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New Azulene-Type Sesquiterpenoids from the Fruiting Bodies of Lactarius deliciosus

Abstract In the 1H NMR-guided fractionation of extracts from the edible mushroom Lactarius deliciosus, two new azulene-type sesquiterpenoids, 7-isopropenyl-4-methyl-azulene-1-carboxylic acid (1) and 15-hydroxy-3,6-dihydrolactarazulene (2), together with seven known compounds were characterized. Their structures were determined on basis of spectroscopic evidence, as well as by comparing with literature data. Amongst the known metabolites, the 13C NMR assignment of 15-hydroxy-6,7-dihydrolactarazulene (3) is reported here for the first time. Moreover, 7-acetyl-4-methylazulene-1-carbaldehyde (5) displayed a moderate antibacterial activity against Staphylococcus aureus. Graphical Abstract *Digital image of L. deliciosus. Retrieved March 17, 2017 from https://upload.wikimedia.org/wikipedia/commons/e/e3/Lactarius_deliciosus_1_(1).jpg . Electronic supplementary material The online version of this article (doi:10.1007/s13659-017-0130-1) contains supplementary material, which is available to authorized users.


Introduction
The genus Lactarius belongs to the family Russulaceae (class Basidiomycetes) and is widely distributed allover the world. Many Lactarius species are edible; chemically, these mushrooms are appreciated for their metabolites, including sesquiterpenes, steroids, nitrogen-containing compounds and other secondary metabolites with e.g. antitumor or antiviral activities [1,2]. Lactarius deliciosus, L. aureus, L. hatsudake, and others are well known for their colorful pigments, some of which are formed as response to injury of the fruiting bodies [3][4][5]. Previous chemical and biological investigation of this genus showed that these pigments belong to the group of azulene-type sesquiterpenoids, some of them possessing potent biological activities [6][7][8][9][10]. This class of metabolites is also considered as chemotaxonomic marker of the genus Lactarius [11].
As part of our search for fungal metabolites, we are reporting herein the structures and complete 1 H and 13 C NMR assignments of two new pigments 1 and 2 from the edible mushroom L. deliciosus collected in the forests near Göttingen (Germany). In addition to these two new compounds, four other pigments (3)(4)(5)(6) and three fatty acids were also isolated. Although 1 H NMR data of the unstable dihydroazulene alcohol 3 have been reported [3,12], to the best of our knowledge, no 13 C NMR data were published for this compound. We also evaluated the antimicrobial activities of the pigments 1 and 4-6.

Results and Discussion
After extraction of the freshly collected mushrooms with methanol and repetitive chromatography on Sephadex LH-20, compound 1 was obtained as a purple amorphous solid, with violet color in solution. Electrospray high resolution mass spectrometry (ESI HRMS) displayed an [M ? H] ? ion peak at m/z 227.1075, suggesting C 15 H 14 O 2 as molecular formula. The 1 H and 13 C NMR data of 1 (Table 1) were very similar to those reported for lactaroviolin (4) [13], a further Lactarius constituent also isolated in the present investigation. that did not show resonances at lower field of an aldehyde group, but exhibited the characteristic signal of a conjugated carboxyl group at d C 170.0. Further comparison of 1 H and 13 C NMR data of 1 with those of lactaroviolin (4) and 7-acetyl-4-methylazulene-1carboxylic acid [13] were in good agreement with the presence of the carboxy group at C-1. This was supported in the HMBC experiment by long-range couplings between the aromatic proton at d H 8.43 (H-2) and the carboxy group (Fig. 2). The HMBC spectrum also exhibited cross-peaks of the olefinic methylene protons at d H 5.46 and 5.33 (H 2 -12) with the carbon atoms at d C 23.3 (C-13), 146.9 (C-11) and 141.2 (C-7), and of the methyl protons at d H 2.96 (H- 14) with the carbons at d C 143.3 (C-10), 148.0 (C-4) and 129.9 (C-5).
From the above data, the structure of 1 was established as 7-isopropenyl-4-methyl-azulene-1-carboxylic acid (Fig. 1). Related azulene-1-carboxylic acids have been characterized previously from L. deliciosus [13] and L. hadsudake [8].  13 C NMR spectra indicated, however, that it was a mixture of two isomeric compounds 2 and 3 in a ratio of 1:3. The 1 H and 13 C NMR spectra of compounds 2 and 3 showed significant differences to those of 1 and 4, notably the signals of two aliphatic methylene groups in 2 and one methylene and one methine group in 3. Carbonyl signals were absent, and the methyl signal of C-14 at the azulene system was shifted upfield. The 1 H NMR spectra of both compounds indicated two additional oxymethylene protons, which were connected with sp 3 carbons at d C 58.1 and 56.6, respectively, indicating the presence of the dihydroazulene alcohols 2 and 3; the latter is known to be very unstable [3,6,12].
We were unable to separate both compounds, however, succeeded to assign all NMR signals in the mixture and elucidated the structures of 2 and 3 unambiguously; the 1 H-1 H COSY spectrum was clearly resolved and indicated three spin systems for each component (see Fig. 2). The different signal intensities in the 1:3 mixture of 2 and 3 allowed us to assign the HSQC and HMBC signals without doubt. For the less intensive peaks (compound 2), the methylene protons at d H 4.33 (H 2 -15) correlated with the carbons at d C 147.4 (C-1) and 125.6 (C-2); the expected correlation with C-9 was observed for compound 3, but not for 2 (Fig. 2). These two carbons in turn correlated with the methylene protons at d H 3.20 (H 2 -3). HMBC correlation was also observed between C-1 and the olefinic proton at d H 6.49 (H-8), which showed further correlations with the carbons at d C 28.5 (C-6), 141.7 (C-11), and 145.7 (C-10). A long-range correlation was seen from the methyl at d H 1.93 (H-14) to C-10, and to the carbons at d C 132.7 (C-4) and 115.8 (C-5). Thereby, the minor component was finally characterized as 15-hydroxy-3,6-dihydrolactarazulene (2).
In a similar way, the main component was identified as 15-hydroxy-6,7-dihydrolactarazulene (3). Its structure was further supported by comparison of the 1 H NMR data with those published by Sterner et al. [3]. To the best of our knowledge, the 13 C NMR data (Table 1) for this compound are reported here for the first time.
Compounds 1 and 4-6 were screened for their antimicrobial activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Mucor miehei. Only 7-acetyl-4-methylazulene-1-carbaldehyde (5) displayed a moderate antibacterial activity against S. aureus in the agar diffusion test, with an inhibition diameter of 15 mm at a concentration of 50 lg/disc.

General Experimental Procedures
The NMR spectra were recorded on a Varian Inova-500 spectrometer at 599.737 MHz ( 1 H) or 150.818 MHz ( 13 C), respectively. The chemical shifts are given in d values with TMS as internal reference, and coupling constants are given in [Hz]. The ESI and ESI HR mass spectra were recorded on a Bruker micrOTOF mass spectrometer. Open column chromatography was done on silica gel 60 (0.063-0.20 mm), and PTLC was performed on silica gel P/UV 254 (both obtained from Macherey-Nagel, Düren, Germany). Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Pre-coated silica gel plates (Polygram SIL G/UV 254 , Macherey-Nagel & Co.) were used for TLC. Spots were visualized at 254 or 365 nm, and sprayed with an anisaldehyde/sulfuric acid reagent followed by heating.

Antimicrobial Test
The antimicrobial test was performed according to a previously described procedure [16].