory [18], and antimicrobial activities [19], among others.Exploiting new alkaloid natural products is of great importance for drug development.
At present, microbial infection is a serious threat to human health [20,21].The rapid increase in the bacterial resistance to the available antibiotics is a serious problem for the treatment of various infections globally.In 2019, 4.95 million people died globally due to drug-resistant infections [22].Not only that, but persistent infection with pathogenic microorganisms can cause inflammatory response in various parts of the body [23,24].Chronic or sustained inflammatory responses can cause tissue damage; potentially trigger acute inflammatory diseases such as acute gastroenteritis, bacteremia, toxemia, and acute renal disease, as well as chronic diseases such as chronic nephritis, chronic hepatitis, chronic bronchitis, and cancer; or even result in death [25,26].As a result, the pursuit of innovative drugs which exhibit enhanced efficacy and fewer side effects and effectively manage inflammatory diseases and microbial infection is crucial [27].
Tetronic acids, which are common fragments among natural products originating from a variety of marine and terrestrial species [28][29][30], are vital in the fields of biology and chemistry [31].Studies on this kind of compound have experienced a renaissance due to these products' wide rang of biological activities, such as their anti-inflammatory, anti-tumor [32], antimicrobial activities [33], and so on.In addition, tetronic acids are also key pharmacophores of pesticides [34].They play an important role in controlling agricultural pathogenic microorganisms and pests, as well as in regulating plant growth, and have a far-reaching influence on searching for environment-benign agrochemicals for humankind.
During our ongoing search for new bioactive natural products from mangrove endophytic fungi, a novel alkaloid, penifuranone A (1), featuring a fantastic piperidine fused tetronic acid ring system and three known compounds were isolated and identified from Penicillium crustosum SCNU-F0006 (Figure 1).Furthermore, a new derivative of penifuranone A, compound 1a, was semi-synthesized to expand the new active compound library for bioassay screening.Compounds 1 and 2 were tested for their anti-inflammatory activity.And all the obtained compounds were used in antimicrobial and antioxidant activity biological assays.The results disclosed that penifuranone A (1) showed strong anti-inflammatory activity.Thus, the isolation, structure elucidation, and bioactivity evaluation of 1-4 and 1a are described herein.
renal disease, as well as chronic diseases such as chronic nephritis, chronic chronic bronchitis, and cancer; or even result in death [25,26].As a result, the innovative drugs which exhibit enhanced efficacy and fewer side effects and e manage inflammatory diseases and microbial infection is crucial [27].
Tetronic acids, which are common fragments among natural products o from a variety of marine and terrestrial species [28][29][30], are vital in the fields and chemistry [31].Studies on this kind of compound have experienced a renais to these products' wide rang of biological activities, such as their anti-inflamma tumor [32], antimicrobial activities [33], and so on.In addition, tetronic acids ar pharmacophores of pesticides [34].They play an important role in controlling ag pathogenic microorganisms and pests, as well as in regulating plant growth, a far-reaching influence on searching for environment-benign agrochemicals fo kind.
During our ongoing search for new bioactive natural products from mangr phytic fungi, a novel alkaloid, penifuranone A (1), featuring a fantastic piperid tetronic acid ring system and three known compounds were isolated and ident Penicillium crustosum SCNU-F0006 (Figure 1).Furthermore, a new derivativ furanone A, compound 1a, was semi-synthesized to expand the new active c library for bioassay screening.Compounds 1 and 2 were tested for their anti-infla activity.And all the obtained compounds were used in antimicrobial and antio tivity biological assays.The results disclosed that penifuranone A (1) showed st inflammatory activity.Thus, the isolation, structure elucidation, and bioactivi tion of 1-4 and 1a are described herein.

Structure of Compound 1
Compound 1 was obtained as a colorless crystal with the molecular formula NO5, with 7 degrees of unsaturation deduced from its HRESIMS ion peak in mode at m/z 264.0877 [M − H] − (calcd for C13H14NO5, 264.0876) (Figure S7).The of compound 1, along with the HSQC data (Table 1, Figure S11), indicated the p

Semi-Synthesis of Compound 1a
Due to the fact that the hexadecanoyl group always benefits the antimicrobial activity, we semi-synthesized a hexadecanoyl derivative of compound 1 with a limited available amount of the isolated compound 1 (8 mg) [31,38].The synthetic route for compound 1a is depicted in Scheme 1.One step reaction of penifuranone A (1) with hexadecanoyl chloride gave 1a in up to 90% yields.The structure of target compound 1a was confirmed by extensive spectroscopic methods, including HRESIMS (Figure S16), 1 H NMR (Figure S17), and 13 C NMR (Figure S18).

Semi-Synthesis of Compound 1a
Due to the fact that the hexadecanoyl group always benefits the antimicrobial activity, we semi-synthesized a hexadecanoyl derivative of compound 1 with a limited available amount of the isolated compound 1 (8 mg) [31,38].The synthetic route for compound 1a is depicted in Scheme 1.One step reaction of penifuranone A (1) with hexadecanoyl chloride gave 1a in up to 90% yields.The structure of target compound 1a was confirmed by extensive spectroscopic methods, including HRESIMS (Figure S16), 1 H NMR (Figure S17), and 13 C NMR (Figure S18).

Semi-Synthesis of Compound 1a
Due to the fact that the hexadecanoyl group always benefits the antimicrobial activity, we semi-synthesized a hexadecanoyl derivative of compound 1 with a limited available amount of the isolated compound 1 (8 mg) [31,38].The synthetic route for compound 1a is depicted in Scheme 1.One step reaction of penifuranone A (1) with hexadecanoyl chloride gave 1a in up to 90% yields.The structure of target compound 1a was confirmed by extensive spectroscopic methods, including HRESIMS (Figure S16), 1 H NMR (Figure S17), and 13 C NMR (Figure S18).Scheme 1.The semi-synthesis of 1a.

Antimicrobial Assay
The antimicrobial activities of compounds 1−4 and 1a were investigated.As shown in Table 2, all the five compounds displayed modest inhibitory activities against Fusarium oxysporum and Penicillium italicum, with MIC values of 0.25 mg/mL, except for 1a, with an MIC of 0.125 mg/mL to Penicillium italicum.The results from Table 3 indicate that compounds 1−4 and 1a can inhibit the growth of Pseudomonas aeruginosa, with MIC values ranging from 0.5 mg/mL to 0.125 mg/mL.The SAR analysis indicated that the antimicrobial potency of compound 1a against Penicillium italicum and Pseudomonas aeruginosa was higher than that of compound 1.As for the other tested strains, these compounds showed no obvious inhibitory activity.

Antimicrobial Assay
The antimicrobial activities of compounds 1-4 and 1a were investigated.As shown in Table 2, all the five compounds displayed modest inhibitory activities against Fusarium oxysporum and Penicillium italicum, with MIC values of 0.25 mg/mL, except for 1a, with an MIC of 0.125 mg/mL to Penicillium italicum.The results from Table 3 indicate that compounds 1-4 and 1a can inhibit the growth of Pseudomonas aeruginosa, with MIC values ranging from 0.5 mg/mL to 0.125 mg/mL.The SAR analysis indicated that the antimicrobial potency of compound 1a against Penicillium italicum and Pseudomonas aeruginosa was higher than that of compound 1.As for the other tested strains, these compounds showed no obvious inhibitory activity.

Escherichia coli
Staphylococcus aureus

Anti-Inflammatory Assay
Initially, we evaluated the cytotoxic effects of all the compounds on RAW 264.7 cells (Table 4).It was observed that compounds 1 and 2 showed low cytotoxicity, with IC 50 values above 50 µM, whereas the IC 50 values for the remaining compounds were below this threshold.Consequently, we focused our subsequent anti-inflammatory activity assessments solely on compounds 1 and 2. The anti-inflammatory activities of compounds 1 and 2 were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.Notably, compounds 1 and 2 showed significant inhibitory activity on NO production, with IC 50 values of 42.22 and 37.01 µM, respectively (Dexamethasone as a control, IC 50 = 58.21µM).In the MTT assay, none of the compounds except for the three compounds of 1a, 3, and 4 exhibited significant cytotoxic effects on the RAW264.7 cells (IC 50 < 50 µM).

DPPH Scavenging Assay
Compounds 1-4 and 1a were evaluated for their antioxidant activity using the DPPH scavenging assay (Table 5).Compounds 1 and 2 showed moderate radical scavenging activity, with IC 50 values of 180.2 µM and 120.5 µM, respectively, while the other compounds exhibited no inhibitory effect at a concentration of 200 µM.Vitamin C was used as a control (IC 50 = 68.4µM).

Molecular Docking Studies
Modulating the excessive production of nitric oxide is commonly pursued by targeting the inducible nitric oxide synthase (iNOS) enzyme to suppress its expression or activity [39,40].To explore the suppressive action of compounds 1 and 2 on nitric oxide (NO) synthesis, the study focused on the interplay and interaction pattern between compounds 1-2 and the inducible nitric oxide synthase (iNOS) enzyme, as depicted in the Protein Data Bank entry 3E6T [41]; thus, a molecular docking study was carried out using AUTODOCK 4.2.6 modeling software.The docking procedure was validated by the docking of the ligand indomethacin (positive drug) in the active site of iNOS and a rootmean-square deviation (RMSD) of 0.12 Å to the X-ray structure.The results showed that the binding energy of compound 1 (−6.32 Kcal/mol) and compound 2 (−5.58Kcal/mol) was comparable to that of the positive drug indomethacin (−7.45 Kcal/mol).Further observations showed that indomethacin formed a hydrogen bond with the key amino acid residue GLU-371, two hydrogen bonds with the amino acid residue ARG-260, and a hydrogen bond with GLN-257 in the iNOS active pocket (Figure 4A) [42,43].Compound 1 formed a hydrogen bond with the key amino acid residue ARG-382 through the ester group, three hydrogen bonds with the residue ASP-379, and one hydrogen bond with the residue HEM-901 in the iNOS active pocket (Figure 4B).Compound 2 formed three hydrogen bonds with the key amino acid residue GLU-371 through two hydroxyl groups, one hydrogen bond with the residue 1A2-905, and one hydrogen bond with the residue HEM-901 in the iNOS active pocket (Figure 4C).As a result, compounds 1 and 2 exhibited comparable inhibitory activity on iNOS to the positive control drug indomethacin (Table 4).
drogen bonds with the amino acid residue ARG-260, and a hydrogen bond with GLN-257 in the iNOS active pocket (Figure 4A) [42,43].Compound 1 formed a hydrogen bond with the key amino acid residue ARG-382 through the ester group, three hydrogen bonds with the residue ASP-379, and one hydrogen bond with the residue HEM-901 in the iNOS active pocket (Figure 4B).Compound 2 formed three hydrogen bonds with the key amino acid residue GLU-371 through two hydroxyl groups, one hydrogen bond with the residue 1A2-905, and one hydrogen bond with the residue HEM-901 in the iNOS active pocket (Figure 4C).As a result, compounds 1 and 2 exhibited comparable inhibitory activity on iNOS to the positive control drug indomethacin (Table 4).

Plant Material
Acanthus ilicifolius L. mangrove plants were collected from the Yangjiang Mangrove Nature Reserve in Guangdong province, China (E 111 5).
The management personnel of the mangrove nature reserve classified and identified various mangrove plants (Mangrove Management Center in Hailing District, Yangjiang City, China).Morphological Features: the leaves of Acanthus ilicifolius L. are opposite, pinnately cleft or shallowly lobed, and often have sharply spiny teeth.In florescence and Flowers: The terminal spike-like inflorescence of Acanthus ilicifolius L. is borne at the top, with large bracts that commonly have spines along the edges and smaller bracteoles that may be absent.The corolla is bilabiate, with the upper lip being extremely reduced and appearing almost like a single lip, while the lower lip is large, spreading, and trifid.The calyx is lobed 4-5 times, and the corolla tube is very short, nearly spherical, or has a very narrow cylindrical shape.Fruit and Seeds: The capsule of Acanthus ilicifolius L. is elliptical, laterally compressed, lustrous, and chestnut brown in color, containing four The management personnel of the mangrove nature reserve classified and identified various mangrove plants (Mangrove Management Center in Hailing District, Yangjiang City, China).Morphological Features: the leaves of Acanthus ilicifolius L. are opposite, pinnately cleft or shallowly lobed, and often have sharply spiny teeth.In florescence and Flowers: The terminal spike-like inflorescence of Acanthus ilicifolius L. is borne at the top, with large bracts that commonly have spines along the edges and smaller bracteoles that may be absent.The corolla is bilabiate, with the upper lip being extremely reduced and appearing almost like a single lip, while the lower lip is large, spreading, and trifid.The calyx is lobed 4-5 times, and the corolla tube is very short, nearly spherical, or has a very narrow cylindrical shape.Fruit and Seeds: The capsule of Acanthus ilicifolius L. is elliptical, laterally compressed, lustrous, and chestnut brown in color, containing four seeds.The seeds are laterally compressed, nearly round, or broadly ovate and possess a funicular hook.

Fungal Material
The fungal strain Penicillium crustosum SCNU-F0006 was isolated from the fresh stems Acanthus ilicifolius L. mangrove plants collected from the Yangjiang Mangrove Nature Reserve in Guangdong province, China (Figure 5).The management personnel of the mangrove nature reserve classified and identified various mangrove plants.On 9 October, 2021, fresh samples collected from the mangrove wetland conservation area were first cleaned with sterile water, with the plant's roots, stems, leaves, flowers, fruits, and seeds being separated and placed individually.Sterilized forceps were used to sequentially transfer the plant tissues through a 3% sodium hypochlorite solution and a 75% ethanol solution for disinfection, followed by rinsing with sterile water to remove residual solvents.The stems of the plant known as Acanthus ilicifolius L. were then cut into regular small pieces (approximately 0.2 × 0.6cm) and transferred onto a Bengal Rose Agar plate that had been sterilized with high-pressure steam.After sealing the plate, it was incubated at room temperature in a sterile environment.After 2-3 days, as colonies emerged, a sterile loop that had been flamed with an alcohol lamp was used to take sections of mycelium from different colonies and transfer them to Potato Dextrose Agar (PDA) medium that had been sterilized with high-pressure steam.The process was repeated until a single pure colony was present in each plate, indicating the completion of purification.The purified strains were then transferred to sterile PDA slant tubes for storage in a refrigerator for future use.The fungus was identified by using ITS gene sequencing (seq: ACCCTCCGTTTAGGGGAACTGCGGAAGGATCATTACCGAGTGAGGGCCCTCTGG GTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGCCTTAA CTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCG AACTCTGTCTGAAGATTGAAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCA

Fungal Material
The fungal strain Penicillium crustosum SCNU-F0006 was isolated from the fresh stems Acanthus ilicifolius L. mangrove plants collected from the Yangjiang Mangrove Nature Reserve in Guangdong province, China (Figure 5).The management personnel of the mangrove nature reserve classified and identified various mangrove plants.On 9 October, 2021, fresh samples collected from the mangrove wetland conservation area were first cleaned with sterile water, with the plant's roots, stems, leaves, flowers, fruits, and seeds being separated and placed individually.Sterilized forceps were used to sequentially transfer the plant tissues through a 3% sodium hypochlorite solution and a 75% ethanol solution for disinfection, followed by rinsing with sterile water to remove residual solvents.The stems of the plant known as Acanthus ilicifolius L. were then cut into regular small pieces (approximately 0.2 × 0.6 cm) and transferred onto a Bengal Rose Agar plate that had been sterilized with high-pressure steam.After sealing the plate, it was incubated at room temperature in a sterile environment.After 2-3 days, as colonies emerged, a sterile loop that had been flamed with an alcohol lamp was used to take sections of mycelium from different colonies and transfer them to Potato Dextrose Agar (PDA) medium that had been sterilized with high-pressure steam.The process was repeated until a single pure colony was present in each plate, indicating the completion of purification.The purified strains were then transferred to sterile PDA slant tubes for storage in a refrigerator for future use.The fungus was identified by using ITS gene sequencing (seq: ACCCTCCGTTTAGGGGAACTGCGGAAGGATCATTACCGAGTGAGGGC-CCTCTGG GTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGGGCCCGC-CTTAA CTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACC-CTCG AACTCTGTCTGAAGATTGAAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCA ACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATACG TAAT-GTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCC CCTGGTA TTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGG CTTGTGTGTT GGGCCCCGTCCCCCGATCTCCGGGGGACGGGCCCGAAAGGCAGC GGCGGCACCGC GTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGG CCCGGCCGGCGCTT GCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCA GGTAGGGATACCCGCT-GAACTTAAGCATATCAATAAGGCGGAGGAAA).The sequence data have been stored in Gen Bank with accession no.MH345907.The BLAST result showed that the sequence was most similar (99%) to the sequence of Penicillium crustosum.A voucher strain was deposited in School of Chemistry, South China Normal University, Guangzhou, China, with the access code SCNU-F0006.

Infrared Spectroscopy
For infrared spectroscopy, the following step-by-step protocol was used: Firstly, use an electronic balance to weigh out the required amounts of the sample to be tested: 1 mg compound and 120 mg of spectroscopic-grade potassium bromide (KBr) that has been dried in an oven.Then, place the dried samples and KBr into an agate mortar and grind them for 2-3 min until a uniform powder is formed.Transfer the ground powder into a pellet press mold.Utilize a specially designed mold to ensure that the sample powder is evenly distributed, with the center slightly higher than the edges.Place the mold containing the sample into the press and apply pressure (typically between 10 and 20 MPa) for 40 s.During the pressing process, connect to a vacuum pump to evacuate air and moisture from both the sample and KBr powder.After pressing, slowly release the pressure and remove the pressed KBr pellet.Place the pressed pellet into an infrared spectrometer for spectral scanning.To eliminate the infrared absorption peaks of the KBr carrier itself, it is usually necessary to first scan a pure KBr pellet as a background.Based on the obtained infrared spectrum, analyze the characteristic absorption peaks of the sample to determine its chemical structure.

X-ray Crystal Data for Compound 1
Compound 1 was filtered through a microporous membrane, and a suitable single crystal was successfully grown using the slow evaporation method from a 2:1 mixed solvent system of methanol and dichloromethane.The crystal structure and absolute configuration of 1 were determined by using data collected at T = 100 K with Cu Kα radiation (λ = 0.0031 Å) on an Agilent Gemini Ultra diffractometer.The structure was solved by direct methods using SHELXS-9729 and refined using full-matrix least-squares difference Fourier techniques [44].Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions and refined using a riding model.Crystallographic data of 1 were saved in Cambridge Crystallographic Data Centre.(Deposition number: CCDC 2337713 for 1) Copies of the data can be obtained, free of charge, upon application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: 44-(0)1223-336033; e-mail: deposit@ccdc.cam.ac.uk).

In Vitro Antifungal Activity
The bioactivity test screened the following four phytopathogenic microorganisms: Fusarium oxysporum; Penicillium italicum; Banana Colletotrichum gloeosporioides; and Colletrichum litchi Trag.The minimum inhibitory concentration (MIC) was determined using the broth dilution method, with a minor modification [45][46][47][48].Fungi were removed from the refrigerator and cultured dynamically at a constant temperature in Potato Dextrose Broth at room temperature for 72 h.The fungal liquid was diluted with broth to 0.5 McFarland turbidity.The sample was prepared into an initial solution of 1 mg/mL with dimethyl sulfoxide (DMSO), and the concentrations of the 1st to 12th wells of the 96-well plate sample group were made by the double dilution method (mg/mL): 1, 0.5, 0.25, 0.125, and 0.0625.Each plate also contained a positive control (ciprofloxacin or carbendazim), as well as a sterile control and growth control (containing broth and fungal liquid, no compound).The plates were incubated at 35 • C for 72 h.The use of 10 µL of a solution with a concentration of 11 mg/mL of Esazurin added to each well of a 96-well plate, followed by incubation at 37 • C for 3 h, allowed for the observation of color changes to determine the minimum inhibitory concentration (MIC) values.A blue or purple color indicates that the inoculated bacteria have been completely inhibited, a purplish-red color suggests the partial inhibition of the fungal, and a pink color indicates fungal growth.

In Vitro Antibacterial Activity
The bioactivity test screened the following four human pathogenic microorganisms: Pseudomonas aeruginosa (ATCC 9027); Salmonella typhimurium (ATCC 6539); Escherichia coli (ATCC 25922); and Staphylococcus aureus (ATCC 12598).The minimum inhibitory concentration was determined using the broth dilution method [46,47], with a minor modification.Bacteria were removed from the refrigerator and cultured dynamically at a constant temperature in a Mueller-Hinton Broth at room temperature for 24 h.The bacterial liquid was diluted with broth to 0.5 McFarland turbidity.The sample was prepared into an initial solution of 1 mg/mL with dimethyl sulfoxide (DMSO), and the concentrations of the 1st to 12th wells of the 96-well plate sample group were made by the double dilution method (mg/mL): 1, 0.5, 0.25, 0.125, and 0.0625.Each plate also contained a positive control (ciprofloxacin or carbendazim), as well as a sterile control and growth control (containing broth and bacterial liquid, no compound).The plates were incubated at 35 • C for 24 h.The use of 10 µL of a solution with a concentration of 11 mg/mL of Esazurin added to each well of a 96-well plate, followed by incubation at 37 • C for 3 h, allowed for the observation of color changes to determine the minimum inhibitory concentration (MIC) values.A blue or purple color indicates that the inoculated bacteria have been completely inhibited, a purplish-red color suggests the partial inhibition of the bacteria, and a pink color indicates bacterial growth [48,49].

Anti-Inflammatory Assay
The MTT method was utilized to assess cell viability [25][26][27].RAW 264.7 cells, at a concentration of 5 × 10 5 cells/mL, were plated in 96-well plates and exposed to lipopolysaccharide (LPS, at 1 µg/mL) in the presence or absence of the test compounds for a duration of 24 h.Following this, 100 µL of a 2 mg/mL MTT solution was introduced into each well, and after a 4 h incubation, the resulting formazan precipitates were dissolved in 100 µL of DMSO.The optical density was then determined at 490 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA).The results were presented as the average percentage of cell viability relative to the untreated control group.
Compounds 1-2 were evaluated for their inhibitory effects on LPS-stimulated NO production in RAW 264.7 cells using the Griess assay [45].The cell line RAW 264.7, obtained from the Cell Bank of the Chinese Academy of Sciences (CBCAS) in Shanghai, China, was propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).In the logarithmic growth phase, the cells were digested with trypsin and inoculated into 96-well plates (1 × 10 5 /well).After being cultured at 37 • C with 5% CO 2 for 12 h, the cells were treated with DMEM containing different concentrations of compounds (0, 3.125, 6.25, 12.5, 25, 50, and 100 µM).Two hours later, the cells were treated with LPS at a concentration of 1 and 2 (1 µg/mL) and incubated under standard cell culture conditions for a period of 24 h.Fifty microliters of cell supernatant was mixed with an equal volume of Griess reagent (1% anhydrous p-aminobenzene sulfonic acid and 0.1% N-1-naphthalene ethylenediamine hydrochloride in 5% concentrated phosphoric acid).Absorbance was measured with a microplate reader at 540 nm.The IC 50 values were calculated using the SPSS 25.0 statistical software analysis package based on the inhibition rate of each group.

DPPH Scavenging Assay
The assay for scavenging 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6trinitrophenyl)hydrazyl (DPPH free radicals) was conducted following a previously established modified protocol [49].The DPPH was dissolved in ethanol to prepare a 0.5 mmol/L DPPH ethanolic solution (10 mL), which was stored away from light.Subsequently, 100 µL of the DPPH solution was added to each well of a 96-well plate.The sample was prepared into an initial solution of 200 µM with ethanol, and the concentrations of the 1st to 12th wells of the 96-well plate sample group were made by the double dilution method (µM): 200,100, 50, 25, and 12.5 µM.The reaction was monitored by measuring the absorbance at 490 nm using a microplate reader (iMark, Bio-Rad, Hercules, CA, USA) after shaking the reaction for 30 min at 37 • C in the dark.The DPPH radical scavenging activity was determined by a comparison of the absorbance of the samples and a blank control.Vitamin C was used as a positive control at the same concentration as the other samples.

Molecular Docking
The AutoDock 4.2.6 software package includes an AutoDock tool that is utilized for conducting virtual docking simulations [39].This is a prevalent approach in docking that provides the ligand with ample flexibility while preserving the target protein's structural integrity.The X-ray crystal structure of iNOS (PDB ID: 3E6T) [41] was obtained from the RCSB protein database (PDB).Prior to the docking simulation, the original ligand and water molecules were removed from the crystal structure using PyMOL, and the resulting protein structure was exported in PDB format as 'receptor.pdb'.The molecular structure was initially sketched in two dimensions using ChemDraw's 2D 17.1 software.Subse-

Figure 3 .
Figure 3.The single-crystal X-ray crystallography structure of compound 1.

Figure 3 .
Figure 3.The single-crystal X-ray crystallography structure of compound 1.

Figure 3 .
Figure 3.The single-crystal X-ray crystallography structure of compound 1.
concentration values (MIC, mg/mL); data are shown as the means from three parallel experiments.b positive control.
concentration values (MIC, mg/mL); data are shown as the means from three parallel experiments.b positive control.
concentration values (MIC, mg/mL); data are shown as the mean from three parallel experiments.b positive control.

Figure 4 .
Figure 4. (A) Docking results regarding the binding positions of the positive drug indomethacin in iNOS.(B) Predicted binding mode of compound 1 docked into iNOS (PDB: 3E6T).(C) Predicted binding mode of compound 2 docked into iNOS (PDB: 3E6T).Indomethacin is shown as the green stick; compound 1 is the yellow stick; compound 2 is the purple stick; the red dashed lines represent H-bonds; the amino acids involved in hydrogen bond interactions are shown as cyan sticks).

Figure 4 .
Figure 4. (A) Docking results regarding the binding positions of the positive drug indomethacin in iNOS.(B) Predicted binding mode of compound 1 docked into iNOS (PDB: 3E6T).(C) Predicted binding mode of compound 2 docked into iNOS (PDB: 3E6T).Indomethacin is shown as the green stick; compound 1 is the yellow stick; compound 2 is the purple stick; the red dashed lines represent H-bonds; the amino acids involved in hydrogen bond interactions are shown as cyan sticks).
a .

Table 3 .
Antimicrobial activities against human pathogenic microorganisms of compounds 1−4 and 1a. a .
a minimum inhibitory concentration values (MIC, mg/mL); data are shown as the mean from three parallel experiments.b positive control.Scheme 1.The semi-synthesis of 1a.

Table 4 .
Anti-inflammatory activity of compounds 1-4 and 1a.cytotoxicity was evaluated with cell viability at 50 µM; data are shown as the means from three parallel experiments.c not tested.
a positive control.b
a positive control.b data are shown as the means from three parallel experiments.