Actinoquinazolinone, a New Quinazolinone Derivative from a Marine Bacterium Streptomyces sp. CNQ-617, Suppresses the Motility of Gastric Cancer Cells

A HPLC-UV guided fractionation of the culture broth of Streptomyces sp. CNQ-617 has led to the isolation of a new quinazolinone derivative, actinoquinazolinone (1), as well as two known compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3). The interpretation of 1D, 2D NMR, and MS spectroscopic data revealed the planar structure of 1. Furthermore, compound 1 suppressed invasion ability by inhibiting epithelial–mesenchymal transition markers (EMT) in AGS cells at a concentration of 5 µM. In addition, compound 1 decreased the expression of seventeen genes related to human cell motility and slightly suppressed the signal transducer and activator of the transcription 3 (STAT3) signal pathway in AGS cells. Together, these results demonstrate that 1 is a potent inhibitor of gastric cancer cells.


Introduction
Cancer is the uncontrolled growth and division of cells in the body [1].Gastric cancer, also known as stomach cancer, is responsible for an estimated 768,793 deaths, making it the fourth leading cause of cancer death in 2020 [2,3].Suppressed metastasis may be a target in gastric cancer therapy [4].During the epithelial-mesenchymal transition (EMT) process, cancer cells take on a mesenchymal cell phenotype to spread to a different part of the body.Therefore, EMT plays a significant role in metastatic cancers [5,6].Furthermore, the signal transducer and activator of transcription 3 (STAT3) is a kind of oncogene that can promote the invasion and migration potential of cancer cells [7].Thus, suppressing the EMT and STAT3 signal pathways is crucial for developing effective cancer therapies.
Marine microorganisms, including actinobacteria, have emerged as a promising source for the discovery of novel bioactive compounds with potential pharmacological properties [8,9].Actinobacteria are Gram-positive bacteria that have been extensively studied for their ability to produce secondary metabolites with a wide range of biological activities.Many of these compounds have demonstrated promising activity against various diseases, including cancer, bacterial infections, and viral infections [10,11].The high biodiversity of marine-derived actinobacteria provides a rich source of novel compounds, which is one of their main advantages [11].Additionally, marine environments offer unique ecological niches, including extreme temperatures, pressures, and salinity, which can produce unique compounds not found in terrestrial environments.Thus, marine-derived actinobacteria are considered a valuable resource for drug discovery and development [10][11][12].
Streptomyces sp.strain CNQ-617 is a marine-derived actinobacterium isolated from sediment samples collected from La Jolla Submarine Canyon in California.This strain has been shown to produce several bioactive compounds with potential therapeutic applications [10].Streptomyces is a well-known genus of actinobacteria known for producing a large number of secondary metabolites, including many clinically important antibiotics.Indeed, Streptomyces species are thought to have produced approximately two-thirds of all known antibiotics [11,13,14].Researchers have also discovered several novel bioactive compounds derived from marine-derived Streptomyces species in recent years.
Therefore, marine-derived Streptomyces and other actinobacteria are regarded as a valuable resource for the discovery of novel bioactive compounds with potential therapeutic applications [15][16][17][18][19][20][21][22][23][24][25][26].Marineosins A and B are two structurally related compounds that were first discovered from this strain.They are analogs of the natural product prodigiosin and have been shown to possess strong and selective anticancer activity against a variety of cancer cell lines by inducing apoptosis, inhibiting angiogenesis, and disrupting microtubule assembly [26,27].
In addition to marineosins A and B, this strain has also been shown to produce deoxyvasicinone, a compound with potential anti-melanogenic properties.Deoxyvasicinone has been shown to inhibit melanin production in murine and human melanoma cells, suggesting that it could be a promising agent for treating hyperpigmentation disorders [10].Moreover, further extensive investigation of crude extracts from Streptomyces sp.strain CNQ-617 has resulted in the isolation of a novel quinazolinone derivative, actinoquinazolinone (1).This study reported the isolation of actinoquinazolinone (1) along with two known compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3) (Figure 1), as well as the structural characterization and bioactivity of 1.
Mar. Drugs 2023, 21, 489 2 of 15 biodiversity of marine-derived actinobacteria provides a rich source of novel compounds, which is one of their main advantages [11].Additionally, marine environments offer unique ecological niches, including extreme temperatures, pressures, and salinity, which can produce unique compounds not found in terrestrial environments.Thus, marine-derived actinobacteria are considered a valuable resource for drug discovery and development [10][11][12].Streptomyces sp.strain CNQ-617 is a marine-derived actinobacterium isolated from sediment samples collected from La Jolla Submarine Canyon in California.This strain has been shown to produce several bioactive compounds with potential therapeutic applications [10].Streptomyces is a well-known genus of actinobacteria known for producing a large number of secondary metabolites, including many clinically important antibiotics.Indeed, Streptomyces species are thought to have produced approximately two-thirds of all known antibiotics [11,13,14].Researchers have also discovered several novel bioactive compounds derived from marine-derived Streptomyces species in recent years.
Therefore, marine-derived Streptomyces and other actinobacteria are regarded as a valuable resource for the discovery of novel bioactive compounds with potential therapeutic applications [15][16][17][18][19][20][21][22][23][24][25][26].Marineosins A and B are two structurally related compounds that were first discovered from this strain.They are analogs of the natural product prodigiosin and have been shown to possess strong and selective anticancer activity against a variety of cancer cell lines by inducing apoptosis, inhibiting angiogenesis, and disrupting microtubule assembly [26,27].
In addition to marineosins A and B, this strain has also been shown to produce deoxyvasicinone, a compound with potential anti-melanogenic properties.Deoxyvasicinone has been shown to inhibit melanin production in murine and human melanoma cells, suggesting that it could be a promising agent for treating hyperpigmentation disorders [10].Moreover, further extensive investigation of crude extracts from Streptomyces sp.strain CNQ-617 has resulted in the isolation of a novel quinazolinone derivative, actinoquinazolinone (1).This study reported the isolation of actinoquinazolinone (1) along with two known compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3) (Figure 1), as well as the structural characterization and bioactivity of 1.

Results and Discussion
Actinoquinazolinone (1) was isolated as a yellowish brown oil with a molecular formula of C13H14N2O6 based on the HR-FAB-MS ion at m/z 295.0924 [M + H] + (calcd for C13H15N2O6, 295.0930).The 1 H NMR spectrum of 1 (    Additional 2D NMR analyses allowed the structure of 1 to be constructed.The quinazolin-4-one unit was assigned by the NMR signals of six aromatic carbons at δC 105.7

Results and Discussion
Actinoquinazolinone (1) was isolated as a yellowish brown oil with a molecular formula of C 13 H 14 N 2 O 6 based on the HR-FAB-MS ion at m/z 295.0924 [M + H] + (calcd for C 13 H 15 N 2 O 6 , 295.0930).The 1 H NMR spectrum of 1 (Table 1, Figures S1-S5) displayed one hetero-aromatic proton at δ H 8.07 ( 1 H, s, H-2), two aromatic protons at δ H 7.46 (1H, s, H-5) and 6.98 (1H, s, H-8), one methoxy group at δ H 3.88 (3H, s, 6-Ome), and one exchangeable proton at δ H 10.35 (1H, s, OH).Based on spectroscopic data from 13 C NMR and HSQC, six quaternary carbons were assigned at δ C 159.7 (C-4), 148.2) and from the observation of the long-range HMBC correlation from H-12 to C-2 and C-4.Additionally, the unassigned hydroxy group was determined to be located at C-7 based on the carbon chemical shift of C-7 (δ C 153.0) and the molecular formula of the compound.Therefore, the final structure of actinoquinazolione (1) was determined to be 7-hydroxy-6-methoxy quinazolinone with a 3-hydroxy butanoic acid moiety, as shown in Figure 2.    Compound 2 was identified as 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one based on a comparison of its NMR data to that of a previously reported synthetic compound [28].However, it was the first report of the compound being isolated from a natural source.
Additionally, compounds 1 and 2 share structural similarities with previously reported compound 4-(7,8-dihydroxy-4-oxoquinazolin-3(4H)-yl) butanoic acid, which was isolated from the leather coral-derived fungus Xylaria sp.FM1005 [66].However, the structures of compounds 1 and 2 differ from that of the previously reported compound.In the quinazolinone moiety, compound 1 has a substituted methoxy group at C-6 and an additional hydroxy group attached at C-12, but no 8-OH group.Meanwhile, compound 2 has a similar structure to compound 1, except for the absence of the 3-hydroxybutanoic acid substituent on the amide moiety.In addition, 1-3 have been tested for their viability of cancer cell lines.The relative cell viability of cancer cell lines A549 (lung cancer), AGS (gastric cancer), and Caco-2 (colorectal cancer) was measured by an MTT assay after treatment with various concentrations of 1-3 for 48 h.The result showed that treatment with 100 µM of compound 1 significantly decreased the cell viability of A549, AGS, and Caco-2.However, 100 µM of compound 2 did not affect the cell viability of A549 and AGS, while suppressing the cell viability of Caco-2.Moreover, 100 µM of compound 3 suppressed the cell viability of AGS and Caco-2 while having no significant effect on the cell viability of A549 (Figure 3).Therefore, these results showed that compound 1 is more effective in reducing the cell viability of A549, AGS, and Caco-2 than the other two compounds.However, it should be noted that the observed cell viability reduction by 1-3 is modest even at 100 µM.
suppressing the cell viability of Caco-2.Moreover, 100 µM of compound 3 suppressed the cell viability of AGS and Caco-2 while having no significant effect on the cell viability of A549 (Figure 3).Therefore, these results showed that compound 1 is more effective in reducing the cell viability of A549, AGS, and Caco-2 than the other two compounds.However, it should be noted that the observed cell viability reduction by 1-3 is modest even at 100 µM.To confirm the concentration-dependent inhibitory effect of compound 1 on cancer motility, we evaluated invasion assays in A549, AGS, and Caco-2 cells.From the results (Figure 4A,B), 1 displayed a dose-dependent inhibitory effect on the invasion ability of AGS at concentrations from 1 to 5 µM, whereas the invasion ability of A549 and Caco-2 cells were not affected by treatment of 1.The results indicated that 1 has a higher suppression activity on the AGS invasion ability than A549 and Caco-2.To confirm the concentration-dependent inhibitory effect of compound 1 on cancer motility, we evaluated invasion assays in A549, AGS, and Caco-2 cells.From the results (Figure 4A,B), 1 displayed a dose-dependent inhibitory effect on the invasion ability of AGS at concentrations from 1 to 5 µM, whereas the invasion ability of A549 and Caco-2 cells were not affected by treatment of 1.The results indicated that 1 has a higher suppression activity on the AGS invasion ability than A549 and Caco-2.
suppressing the cell viability of Caco-2.Moreover, 100 µM of compound 3 suppressed the cell viability of AGS and Caco-2 while having no significant effect on the cell viability of A549 (Figure 3).Therefore, these results showed that compound 1 is more effective in reducing the cell viability of A549, AGS, and Caco-2 than the other two compounds.However, it should be noted that the observed cell viability reduction by 1-3 is modest even at 100 µM.To confirm the concentration-dependent inhibitory effect of compound 1 on cancer motility, we evaluated invasion assays in A549, AGS, and Caco-2 cells.From the results (Figure 4A,B), 1 displayed a dose-dependent inhibitory effect on the invasion ability of AGS at concentrations from 1 to 5 µM, whereas the invasion ability of A549 and Caco-2 cells were not affected by treatment of 1.The results indicated that 1 has a higher suppression activity on the AGS invasion ability than A549 and Caco-2.Cancer metastasis accounts for approximately 90% of cancer deaths [67,68].A poor prognosis can be caused by the EMT, which is linked to metastasis [69].E-cadherin is known to suppress cancer metastasis; the loss of its expression promotes the EMT markers Mar. Drugs 2023, 21, 489 6 of 14 E-cadherin, Snail, Slug, and Twist [69].We assessed whether compound 1 decreased the motility associated with EMT by using qPCR assays and Western blot assays (Figure 5A).Based on the result, treatment with 5 µM of 1 significantly promoted the protein and mRNA expression level of E-cadherin in AGS cells (Figure 5B,C).Furthermore, the protein and mRNA expression levels of the EMT effector N-cadherin and the EMT transcription factors Snail, Slug, and Twist were decreased in AGS by treatment with 5 µM of 1 (Figure 5B,C).
Cancer metastasis accounts for approximately 90% of cancer deaths [67,68].A poor prognosis can be caused by the EMT, which is linked to metastasis [69].E-cadherin is known to suppress cancer metastasis; the loss of its expression promotes the EMT markers E-cadherin, Snail, Slug, and Twist [69].We assessed whether compound 1 decreased the motility associated with EMT by using qPCR assays and Western blot assays (Figure 5A).Based on the result, treatment with 5 µM of 1 significantly promoted the protein and mRNA expression level of E-cadherin in AGS cells (Figure 5B,C).Furthermore, the protein and mRNA expression levels of the EMT effector N-cadherin and the EMT transcription factors Snail, Slug, and Twist were decreased in AGS by treatment with 5 µM of 1 (Figure 5B,C).Previous studies have shown that STAT3 and EMT interact with each other to promote cancer metastasis.EMT is the downstream mediator of STAT3 and, therefore, the upregulation of the positive effect of STAT3 on the EMT process [70].Western blot assays were performed to assess whether treatment with 1 affects the STAT3 protein level.The results (Figure 6A,B) indicate that 1 did not affect the total STAT3 protein level, whereas 5 µM of 1 slightly decreased the protein level of phosphorylated STAT3 (p-STAT3) in AGS cells.
Previous studies have shown that STAT3 and EMT interact with each other to promote cancer metastasis.EMT is the downstream mediator of STAT3 and, therefore, the upregulation of the positive effect of STAT3 on the EMT process [70].Western blot assays were performed to assess whether treatment with 1 affects the STAT3 protein level.The results (Figure 6A,B) indicate that 1 did not affect the total STAT3 protein level, whereas 5 µM of 1 slightly decreased the protein level of phosphorylated STAT3 (p-STAT3) in AGS cells.Finally, the Human Cell Motility RT2 Profiler PCR Array was used to examine the effect of compound 1 on the mRNA expression level of cell motility-related genes.The results (Figure 7) showed that treatment with 5 µM of 1 decreased the expression of ARP2/3 actin-related protein 2/3 (ACTR2/3), rho guanine nucleotide exchange factor (GEF) 7 (ARHGEF7), cell division cycle 42 (CDC42), V-crk sarcoma virus CT10 oncogene homolog (CRK), cortactin (CTTN), enabled homolog (ENAH), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF1R), mitogen-activated protein kinase 1 (MAPK1), Met proto-oncogene (hepatocyte growth factor receptor (MET), Phosphoinositide-3-kinase, catalytic (PIK3CA), rho family GTPase 3 (RND3), rho-associated, coiled-coil containing protein kinase 1 (ROCK1), Vimentin (VIM), WAS protein family 1 (WASF1), and walkout-Aldrich syndrome protein-like (WASL).These genes are related to cancer cell motility and could be used to treat cancer.ACTR2/3 regulates cell motility by playing an important role in actin dynamics and cytoskeleton organization [71].The ARHGEF7 gene stimulates cancer cell motility and invasiveness by modifying the cytoskeleton [72].CDC42 is as known a structural homolog of the Rho GTPase family; inhibition of CDC42 can reduce cancer progression by suppressing distinct GEFs [73].CRK is a regulator of kinase and overexpression of CRK is associated with adenocarcinomas of the stomach [74].CTTN is a regulator of actin polymerization by binding the actin-regulated protein complex ACTR2/3 [75].ENAH includes a member of the ENAH/VASP family which can modify cell morphology and adhesion in the metastasis process [76].IGF1 promotes cell proliferation by suppressing apoptosis in cancer [77].MAPK1 is closely related to invasion and metastasis via modulated EMT [78].The invasiveness and metastasis of Finally, the Human Cell Motility RT2 Profiler PCR Array was used to examine the effect of compound 1 on the mRNA expression level of cell motility-related genes.The results (Figure 7) showed that treatment with 5 µM of 1 decreased the expression of ARP2/3 actin-related protein 2/3 (ACTR2/3), rho guanine nucleotide exchange factor (GEF) 7 (ARHGEF7), cell division cycle 42 (CDC42), V-crk sarcoma virus CT10 oncogene homolog (CRK), cortactin (CTTN), enabled homolog (ENAH), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF1R), mitogen-activated protein kinase 1 (MAPK1), Met proto-oncogene (hepatocyte growth factor receptor (MET), Phosphoinositide-3-kinase, catalytic (PIK3CA), rho family GTPase 3 (RND3), rho-associated, coiled-coil containing protein kinase 1 (ROCK1), Vimentin (VIM), WAS protein family 1 (WASF1), and walkout-Aldrich syndrome protein-like (WASL).These genes are related to cancer cell motility and could be used to treat cancer.ACTR2/3 regulates cell motility by playing an important role in actin dynamics and cytoskeleton organization [71].The ARHGEF7 gene stimulates cancer cell motility and invasiveness by modifying the cytoskeleton [72].CDC42 is as known a structural homolog of the Rho GTPase family; inhibition of CDC42 can reduce cancer progression by suppressing distinct GEFs [73].CRK is a regulator of kinase and overexpression of CRK is associated with adenocarcinomas of the stomach [74].CTTN is a regulator of actin polymerization by binding the actin-regulated protein complex ACTR2/3 [75].ENAH includes a member of the ENAH/VASP family which can modify cell morphology and adhesion in the metastasis process [76].IGF1 promotes cell proliferation by suppressing apoptosis in cancer [77].MAPK1 is closely related to invasion and metastasis via modulated EMT [78].The invasiveness and metastasis of aggressive cancer cells link to the overexpression of oncogene MET [79].PIK3CA has been related to cancer cell motility, which is the second most frequent mutant oncogene.A statistical analysis showed that mutation of PIK3CA is the reason for more than 10% of cancer cases [80].RND3 may serve as a predictor of EMT upregulation [81].ROCK1 is a small GTPase Rho downstream effector that is crucial in cancer metastasis [82].VIM has been related to cancer metastasis by promoting the EMT process [83].WASF1, also known as WAVE1 (WASP family verprolin homologous protein 1), is associated with regulating actin cytoskeleton dynamics for cancer cell invasion and migration [84].
tistical analysis showed that mutation of PIK3CA is the reason for more than 10% of cancer cases [80].RND3 may serve as a predictor of EMT upregulation [81].ROCK1 is a small GTPase Rho downstream effector that is crucial in cancer metastasis [82].VIM has been related to cancer metastasis by promoting the EMT process [83].WASF1, also known as WAVE1 (WASP family verprolin homologous protein 1), is associated with regulating actin cytoskeleton dynamics for cancer cell invasion and migration [84].

General Experimental Procedures
Optical rotations were measured using a Kruss Optronic P-8000 polarimeter with a 5 cm cell.Infrared spectra were measured with a Varian Scimitar Series FT-IR spectrometer in methanol (MeOH).The NMR spectra were established by an Agilent NMR spectrometer (Agilent, Santa Clara, CA, USA, at 400 MHz for 1 H and at 100 MHz for 13 C) equipped at the Drug Development Research Core Center using the signals of the residual solvent as internal references (δH 2.50 ppm and δC 39.5 ppm for dimethyl sulfoxide-d6 (DMSO-d6) and δH 3.31 ppm and δC 4.91 ppm for deuterated methanol (CD3OD).The low-resolution LC/MS measurements were recorded on the Agilent Technologies 1260 quadrupole and Waters Micromass ZQ LC/MS system using a reversed-phase column (Phenomenex Luna C18 (2) 100 Å, 50 mm × 4.6 mm, 5 µm) at a flow rate of 1.0 mL/min at the National Research Facilities and Equipment Center (NanoBioEnergy Materials Center) at Ewha Womans University.Open column chromatography was performed using silica (40-63 µm, Merck silica gel 60, Kenilworth, NJ, USA) eluting with a gradient solvent of dichloromethane (CH2Cl2) and methanol (MeOH).The fractions were purified via semi-preparative HPLC using a Waters 996 Photodiode Array Detector HPLC coupled with a reversed-phase Phenomenex Luna C18 (2) (100 Å, 250 nm × 10 mm, 5 µm) column with a mixture of acetonitrile and H2O at a flow rate of 2.0 mL/min.High-resolution mass spectra were recorded on a JMS-700 mass spectrometer (JEOL Ltd., Tokyo, Japan) at Seoul National University.

Collection and Phylogenetic Analysis of the CNQ-617 Strain
The marine actinomycete strain CNQ-617 was isolated from a marine sediment sample collected offshore of La Jolla, California.The strain was specified as the MAR3 clade based on 16S rDNA analysis.The phylogenetic analysis revealed that this strain showed 99.7% similarity to Streptomyces cacaoi based upon the result of NCBI blast analysis of the

General Experimental Procedures
Optical rotations were measured using a Kruss Optronic P-8000 polarimeter with a 5 cm cell.Infrared spectra were measured with a Varian Scimitar Series FT-IR spectrometer in methanol (MeOH).The NMR spectra were established by an Agilent NMR spectrometer (Agilent, Santa Clara, CA, USA, at 400 MHz for 1 H and at 100 MHz for 13 C) equipped at the Drug Development Research Core Center using the signals of the residual solvent as internal references (δ H 2.50 ppm and δ C 39.5 ppm for dimethyl sulfoxide-d 6 (DMSO-d 6 ) and δ H 3.31 ppm and δ C 4.91 ppm for deuterated methanol (CD 3 OD).The low-resolution LC/MS measurements were recorded on the Agilent Technologies 1260 quadrupole and Waters Micromass ZQ LC/MS system using a reversed-phase column (Phenomenex Luna C18 (2) 100 Å, 50 mm × 4.6 mm, 5 µm) at a flow rate of 1.0 mL/min at the National Research Facilities and Equipment Center (NanoBioEnergy Materials Center) at Ewha Womans University.Open column chromatography was performed using silica (40-63 µm, Merck silica gel 60, Kenilworth, NJ, USA) eluting with a gradient solvent of dichloromethane (CH 2 Cl 2 ) and methanol (MeOH).The fractions were purified via semi-preparative HPLC using a Waters 996 Photodiode Array Detector HPLC coupled with a reversed-phase Phenomenex Luna C18 (2) (100 Å, 250 nm × 10 mm, 5 µm) column with a mixture of acetonitrile and H 2 O at a flow rate of 2.0 mL/min.High-resolution mass spectra were recorded on a JMS-700 mass spectrometer (JEOL Ltd., Tokyo, Japan) at Seoul National University.

Collection and Phylogenetic Analysis of the CNQ-617 Strain
The marine actinomycete strain CNQ-617 was isolated from a marine sediment sample collected offshore of La Jolla, California.The strain was specified as the MAR3 clade based on 16S rDNA analysis.The phylogenetic analysis revealed that this strain showed 99.7% similarity to Streptomyces cacaoi based upon the result of NCBI blast analysis of the partial 16S rDNA.The gene sequence data are available from GenBank (deposit #EU161093).

Cultivation and Extraction
Streptomyces strain CNQ-617 was cultured in 160 of 2.5-L Ultra Yield Flasks each containing 1 L of the medium (10 g/L of soluble starch, 2 g/L of yeast, 4 g/L of peptone, 10 g/L of CaCO 3 , 20 g/L of KBr, 8 g/L Fe 2 (SO 4 ) 3 •4H 2 O dissolved in 750 mL of natural seawater and 250 mL of distilled water) at 27 • C with constant shaking at 120 rpm.After Mar.Drugs 2023, 21, 489 9 of 14 15 days, the culture broth was with ethyl acetate (EtOAc; 160 L in total) to obtain 16.0 g of EtOAc extract.

Invasion Assay
Transwell containing polycarbonate membranes with 8 µm pores coated with 1% gelatin-coated polycarbonate was used to measure the invasion ability of AGS cells.The AGS cells were seeded in media containing 0.2% bovine serum albumin (BSA), then treated with 1 and 5 µM concentrations of compound 1 for 24 h and DMSO as a control.The lower chamber was filled with 600 µL RPMI containing 0.2% BSA and 6 µg/mL fibronectin (EMD Millipore Corp., Billerica, MA, USA) as a chemoattractant.Diff-Quik kit (Sysmex, Kobe, Japan) was used for the fixation and dying of AGS cells after 24 h the cells in the upper chambers were quantified using a K1-Fluo Confocal Microscope (Nanoscope Systems, Republic of Korea) and i-Solution FL Auto Software (IMT i-Solution Inc., Vancouver, QC, Canada) [88,89].

qPCR
Total RNA of AGS cells was extracted by RNAiso Plus (Takara, Otsu, Japan) according to the manufacturer's suggestions.Using a Moloney Murine Leukemia Virus (M-MLV) kit, total RNA (1 µg) from DMSO as well as 1 and 5 µM of treatment with compound 1 groups were converted to cDNA.SYBR Green (Enzynomics, Seoul, Republic of Korea) was used to evaluate relative gene expression and CFX (Bio-Rad, Hercules, CA, USA) was applied for analysis [90,91].

Western Blots
The AGS cells were seeded in 6-well plates for gown overnight, and then treated with DMSO as well as 1 and 5 µM concentrations of 1.After 24 h treatment, rinsed with PBS, then extracted using lysis buffer.The extracted protein was separated by SDS-PAGE.The membranes were incubated in blocking buffer (20 mmol/l Tris-HCl, 137 mmol/l NaCl, pH 7.6, 492 containing 0.1% Tween and 3% nonfat dry milk) for one hour and antibodies against E-cadherin and N-cadherin (BD Bioscience, San Jose, CA, USA); Snail/Slug and Twist (Abcam, London, UK); α-tubulin, STAT3, and p-STAT3 (Cell Signaling Technology, Danvers, MA, USA) were detected by horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) using the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Billerica, MA, USA).The Multi Gauge 3.0 (Fujifilm, Tokyo, Japan) software was used for analyzing the density of the bands [92,93].

Statistical Analysis
Data is represented as means ± standard deviation.All statistical analyses were carried out using the Sigma Plot software.Student's t-test was used to compare statistically significant differences between two groups.A p-value less than 0.05 was considered statistically significant.

Conclusions
In summary, the exploration of marine natural products from marine sediment-derived Streptomyces sp.CNQ-617 has led to the discovery of a novel compound, actinoquinazolinone (1), along with two previously reported compounds, 7-hydroxy-6-methoxy-3,4dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3).Furthermore, compound 1 exhibited moderate antibacterial activity against the Gram-positive bacteria K. rhizophila KCTC 1915 and weak inhibitory activities against B. subtilis KCTC 1021 and S. aureus KCTC 1927.In addition, compound 1 showed higher activity than compounds 2 and 3 in decreasing the cell viability of cancer cells.Moreover, compound 1 suppressed the invasion ability of the EMT and STAT3 signal pathways, and some of the cell motility-related genes.These findings highlight the potential of marine sedimentderived Streptomyces sp.CNQ-617 as a source of novel bioactive compounds that may have therapeutic applications.Further studies are required to determine the potential therapeutic effect and the potential side effect of 1.
Review and editing; S.-J.N.: Conceptualization, Methodology, and editing, Supervision; W.F.: Writing-Review and editing, Resources.All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Research Foundation of Korea, grant funded by the Korean Government (Ministry of Science and ICT; no.2021R1A4A2001251 to S.-J.N.).Isolation of the bacterium was a result of the financial support from the US National Cancer Institute (grant CA R37044848 to W.F.).
Institutional Review Board Statement: Not applicable.
Additional 2D NMR analyses allowed the structure of 1 to be constructed.The quinazolin-4-one unit was assigned by the NMR signals of six aromatic carbons at δ C 105.7 (C-5), 148.2 (C-6), 153.0 (C-7), 110.9 (C-8), 143.9 (C-9), and 113.8 (C-10), and two heteroaromatic carbons at δ C 147.1 (C-2) and 159.7 (C-4), along with the HMBC correlations from H-2 to C-4, C-9 and C-10; from H-5 to C-4, C-6, C-7 and C-10; and from H-8 to C-7, C-9 and C-10.In addition, HMBC correlations from 6-Ome to C-6 provided the attachment of the methoxy group at C-6.Furthermore, COSY cross peaks [H-11/H-12 and H-12/H-13] and long-range HMBC correlations of H-12 and H-13 with C-14 (δ C 172.2) as well as the carbon chemical shift of C-12 (δ C 65.0) allowed the construction of the 3-hydroxybutanoic acid moiety.This moiety was linked to the quinazolinone moiety through the nitrogen atom between C-2 and C-4 from the consideration of the carbon chemical shift of C-11 (δ C 51. 5), 148.2 (C-6), 153.0 (C-7), 110.9 (C-8), 143.9 (C-9), and 113.8 (C-10), and two heteroaromatic carbons at δC 147.1 (C-2) and 159.7 (C-4), along with the HMBC correlations from H-2 to C-4, C-9 and C-10; from H-5 to C-4, C-6, C-7 and C-10; and from H-8 to C-7, C-9 and C-10.In addition, HMBC correlations from 6-Ome to C-6 provided the attachment of the methoxy group at C-6.Furthermore, COSY cross peaks [H-11/H-12 and H-12/H-13] and long-range HMBC correlations of H-12 and H-13 with C-14 (δC 172.2) as well as the carbon chemical shift of C-12 (δC 65.0) allowed the construction of the 3-hydroxybutanoic acid moiety.This moiety was linked to the quinazolinone moiety through the nitrogen atom between C-2 and C-4 from the consideration of the carbon chemical shift of C-11 (δC 51.2) and from the observation of the long-range HMBC correlation from H-12 to C-2 and C-4.Additionally, the unassigned hydroxy group was determined to be located at C-7 based on the carbon chemical shift of C-7 (δC 153.0) and the molecular formula of the compound.Therefore, the final structure of actinoquinazolione (1) was determined to be 7hydroxy-6-methoxy quinazolinone with a 3-hydroxy butanoic acid moiety, as shown in Figure2.

Figure 4 .
Figure 4. Effect of actinoquinazolione (1) on the invasion ability of AGS, A549, and Caco2 cells.(A) representative images of each insertion in the invasion assay.(B) relative percentage of invaded

Figure 4 . 4 .
Figure 4. Effect of actinoquinazolione (1) on the invasion ability of AGS, A549, and Caco2 cells.(A) representative images of each insertion in the invasion assay.(B) relative percentage of invadedFigure 4. Effect of actinoquinazolione (1) on the invasion ability of AGS, A549, and Caco2 cells.(A) representative images of each insertion in the invasion assay.(B) relative percentage of invaded cells.Data are presented as the mean ± SD and analysis was performed by Student's t-test (n = 5).* p < 0.05, *** p < 0.001.

Figure 5 .
Figure 5.Effect of actinoquinazolione (1) on EMT markers in AGS cells.(A) Western blot analysis of E-cadherin, N-cadherin Snail/Slug, and Twist.(B) relative protein levels of E-cadherin, N-cadherin Snail/Slug, and Twist.(C) relative mRNA expression levels of E-cadherin, N-cadherin Snail, Slug, and Twist.mRNA expression levels were normalized against the glyceraldehyde 3-phosphate

Figure 6 .
Figure 6.Effect of actinoquinazolione (1) on STAT3 in AGS cells.(A) Western blot analysis of STAT3 and p-STAT3.(B) relative protein levels of STAT3 and p-STAT3.Data are presented as mean ± SD and analysis was performed by Student's t-test (n = 3).* p < 0.05.

Figure 6 .
Figure 6.Effect of actinoquinazolione (1) on STAT3 in AGS cells.(A) Western blot analysis of STAT3 and p-STAT3.(B) relative protein levels of STAT3 and p-STAT3.Data are presented as mean ± SD and analysis was performed by Student's t-test (n = 3).* p < 0.05.

d C , mult. b d H (J in Hz) COSY HMBC
400 MHz for 1 H NMR and 100 MHz for13C NMR.b Numbers of attached protons were determined by analysis of 2D spectra. a