Cytotoxic Pentaketide-Sesquiterpenes from the Marine-Derived Fungus Talaromyces variabilis M22734

Talaromyces, a filamentous fungus widely distributed across terrestrial and marine environments, can produce a diverse array of natural products, including alkaloids, polyketones, and polyketide-terpenoids. Among these, chrodrimanins represented a typical class of natural products. In this study, we isolated three previously undescribed pentaketide-sesquiterpenes, 8,9-epi-chrodrimanins (1–3), along with eight known compounds (4–11). The structures of compounds 1–3 were elucidated using nuclear magnetic resonance (NMR) and mass spectrometry (MS), while their absolute configurations were determined through X-ray crystallography and electronic circular dichroism (ECD) computations. The biosynthetic pathways of compounds 1–3 initiate with 6-hydroxymellein and involve multiple stages of isoprenylation, cyclization, oxidation, and acetylation. We selected four strains of gastrointestinal cancer cells for activity evaluation. We found that compound 3 selectively inhibited MKN-45, whereas compounds 1 and 2 exhibited no significant inhibitory activity against the four cell lines. These findings suggested that 8,9-epi-chrodrimanins could serve as scaffold compounds for further structural modifications, potentially leading to the development of targeted therapies for gastric cancer.


Introduction
The genus Talaromyces, a member of the Trichocomaceae family, was first identified in 1955 by the American mycologist Chester Ray Benjamin.Species within this genus produce ascocarps characterized by soft, cotton-like structures with densely woven hyphal walls.These ascocarps often display a yellowish tint or are surrounded by similarly colored granules [1].Among the secondary metabolites produced by Talaromyces are alkaloids, peptides, lactones, polyketides, and polyketide-terpenoids.Polyketide-terpenoids, a unique class of natural products, feature a biosynthetic pathway that combines polyketides and terpenes, exhibiting a broad range of biological activities [2].In an investigation involving the Talaromyces sp.YO-2 strain, researchers discovered a novel pentaketide-drimane named chrodrimanin C and reisolated the known pentaketide-drimanes, chrodrimanins A and B. Among these, chrodrimanin B demonstrated significant insecticidal properties with an LD 50 of 10 µg/g in dietary tests, while chrodrimanins A and C showed no activity.Subsequent studies uncovered four additional pentaketide-drimanes, designated as chrodrimanins D to G, together with the previously identified chrodrimanin H. Importantly, chrodrimanins D, E, and F exhibited insecticidal effectiveness against silkworms, with LD 50 values of 20, 10, and 50 µg/g of diet, respectively [3][4][5].
In recent years, obtaining microbial resources from unique habitats to develop natural drugs has been a hot trend in drug development.The distinct environmental factors of the marine environment, such as high pressure, low temperature, the absence of light, and partially oligotrophic conditions, have shaped unique metabolic mechanisms in microbes.These mechanisms are crucial for the production of natural products with new structures, meaning that marine-derived microbes are considered significant sources of these novel natural products [6,7].
In our exploration of bioactive compounds originating from the marine environment, we isolated three previously undescribed 8,9-epi-chrodrimanin derivatives (Figure 1) from a marine-derived strain of Talaromyces variabilis M22734.Their absolute configurations were determined through spectral analysis, X-ray crystallography, and ECD calculation.Furthermore, their biosynthetic pathways were deduced, and their cytotoxic activities were preliminarily evaluated.
In recent years, obtaining microbial resources from unique habitats to develop n ral drugs has been a hot trend in drug development.The distinct environmental facto the marine environment, such as high pressure, low temperature, the absence of light, partially oligotrophic conditions, have shaped unique metabolic mechanisms in micro These mechanisms are crucial for the production of natural products with new structu meaning that marine-derived microbes are considered significant sources of these n natural products [6,7].
In our exploration of bioactive compounds originating from the marine environm we isolated three previously undescribed 8,9-epi-chrodrimanin derivatives (Figure 1) f a marine-derived strain of Talaromyces variabilis M22734.Their absolute configurat were determined through spectral analysis, X-ray crystallography, and ECD calcula Furthermore, their biosynthetic pathways were deduced, and their cytotoxic activ were preliminarily evaluated.

Results
Compound 1 was characterized by its proton adduct ion (m/z 471.2369, Figure S1), from which the molecular formula C 27 H 34 O 7 was deduced.This formula suggested eleven degrees of unsaturation.Extensive spectroscopic analysis including 1 H and 13 C NMR, HSQC, and 13 C/DEPT (Figures S2−S5) revealed distinct structural elements (Table 1): one ketone carbonyl (δ C 208.0), three quaternary sp 3 carbons with one oxygenated (δ C 48.5, 77.2, 37.5), two ester carbonyls (δ C 170.2, 170.0), five aromatic sp 2 carbons (δ C 101.9, 139.S7) correlation analysis revealed four spin systems: C-1/C-2, C-5/C-6/C-7, C-9/C-11, and C-7 ′ /C-8 ′ /C-9 ′ , as depicted in Figure 2B.HMBC (Figure S6) correlations allowed for the assignment of subunit I from correlations such as H-2 to C-4/C-10, H 3 -13 (14) to C-5, H 2 -6 to C-8/C-10, H 2 -7 to C-9, H 3 -12 to C-7/C-9, and H 3 -15 to C-1/C-5/C-9 (Figure 2A).Partial subunit II was resolved through HMBC correlations of H 3 -9 2A).From the chemical shift δ H 11.11, it was inferred that a hydroxyl group formed a hydrogen bond with a carbonyl group, causing a downfield shift.This indicated the presence of one ester carbonyl in subunit II at a meta position relative to OH-4 ′ (Table 1).Another ester carbonyl was contributed by the acetyl group attached to C-2, as derived from the HMBC correlation of H-2 with C-16 (Figure 2B).HMBC correlations of H-9 with C-1 ′ and H 2 -11 to C-2 ′ (Figure 2B) indicated the linkage of subunits I and II to C-11-C-1 ′ .Based on the structural data available, the combination of subunits I and II resulted in a total molecular composition of 27 carbons, 34 hydrogens, and 6 oxygens.Subunit I exhibited an unsaturation degree of four, while subunit II displayed a degree of six.Given the molecular formula C 27 H 34 O 7 , there likely existed an additional oxygen bridge that contributed to the final degree of unsaturation.Analysis of the downfield chemical shifts at C-8 and C-6 ′ led to the deduction that these carbons were connected via an oxygen bridge.This connection, along with C-9, C-11, and C-1 ′ , formed an oxygen-containing six-membered ring that linked subunits I and II.
Compound 2 was characterized by a proton adduct ion, m/z 429.2280 (Figure S9), from which the molecular formula C 25 H 32 O 6 was inferred, indicating a total of ten degrees of unsaturation.Through careful comparison of the NMR data of compounds 2 (Figures S10−S13) and 1, it was observed that compound 2 lacked the signal for one acetyl group and showed differences at C-1, C-2, C-3, C-6, C-7, and C-14, suggesting that the primary structural differences between compounds 2 and 1 reside in rings A and B (Table 1).The 2D NMR data of compounds 2 and 1 were very similar; notably, COSY (Figure S15) correlations of H-5/H-6/H 2 -7 and HMBC (Figure S14) correlations of H-6 with C-8/C-10 allowed the deduction of an OH group attached to C-6 (δ C/H 68.4/4.61)(Figure 2B). 3 J HH values for H b -11/H-9 and H b -7 ′ /H-8 ′ were 13.1 and 11.5 Hz, respectively, indicating the axial orientation for H-11b and H-7 ′ b, and tentatively assigned a β orientation (Table 1).The NOESY (Figure S16) correlations of H 3 -15/H a -2, H a -2/H 3 -14, H a -7/H 3 -12, H 3 -12/H b -11, H b -11/H 3 -15, and H 3 -9 ′ /H b -7 ′ indicated that these protons were oriented on the same face of the ring system, tentatively assigned a β orientation, whereas H 3 -13/H-6 and H-5/H b -7 indicated an orientation on the opposite face, tentatively assigned an α orientation (Figure 2B).Subsequently, the conformer 5R,6R,8R,9R,10S,8 ′ R-2 and its epimer underwent ECD calculations at the CAM-B3LYP/6-311G(d) level (Tables S1−S13).It was found that the experimental results corresponded more closely with the structure of 5R,6R,8R,9R,10S,8 ′ R-2.Consequently, the absolute configuration was determined to be 5R,6R,8R,9R,10S,8 ′ R. (Figure 3).Compound 3 was characterized by a sodium adduct ion, m/z 509.2156 (Figure S17), from which the molecular formula C 27 H 34 O 8 was inferred, indicating a total of eleven degrees of unsaturation.Through careful comparison of the NMR data between compounds 3 (Figures S18−S21) and 2, it was observed that compound 3 displayed an additional signal for an acetyl group, while other chemical shifts were very close.The 2D NMR data for compounds 3 and 2 were also very similar; however, notable distinctions included the COSY (Figure S23) correlations of H 2 -1 with H-2 and the HMBC (Figure S22) correlations of H-2 with C-4, C-10, and C-16, which elucidated that C-2 (δ C/H 71.2/5.79) was linked to an Mar.Drugs 2024, 22, 274 5 of 10 acetyl group (Figure 2B).The NOESY (Figure S24) correlations involving H 3 -15 with H-2, H-2 with H 3 -14, H a -7 with H 3 -12, H 3 -12 with H b -11, H b -11 with H 3 -15, and H 3 -9 ′ with H b -7 ′ indicated that these protons were oriented on the same face of the ring system.In contrast, the correlations between H 3 -13 and H-6 and H-5 and H b -7 showed that these protons were oriented on the opposite face, similar to compound 1 (Figure 2B).The orientation of the OH group connected to C-6 was consistent with that in compound 2. From the highly consistent ECD spectra of compounds 1 to 3, it was determined that the absolute configuration of compound 3 at the corresponding positions was consistent with compounds 1 and 2 as 2R,5R,6R,8R,9R,10S,8 ′ R (Figure 3).
The biosynthetic pathways for compounds 1-3 were proposed to have originated from 6-hydroxymellein (5, I).Through isoprenylation, intermediate III was formed, followed by cyclization and oxidation to yield intermediate V.A part of intermediate V The biosynthetic pathways for compounds 1-3 were proposed to have originated from 6-hydroxymellein (5, I).Through isoprenylation, intermediate III was formed, followed by cyclization and oxidation to yield intermediate V.A part of intermediate V underwent further oxidation to produce compound 2, while another portion underwent additional oxidation and acetylation, proceeding through intermediates VI and VII, to yield compounds 1 and 3 (Figure 4).At 20 µM, compound 3 exhibited selective inhibitory activity against four strains of gastrointestinal cancer cells, achieving a 55.3% inhibition rate in MKN-45 cells.In contrast, the positive control drug, doxorubicin (Dox), demonstrated a non-selective inhibition rate exceeding 75% for all tested strains (Table 2).Compounds 1 and 2 displayed significantly lower inhibition rates, below 20% for all strains.Subsequent IC50 determinations for com- the positive control drug, doxorubicin (Dox), demonstrated a non-selective inhibition rate exceeding 75% for all tested strains (Table 2).Compounds 1 and 2 displayed significantly lower inhibition rates, below 20% for all strains.Subsequent IC 50 determinations for compounds 1-3 and Dox on MKN-45 cells revealed that compound 3 had an IC 50 of 18.0 µM, whereas compound 1 had an IC 50 of 79.2 µM, and compound 2 did not achieve a 50% inhibition rate even at 100 µM.The IC 50 for Dox was 62.5 nM (Figure 5).

Discussion
We isolated three previously undescribed 8,9-epi-chrodrimanins and established the orientation of two magnetically inequivalent hydrogen atoms on C-11 and C-7′ by analyzing coupling constants and further determined the relative configurations of rings A and B, as well as C-8′, through NOESY.For compound 1, its absolute configuration was resolved using X-ray crystallography.For compound 2, the absolute configuration was determined via NMR elucidation combined with ECD calculations.Regarding compound 3, we first compared its NMR data with those of compounds 1 and 2 to identify structural similarities and differences, and we then determined its relative configuration with the aid of NOESY.The consistency between the ECD spectra of compound 3 and those of compounds 1 and 2 further confirmed its absolute configuration.Furthermore, we assessed the cytotoxic activities of compounds 1-3 against four gastrointestinal cancer cell lines: MKN-45, HCT 116, TE-1, and PATU8988T.Compound 3 exhibited selective inhibitory activity against MKN-45 cells, in contrast to the non-selective inhibitory activity of the positive control drug.Compounds 1 and 2 demonstrated weaker inhibitory effects across all tested cell lines.

General Experimental Procedures
NMR data were measured on a Bruker Avance 600 MHz spectrometer (Billerica, MA, USA).HRESIMS were determined using a Xevo G2 Q-TOF mass spectrometer (Waters,

Discussion
We isolated three previously undescribed 8,9-epi-chrodrimanins and established the orientation of two magnetically inequivalent hydrogen atoms on C-11 and C-7 ′ by analyzing coupling constants and further determined the relative configurations of rings A and B, as well as C-8 ′ , through NOESY.For compound 1, its absolute configuration was resolved using X-ray crystallography.For compound 2, the absolute configuration was determined via NMR elucidation combined with ECD calculations.Regarding compound 3, we first compared its NMR data with those of compounds 1 and 2 to identify structural similarities and differences, and we then determined its relative configuration with the aid of NOESY.The consistency between the ECD spectra of compound 3 and those of compounds 1 and 2 further confirmed its absolute configuration.Furthermore, we assessed the cytotoxic activities of compounds 1-3 against four gastrointestinal cancer cell lines: MKN-45, HCT 116, TE-1, and PATU8988T.Compound 3 exhibited selective inhibitory activity against MKN-45 cells, in contrast to the non-selective inhibitory activity of the positive control drug.Compounds 1 and 2 demonstrated weaker inhibitory effects across all tested cell lines.

General Experimental Procedures
NMR data were measured on a Bruker Avance 600 MHz spectrometer (Billerica, MA, USA).HRESIMS were determined using a Xevo G2 Q-TOF mass spectrometer (Waters, Milford, MA, USA) in ESI mode.ECD spectra were recorded using a J-715 spectropolarimeter (JASCO Corporation, Tokyo, Japan).The optical rotations were determined via an Anton Paar MCP 500 polarimeter (Graz, Austria).

Fungal Material
The identity of the strain was confirmed through ITS sequencing, revealing that this strain has a 100% similarity to the Talaromyces variabilis strain ZHKUCC 23-1032 (Genbank accession No. PP029409.1).The original designation of the strain was R3-HSHG1, and after deposition, it was cataloged under the preservation number M22734.

Cytotoxic Activity Assay
The cancer cell lines MKN-45, HCT 116, TE-1, and PATU8988T were obtained from Wuhan Pricella Biotechnology Co., Ltd (Wuhan, China).The basis of detection involved the CCK-8 reagent, which includes WST-8.This component is converted into a water-soluble yellow formazan product by mitochondrial dehydrogenases, facilitated by the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-methoxy PMS).The quantity of formazan generated directly correlates with the number of viable cells.

NMR and ECD Calculations
The conformers were first produced using CREST software (V 2.11) [15,16] and further refined via DFT optimization at the B3LYP/6-31G(d) level with Gaussian 16 (Gaussian, Wallingford, CT, USA) [17].We focused on conformers that lay within a 10 kcal/mol energy window.Frequency analysis was conducted on each conformer to verify its status as a local minimum.The electronic energies were then recalculated at the higher M062X/6-311+G(2d,p) level.By applying a Boltzmann distribution, we assessed the conformer populations, especially those accounting for more than 2% of the total, for comprehensive examination.Excited state calculations were executed at the Cam-B3LYP/6-311G(d) level using TDDFT, targeting 36 excited states for each conformer [18].ECD curves were subsequently produced using Multiwfn 3.6 software [19].

Conclusions
This study deepens our understanding of the chemical diversity and biological activities of secondary metabolites from marine-derived Talaromyces strains.The discovery of three novel 8,9-epi-chrodrimanin derivatives from Talaromyces variabilis M22734 illustrates the vast chemical potential of fungi.The selective cytotoxic effect of compound 3 against MKN-45 cells underscores its potential as a lead compound for new gastrointestinal cancer treatments.This selectivity is particularly crucial compared to the non-selective cytotoxicity of traditional chemotherapeutics like doxorubicin.Furthermore, the minimal activities in compounds 1 and 2 highlight the importance of subtle structural differences in bioactivity, suggesting that minor modifications can lead to significant biological impacts.Future research should focus on optimizing the antitumor properties of these compounds through structural modifications.Additionally, exploring their biosynthetic pathways may provide insights into the genetic and enzymatic mechanisms that microbes use to produce complex molecules, offering potential new targets and strategies for synthetic biology.

Data Availability Statement:
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
Mar. Drugs 2024, 22, x FOR PEER REVIEW 7 of 11underwent further oxidation to produce compound 2, while another portion underwent additional oxidation and acetylation, proceeding through intermediates VI and VII, to yield compounds 1 and 3 (Figure4).

Funding:
This research was supported by the Scientific Research Foundation of the Third Institute of Oceanography, MNR (2022007); the Natural Science Foundation of Xiamen (3502Z20227227); the Xiamen Southern Oceanographic Center Project (22GYY007HJ07); the National Key Research and Development Program of China (2022YFC2804100), the Natural Science Foundation of Fujian Province (2022J011401, 2021J01509); the COMRA program (DY135-B2-01), the Open Project of the Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, Ministry of Natural Resources (HY202303, HY202307); and the Research Project of Xiamen Medical College (K2023-25).

Table 2 .
The inhibition rates of compounds 1-3 against four different gastrointestinal cancer cell lines at a concentration of 20 µM.