Published online: 2019-11-18 Original Papers Thieme Laccaridione C, a Bioactive Polyketide from the Fungus Montagnula sp. Authors Celso Almeida1, Thomas Andrew Mackenzie2, Víctor González-Menéndez1, Nuria de Pedro2, Ignacio Pérez-Victoria3, Gloria Crespo3, Jesús Martín3, Olga Genilloud1, Francisca Vicente2, Bastien Cautain2, Fernando Reyes3 Affiliations Dr. Celso Almeida 1 Fundación MEDINA, Microbiology, Armilla, Spain Fundación MEDINA 2 Fundación MEDINA, Screening and Target Validation, Avenida del Conocimiento 34 Armilla, Spain 18016 Armilla, Granada 3 Fundación MEDINA, Chemistry, Armilla, Spain Spain Key words [email protected] cytotoxicity, melanoma, bioassay-guided fractionation, laccaridiones, fungal natural products, Montagnula sp., Supporting Information for this article available structural elucidation online at http://www.thieme-connect.de/products. received 28.08.2019 Abstract revised 14.10.2019 The new polyketide laccaridione C (1) was obtained by bioas- accepted 21.10.2019 say-guided isolation of organic extracts of the fungal strain CF-223743, isolated from dung collected in a forest of Grand Bibliography Comoros Island. Its structure was established using spectro- DOI https://doi.org/10.1055/a-1032-3346 scopic methods, namely HRMS and 1D and 2D NMR. The new Published online: 2019 compound was tested against seven cancer cell lines, evidenc- Planta Med Int Open 2019; 6: e57–e62 ing effective activity against melanoma (A2058) with an IC © Georg Thieme Verlag KG Stuttgart · New York 50 of 13.2 µM, and an increased activity against breast cancer ISSN 2509-9264 (MCF-7) with an IC50 of 3.7 µM. The strain CF-223743 was taxonomically identified asMontagnula sp. based on ITS/28S Correspondence analysis Dr. Fernando Reyes Fundación MEDINA Avenida del Conocimiento 34 18016 Armilla, Granada Spain Tel : + 34 958993965, Fax : + 34 958846710 [email protected] Introduction tomeninges [3]. Malignant melanoma is the most aggressive form Natural products are important sources of anticancer lead mole- of skin cancer and accounts for about 3 % of all cases of malignant cules [1]. More than 60 % of the current compounds with antineo- tumors. Its incidence is increasing worldwide, and it is becoming plastic activity were originally isolated as natural products or are resistant to current therapeutic agents [4]. In a recent report, 30 natural product derivatives, and microbial metabolites are among in vivo and in vitro natural active principles were reviewed for their the most important of these chemotherapeutic agents [2]. Mela- pharmacological effects on migration and/or metastasis of mela- noma is a malignancy of pigment producing cells (melanocytes), noma cells, mapping the mechanisms of action for these underex- which are located primarily in the skin, but are also found in the ploited properties. They were described as acting mainly through ears, gastrointestinal tract, eyes, oral and genital mucosa, and lep- their antagonistic effects upon the TNF-α and EP2 receptors or the Almeida C et al. Laccaridione C, a Bioactive … Planta Med Int Open 2019; 6: e57–e62 e57 Original Papers Thieme suppression of several protein kinases involved in metastatic path- leptosphaerodione (4) [7]. The closely related molecular formulae, ways such as RAS, PI3K, ERK, and FAK. Some were able to reduce UV and NMR spectra reported for these compounds unequivocally the level of mesenchymal biomarkers such as N-cadherin and/or indicated that 1 belonged to the same structural class. The UV elevate the expression of other molecules such as E-cadherin [5]. (DAD) spectrum of 1 (Supporting Information) displayed the char- Herein, we report the isolation and structural elucidation of the acteristic bands of the ortho-benzoquinone ring system (λmax 308 cytotoxic polyketide laccaridione C (1) (▶Fig. 1) obtained from cul- and 490 nm) responsible for the strong red coloration of the com- ture broths of the fungal strain CF-223743 after fractionation of pound. Further analysis of all key 1H-1H and 1H-13C long-range cor- ethyl methyl ketone extracts based on melanoma targeted activi- relations observed in the COSY and HMBC spectra of 1 (▶Fig. 3) al- ty. A phylogenetic placement of the strain CF-223743, based on its lowed for establishing its full structure, which turned out to be ITS/28S, identified the fungus as a species of the genusMontagnula , identical to that of reported laccaridiones A (2) and B (3), but lack- an ascomycete of the order Pleosporales (▶Fig. 2). The related ing the alkyl acetalic substituent and thus being a hemiacetal. The poly ketides laccaridione A (2), laccaridione B (3) [6], and lepto- E stereochemistry of the double bond in the side chain was likewise sphaerodione (4) [7] were also detected as minor components in established based on comparison of chemical shifts. Since 1 con- the extract after LC-DAD-HRMS-based dereplication [8]. tains a chiral center at position C-3’of the fixed configuration, the two possible configurations at the hemiacetal carbon (two epimers at C-11) render two possible diastereomers with almost identical Results and Discussion chemical shifts. The presence of a hemiacetal explains the splitting Compound 1 was isolated from the ethyl methyl ketone extract of observed in the 1H NMR spectrum for some signals of the lateral a culture of Montagnula sp. (CF-223743) by melanoma activity- side chain. Two epimers at the chiral center at position 3’ in an equi- guided fractionation on SP207SS resin followed by repeated semi- molar ratio were present in compound 1, as indicated by the inten- preparative HPLC. The molecular formula of 1 was inferred to be sity of the split NMR signals. This is not surprising considering that C21H22O6 by accurate mass measurements (ESI-TOF, see Support- formally the hemiacetal is the product of the intramolecular nucle- + + ing Information) (m/z 371.1489, [M + H] , calcd. for C21H23O6 , ophilic addition of an enolic hydroxy group at position C-9 over an 371.1479), which translates into 11 degrees of unsaturation. The aldehyde at position C-11. Since such an attack may take place over structure of 1 was established by 1D and 2D NMR spectroscopy both faces of the carbonyl with equal probability, two epimeric (▶Table 1 and Supporting Information). The 1H NMR and HSQC hemiacetals in an equimolar ratio are formed. Thus, 1 was isolated spectra displayed signals corresponding to four sp2 methines, two as a mixture of diastereomers, and for this reason, its optical rota- aliphatic methines, one of them oxygenated, one aliphatic meth- tion was not measured. ylene, a methoxy group, and three aliphatic methyl groups with The presence in the fungal extract of the related polyketides lac- singlet, doublet, and triplet multiplicities. Two hydroxy protons caridione A (2), laccaridione B (3), and leptosphaerodione (4), de- were also observed, with one of them corresponding to a phenol tected as trace components in the enriched fractions after LC-DAD- involved in intramolecular hydrogen bonding according to its sharp HRMS-based dereplication, indicates their obvious biosynthetic and remarkably deshielded singlet signal at δH 12.4 ppm. Three of relationship. the four sp2 protons constituted isolated spin systems. The pres- Compound 1 was tested against the melanoma (A2058) cell line ence of a key substructural moiety corresponding to a 1,3-dimeth- used for its bioassay-guided isolation, exhibiting an IC50 of ylpentenyl unit directly bonded to a quaternary oxygenated sp2 13.2 ± 2.9 µM. It was also further tested against an extended panel carbon was established by analysis of COSY and HMBC spectra of six human cell lines, exhibiting potent activity after MTT assays (Supporting Information). Searching for such a substructure in against the breast cancer MCF7 cell line with an IC50 value of the Chapman & Hall Dictionary of Natural Products Database 3.7 ± 1.5 µM, neuroblastoma (SH-SY5Y) with an IC50 of 8.8 ± 3.0 µM, (v25.1) revealed its presence in laccaridiones A (2) and B (3) [6] and liver carcinoma (Hep G2) with an IC50 of 11.6 ± 4.2 µM, pancreas car- cinoma (MIA PaCa-2) with an IC50 of 18.7 ± 2.3 µM, lung carcinoma (A549) with an IC50 of 29.0 ± 0.7 µM, and skin fibroblasts (CCD-25Sk, 6' 7' derived from normal skin of a patient that died from a high-grade 4 6 8 5' 1' glioma) with an IC50 of 20.5 ± 1.7 µM. Doxorubicin gave IC50 values 15 O 5 7 9 4' 3' of 1.79 ± 0.06 (A2058), 6.99 ± 0.07 (MCF7), 0.69 ± 0.07 (SH-SY5Y), 3 2' 2 1.95 ± 0.09 (Hep G2), 2.97 ± 0.73 (MIA PaCa-2), 10.51 ± 0.28 (A549), 1 11 O 14 12 and 0.86 ± 0.05 µM (CCD-25Sk) when tested as a positive control O 13 under the same conditions. Methyl methanesulfonate (MMS) was O OH R used as a second positive control at a concentration of 4 μM, which caused a 100 % inhibition of all cell lines tested. (1) R=OH In conclusion, we report herein the isolation and cytotoxic prop- (2) R=OCH3 erties of a new bioactive polyketide, laccaridione C (1), from Mon- (3) R=OCH2CH3 (4) R=H tagnula sp., an ascomycete from the Pleosporales order. The inter- est in the bioactivity of laccaridione derivatives can be perceived by the previously published patents concerning their use as anti- ▶Fig. 1 Structure of laccaridione C (1), the related laccaridiones A (2) and B (3), and leptosphaerodione (4). tumor, antifungal, antibacterial, and anti-HIV agents [9–11]. A fur- ther literature review confirmed these properties and indicates that e58 Almeida C et al. Laccaridione C, a Bioactive … Planta Med Int Open 2019; 6: e57–e62 Coniothyrium carteri CBS10591