Heasquinazolinone, a New Heat-Shock Metabolite Produced by Thermotolerant Streptomyces sp. HR41

Abstract

We previously discovered metabolites that are either enhanced or specifically produced by actinomycetes in high-temperature cultures, which were designated as heat-shock metabolites (HSMs). In this study, we investigated HSMs produced by thermotolerant Streptomyces sp. HR41. HPLC-UV analysis revealed the presence of a peak, designated HSM (1), with maximum UV absorptions at 204, 246, and 282 nm, in ethyl acetate extract of Streptomyces sp. HR41. NMR and MS analyses indicated that the planar structure of 1 was 3,4-dihydro-4-methyl-2(1H)-quinazolinone. The chiral-phase HPLC analysis and comparison of the optical rotation value with that of related compounds were performed to examine the stereochemistry of 1 at C4. Thus, 1 was estimated to be an enantiomeric mixture of (S)- and (R)-3,4-dihydro-4-methyl-2(1H)-quinazolinone (1a and 1b), with an approximate enantiomer ratio of S/R = 18 : 82. This is the first discovery of 1 as a natural product, and it was therefore designated (S)- and (R)-heasquinazolinone. The biological activity of 1a and 1b was evaluated using several bioassay systems. The compounds selectively suppressed the growth of HT29 cells in 3-dimensional (3D) culture, with 50% inhibitory concentrations of 19 and 30 µM, respectively, indicating no apparent cytotoxicity in 2D culture.

Introduction

Actinomycetes produce secondary metabolites with diverse chemical structures under a range of culture conditions. Because these secondary metabolites exhibit a wide variety of biological activities, they have been applied in multiple fields.^1,2) Many recent studies have reported that when cultured under certain conditions, actinomycetes can generate secondary metabolites that are not produced under normal conditions.³⁾ For example, a study examining the effect of varying culture conditions on a specific actinomycete strain proposed a method known as “One Strain Many Compounds” as a way to induce the production of secondary metabolites.⁴⁾ The importance of specific culture conditions such as the presence of metals, medium pH, light intensity, and typical nutrient sources has been reported.^5,6) In this context, we have examined the effect of culture temperature on the activation of secondary metabolism in actinomycetes, particularly when cultured at high temperature.

Actinomycetes are typically cultured at 28–30°C. We previously identified 57 actinomycete strains capable of growing at 45 and 30°C from an in-house actinomycete library of 3160 strains. We then attempted to analyze the metabolites produced during high-temperature culture of these actinomycetes. In a previous study, we found that production of metabolites could either be specifically induced or upregulated by culturing the actinomycetes at high temperature. The compounds generated in this manner were designated heat-shock metabolites (HSMs).⁷⁾ We confirmed that approximately 60% of actinomycetes that can be cultured at 45°C produced HSMs. The isolation and structural determination of these metabolites led to the identification of the new compounds noaoxazole, dihydromaniwamycin E, and streptolactam D^8–10) (Fig. 1a). These compounds exhibited various useful biological effects, such as the suppression of cancer malignancy and antiviral activity. Because high-temperature culture of actinomycetes is useful in the search for new compounds, we have attempted to identify additional HSMs. In the present study, we conducted a detailed examination of the metabolites produced by the thermotolerant actinomycete Streptomyces sp. HR41, a noaoxazole-producing strain. We identified (S)- and (R)-3,4-dihydro-4-methyl-2(1H)-quinazolinone, heasquinazolinone (1a and 1b), as HSMs produced by this organism (Fig. 1b). Here, we report the isolation, structure determination, and biological activity of 1a and 1b and investigate quinazoline-containing compounds as secondary metabolites produced by actinomycetes.

Fig. 1. Structures of HSMs Produced by Thermotolerant Streptomyces Strains

(a) New compounds previously discovered as HSMs. (b) First natural product compound identified as an HSM in this study. HSM: heat-shock metabolites.

Results and Discussion

Isolation of the HSM

Previously, we examined the HSM productivity of Streptomyces sp. HR41 and found no phenotypic differences between organisms cultured at 30 and 45°C on agar plates.⁸⁾ HPLC-UV analysis of an ethyl acetate (EtOAc) extract of Streptomyces sp. HR41 showed a peak of an HSM with maximum UV absorptions at 204, 246, and 282 nm (highlighted in red in Supplementary Fig. S1). We then focused on determining the structure of this HSM.

The producing strain, Streptomyces sp. HR41, was cultured in ISP2 liquid medium at 45°C for 3 d with shaking, and the entire culture broth was then treated with acetone to disrupt the cells, followed by extraction with EtOAc. The resulting crude extract (0.6 g from 2 L) was fractionated using silica gel column chromatography, followed by octa decyl silyl (ODS) column chromatography and reversed-phase HPLC separation. Final purification was achieved using normal-phase TLC separation to yield compound 1 (1.4 mg).

Determination of the Planar Structure of the HSM

Compound 1 was obtained as a white powder. The molecular formula, deduced from a pseudomolecular ion at m/z 163.0862 [M + H]⁺ obtained via high-resolution electrospray ionization time-of-flight MS (HR-ESI-TOF-MS) analysis, was C₉H₁₀N₂O, corresponding to 6 degrees of unsaturation. The IR spectrum indicated the presence of exchangeable proton(s) (3230 cm⁻¹), which were corroborated by ¹H-NMR signals for 2 broad singlet exchangeable protons (δ_H 6.88 and 9.04) and carbonyl (1682 cm⁻¹), and aromatic (1489 cm⁻¹) functional groups. Data from ¹³C-NMR and heteronuclear single quantum coherence spectra confirmed the presence of 9 carbons assignable to 1 carboxyl or amide carbon, 6 olefinic or aromatic carbons (of which 4 were proton bearing), 1 sp³ methine, and 1 methyl group.

¹H–¹H homonuclear correlation spectroscopy analysis revealed 2 partial proton–proton connections: (a) H4/H9 and (b) H5/H6/H7/H8 (Fig. 2). These fragments were joined through C4a and C8a to form an aromatic functional group based on heteronuclear multiple bond correlations (HMBCs) from H4, H5, and H7 to C8a; H4 to C5; and H6, H8 and H9 to C4a (Fig. 2 and Table 1). These molecular constituents accounted for 4 of the 6 degrees of unsaturation, leaving 2 degrees for a carbonyl group and a ring structure. The remaining CH₂N₂O in the molecular formula formed a quinazolinone nucleus that satisfied the degrees of unsaturation based on HMBC correlations from H4 to a carbonyl carbon (C2; δ_C 153.7) and from a broad singlet exchangeable proton (NH1; δ_H 9.04) to C4a. These spectral data showed that the planar structure of 1 is identical to that of 3,4-dihydro-4-methyl-2(1H)-quinazolinone.

Fig. 2. COSY and Key HMBC Correlations of Compound 1

Table 1. ¹H- and ¹³C-NMR Data for 1 in DMSO-d₆

No.	δC, typea)	δH (J in Hz)b)	HMBCc)
1		9.04 (1H, brs)	4a
2	153.7, C
3		6.88 (1H, brs)
4	48.5, CH	4.49 (1H, ddd, 13.6, 6.5, 1.9)	2, 4a, 5, 8a
4a	123.1, C
5	125.3, CH	7.09 (1H, d, 7.6)	6, 8a
6	121.0, CH	6.86 (1H, td, 1.0, 7.6)	4a, 5, 8
7	127.6, CH	7.10 (1H, t, 7.6)	8, 8a
8	113.6, CH	6.76 (1H, dd, 1.0, 7.6)	4a, 7
8a	137.1, C
9	24.3, CH3	1.30 (3H, d, 6.5)	4, 4a

^a) 125 MHz. ^b) 500 MHz. ^c) From proton to indicated carbon(s).

Determination of the Absolute Structure of the HSM

Since compound 1 has a chiral center at C4 and could exist as a mixture of enantiomers, chiral-phase HPLC analysis was performed. Chromatography on an amylose tris(3,5-dimethylphenylcarbamate)‐coated chiral-phase column gave 2 separate peaks (1a: t_R = 10 min; 1b: t_R = 12 min) exhibiting the UV spectrum of 1 (Fig. 3). The 2 peaks were purified (1a: 0.2 mg; 1b: 0.9 mg), and their respective optical rotation values were determined (1a: ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +4.4, c 0.01, CH₃OH, 1b: ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −5.0, c 0.01, CH₃OH). As it was not possible to assign S- and R-enantiomers in the chromatogram, we attempted a comparative analysis using structurally similar compounds. For example, (S) and (R)-4-isopropyl-3,4-dihydroquinazolin-2(1H)-one and (S) and (R)-4-cyclohexyl-3,4-dihydroquinazolin-2(1H)-one have been reported as the synthetic compounds, respectively.^11,12) In addition, these optical rotation values have already been measured (Fig. 4). Therefore, 1a and 1b were estimated to be (S)- and (R)-3,4-dihydro-4-methyl-2(1H)-quinazolinone. To date, no reports have identified 3,4-dihydro-4-methyl-2(1H)-quinazolinone as a natural product. Furthermore, although the (R)-form of 1 has been synthesized and patented,¹³⁾ the (S)-form of 1 has not been reported. Thus, the present study is the first to report 3,4-dihydro-4-methyl-2(1H)-quinazolinone as a natural product, which we designated (S)- and (R)-heasquinazolinone.

Fig. 3. UV Chromatogram of Compound 1 Analyzed on a Chiral-Phase Column

Fig. 4. Select Examples of Compounds Related to Compound 1

Biological Activity of the HSM

The biological activity of (S)- and (R)-heasquinazolinone (1a and 1b) was first evaluated by assessing the cytotoxicity against HT29 human colon cancer cells in 2-dimensional (2D) and 3D culture systems. Because the 3D culture system employed in this study uses medium for stem cell generation, this can affect cytotoxicity measurements. For example, although the IC₅₀ of the anticancer drug camptothecin against cells in 2D culture was 0.18 µM, camptothecin was less toxic to cells in 3D culture. This suggests that 3D-generated cells are more resistant to anticancer drugs, which is a property of cancer stem cells. However, compounds 1a and 1b selectively suppressed the growth of 3D-cultured cells, exhibiting IC₅₀ values of 19 and 30 µM, respectively, concentrations at which the compounds exhibited no apparent cytotoxicity against 2D-cultured cells (Fig. 5). The cytotoxicity of compounds 1a and 1b was thus 3D culture selective, which could affect cancer stem cell viability, although elucidation of the mechanism will require further investigation.

Fig. 5. Biological Activities of Compounds 1a and 1b

Blue bars show the viability of HT29 cells cultured in 2D, and red bars show the viability of HT29 cells cultured in 3D. Values were normalized to the activity in control reactions (in which the ctrl was defined as 100%) and presented as the mean ± S.D. of triplicate biological replicates.

Conclusion

In this study, we identified 3,4-dihydro-4-methyl-2(1H)-quinazolinone, heasquinazolinone (1), as an HSM produced by thermotolerant Streptomyces sp. HR41 and successfully separated it into its enantiomers, 1a [(S)-form] and 1b [(R)-form]. The quinazolinone structure is often found in natural products, and several such compounds have been isolated from actinomycetes (Fig. 6). For example, there are several reported examples of 4-quinazolinone‐type natural products. The newly discovered quinazolinone analogue actinoquinazolinone and the known compound 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one were isolated from Streptomyces sp. CNQ-617.¹⁴⁾ In addition, new quinazolinone analogues with a glycerol structure, quinazolinones A and B, were isolated from Streptomyces sp. MBT27.¹⁵⁾ However, there have been no reports of natural products of the 2-quinazolinone type, such as compound 1 discovered in this study, according to our database search using the Dictionary of Natural Products.¹⁶⁾ With regard to natural products with a 4-aminoquinazoline structure, 4-methyl-2-quinazolinamine from Streptomyces sp. GS DV232 has been reported, and its biosynthesis has been analyzed.¹⁷⁾ Tryptophan was proposed as the biosynthetic origin of this compound, but elucidating details of the biosynthesis of 1 will require further study. Although this study did not lead to the discovery of analogues of 1, natural products with the 4-quinazolinone structure may be widely distributed.

Fig. 6. Structures of Secondary Metabolites with the Quinazolinone Structure Produced by Actinomycetes

Experimental

General Experimental Procedures

UV spectra were recorded on a DU530 UV/VIS spectrophotometer: Beckman Coulter, Inc., Brea, CA, U.S.A. IR spectra were recorded on a Bruker FT-IR ALPHA: Bruker Corporation, Billerica, MA, U.S.A. NMR spectra were obtained using a JEOL JNM-ECA500 spectrometer: JEOL Ltd., Tokyo, Japan, in dimethyl sulfoxide-d₆ with reference to the residual solvent signals (δ_H 2.50 and δ_C 39.5). HR-ESI-TOF-MS spectra were recorded on a timsTOF Pro 2 mass spectrometer (Bruker Corp., Billerica, MA, U.S.A.). Silica gel 60 0.040–0.063 mm (Merck Millipore Co., Darmstadt, Germany) was used for silica gel column chromatography. Sep-Pak C18 35 cc (Waters Corp., Milford, MA, U.S.A.) was used for ODS column chromatography. HPLC separation was performed using a 20 × 250 mm CAPCELL PAK C18 MGII packed column (OSAKA SODA Co., Ltd., Japan) on a Hitachi ELITE LaChrom system: Hitachi, Ltd., Tokyo, Japan. TLC purification was conducted using a glass thin-layer plate and silica gel coated with the fluorescent indicator F254 (Merck Millipore Co., Darmstadt, Germany).

Actinomycete Strain

The actinomycete strain was isolated from a soil sample collected in Japan and stored in 20% glycerol solution at −80°C before use in experiments. Strain HR41 was identified as a member of the genus Streptomyces based on 99.5 and 99.3% similarity in the 16S rRNA gene sequence (1395 nucleotides; GenBank accession number LC647575) to Streptomyces sp. RAI364 (accession number LC057380) and Streptomyces sp. MJM15682 (accession number MF344802), respectively. The strain HR41 is preserved at the Chemical Biology Laboratory, Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University for future reference.

Fermentation

Streptomyces sp. HR41 was maintained on ISP2 agar medium (yeast extract 0.2%, malt extract 0.5%, glucose 0.2%, and agar 2%, pH adjusted to 7.3) and subsequently inoculated into 500-mL Erlenmeyer flasks, each containing 100 mL of ISP2 liquid medium (yeast extract 0.2%, malt extract 0.5%, and glucose 0.2%, pH adjusted to 7.3). The flasks were shaken on a rotary shaker (160 rpm) at 30°C for 2 d. Aliquots of the seed culture (5 mL each) were then transferred into 500-mL Erlenmeyer flasks, each containing 100 mL of ISP2 liquid medium. The flasks were shaken on a rotary shaker (160 rpm) at 45°C for 3 d.

Isolation

At the end of fermentation, 100 mL of acetone was added to each flask, and the flasks were shaken for 1 h. The mixture was centrifuged at 6000 rpm for 10 min, and the mycelia were removed. The acetone was evaporated, and the aqueous layer was extracted with EtOAc. The organic layer was concentrated in vacuo to give 0.6 g of extract from a 2-L culture. The extract was subjected to silica gel column chromatography with a solvent gradient of CHCl₃/MeOH (1 : 0, 20 : 1, 10 : 1, 4 : 1, 2 : 1, 1 : 1, and 0 : 1 [v/v]). Fraction 2 (20 : 1) was further fractionated using ODS column chromatography with a gradient of MeCN (2 : 8, 3 : 7, 4 : 6, 5 : 5, 6 : 4, 7 : 3, and 8 : 2 [v/v]). ODS fraction 1 (2 : 8) was evaporated and further purified using preparative HPLC under isocratic MeCN elution (MeCN concentration: 25% over 45 min) at 10 mL/min. The fraction containing the target HSM was evaporated, and final purification was achieved using normal-phase TLC separation under isocratic CHCl₃/MeOH (10 : 1) elution, yielding heasquinazolinone (1, 1.4 mg).

Heasquinazolinone (1), S/R = 18 : 82: white powder; UV (MeOH) λ_max nm (log ε): 204 (4.41), 246 (4.01), 282 (3.25); IR (ATR) ν_max cm⁻¹: 3230, 3073, 1682, 1602, 1489, 1320, 1294; for ¹H- and ¹³C-NMR data, see Table 1 and Supplementary Figs. S1 and S2; HR-ESI-TOF-MS m/z: 163.0862 [M + H]⁺ (Calcd for C₉H₁₁N₂O: 163.0866).

Chiral-Phase HPLC Analysis

Compound 1 was analyzed using chiral-phase HPLC under the following conditions on a JASCO EXTREMA system (JASCO Corp., Tokyo, Japan). Column: CHIRAL PAK IA (4.6 mm i.d. × 150 mm) (Daicel Corp., Japan); mobile phase: n-hexane : isopropanol = 80 : 20 for 45 min; flow rate: 1.0 mL/min; detection at 254 nm. Two separate peaks were eluted at 6.5 and 9.0 min, demonstrating an enantiomeric composition. The optical rotation values were as follows: (S)-heasquinazolinone (1a): ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +4.4, c 0.01, CH₃OH); (R)-heasquinazolinone (1b): ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −5.0, c 0.01, CH₃OH).

Cell Culture

HT29 cells were purchased from the American Type Culture Collection and cultured in Roswell Park Memorial Institute (Buffalo, NY, U.S.A.)-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 unit/mL), and streptomycin (100 µg/mL) at 37°C in a humidified 5% CO₂ atmosphere. To generate cancer stem cells, HT29 cells were seeded at 1 × 10⁵ cells/well in 2 mL in the wells of a 6-well plate coated with poly(2-hydroxyethyl methacrylate) using 3D Tumorsphere Medium XF (PromoCell GmbH, Heidelberg, Germany) and cultured for 7 d. One-half of the volume was removed and replaced with fresh medium every 2 d during the 7-d culture.

Cytotoxicity Assay

HT29 cells cultured using 2D or 3D systems were seeded in a 96-well white plate at 1 × 10⁴ cells/well and incubated for 24 h. The cells were then treated with compounds 1a and 1b. After 72 h, CellTiter-Glo® (Promega Corp., Madison, WI, U.S.A.) was added (30 µL per well). After shaking and incubation at room temperature for 15 min, luminescence was measured using a SpectraMax i3x microplate reader (Molecular Devices LLC, San Jose, CA, U.S.A.), and cell viability was evaluated.

Acknowledgments

This work was supported by Grants-in-Aid for Early-Career Scientists (No. 23K13897 to S.S.) from the Japan Society for the Promotion of Science (JSPS), and Grants-in-Aid for Transformative Research Area (A) “Latent Chemical Space” (Nos. 23H04880 and 23H04884 to M.A.A.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. S.S. was also the recipient of the Noda Institute for Scientific Research (NISR) Young Investigator Research Grant.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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