New Bioactive β-Resorcylic Acid Derivatives from the Alga-Derived Fungus Penicillium antarcticum KMM 4685

Five new β-resorcylic acid derivatives, 14-hydroxyasperentin B (1), β-resoantarctines A-C (3, 5, 6) and 8-dehydro-β-resoantarctine A (4), together with known 14-hydroxyasperentin (5′-hydroxyasperentin) (2), were isolated from the ethyl acetate extract of the fungus Penicillium antarcticum KMM 4685 associated with the brown alga Sargassum miyabei. The structures of the compounds were elucidated by spectroscopic analyses and modified Mosher’s method, and the biogenetic pathways for compounds 3–6 were proposed. For the very first time, the relative configuration of the C-14 center of a known compound 2 was assigned via analyses of magnitudes of the vicinal coupling constants. The new metabolites 3–6 were biogenically related to resorcylic acid lactones (RALs); however, they did not possess lactonized macrolide elements in their structures. Compounds 3, 4 and 5 exhibited moderate cytotoxic activity in LNCaP, DU145 and 22Rv1 human prostate cancer cells. Moreover, these metabolites could inhibit the activity of p-glycoprotein at their noncytotoxic concentrations and consequently synergize with docetaxel in p-glycoprotein-overexpressing drug-resistant cancer cells.


Introduction
Marine-derived fungi are a rich source of promising lead molecules with various bioactive proprieties [1,2]. Penicillium species are among the most widespread fungal organisms on our planet. Penicillium antarcticum is a common species of micromycetes belonging to subgenus Aspergilloides, section Canescentia. Along with other representatives of the section Canescentia, P. antarcticum is a characteristic species of terrestrial and marine fungal assemblages and is often isolated from soils and substrates of plant and animal origin [3,4]. Currently, section Canescentia includes 23 species, of which 6 belong to the series of Atroveneta, including P. antarcticum [5]. Fungi of the Canescentia section are widespread and colonize various terrestrial and marine substrates, which was suggested to be due to their high metabolic activity, making representatives of this group promising sources of biologically active compounds [6]. Various compounds have been reported to be isolated from P. canescens. Among them, there are multioxidized aromatic polyketides penicanesins and colonize various terrestrial and marine substrates, which was suggested to be due to their high metabolic activity, making representatives of this group promising sources of biologically active compounds [6]. Various compounds have been reported to be isolated from P. canescens. Among them, there are multioxidized aromatic polyketides penicanesins A-G [7], penicanesones A-C [8], canescones A-E [9], brominated azaphilones [10], tetrapeptide D-Phe-L-Val-D-Val-L-Tyr [11], polyketides antarones A and B [12] and cladomarine (asperentin B) [13,14].
Resorcylic acid lactones (RALs) are structurally diverse polyketides, which usually consist of condensed resorcylic and macrolide cycles [15]. Very recently, Bang and colleagues reported the very first non-lactonized RALs, i.e., possessing an opened macrolide cycle [16]. These compounds were isolated from the halophyte-associated marine fungus Colletotrichum gloeosporioides JS0419 [16]. RALs exhibit a broad range of biological activities, including anticancer activities [17,18]. To date, there are around 50 molecules belonging to this group for which a significant (IC50 < 10 µM) in vitro anticancer activity in human cancer cells has been shown (reviewed in [17]). Moreover, the main molecular targets of RALs in mammalian cells known to date are Hsp90, protein kinases and NF-kB [17]. Thus, zearalenone was shown to inhibit activity of the NF-κB transcriptional factor [19]; radicicol, pochonin D and other RALs could suppress Hsp-90 [18,20]. Various RALs were reported to irreversibly inhibit activity of several important protein kinases, which play a critical role in cancer-related MEK, ERK, RAS and RAF pathways [17]. Thus, activities of MEK, ERK, TAK, AKT, SRC, Aurora A and other kinases were reported to be suppressed by naturally derived RALs and their synthetic derivatives (reviewed in [17]). Additionally, radicicol A was identified as inhibitor of IL-1β, and some derivatives of pochonin D were shown to be VEGFR suppressors [17].
Recently, our group reported the isolation and characterization of three new bioactive meroterpenoids with rearranged skeletons, meroantarctines A-C, from the Penicillium antarcticum KMM 4685 [21]. Further searches for of new metabolites from this fungus strain resulted in the isolation of six β-resorcylic acid derivatives (1-6) ( Figure 1). Herein, we report their isolation, structure elucidation, and activity in mammalian cells as well as the identification of the new type of biological activity for these RALs.

Structure Determination
The molecular formula of 2 was established as C 16 H 20 O 6 from an HRESIMS peak at m/z 331.1153 [M + Na] + with seven degrees of unsaturation. Using this result combined with the results of careful inspection of 13 C and 1 H NMR spectra (Table 1), including HSQC, HMBC, COSY and DEPT experiments, as well as comparison with literature data [22,23], the compound 2 was established as known 14-hydroxyasperentin (5 -hydroxyasperentin). Table 1. 13  According to the previously published data, the relative configuration of C-14 (C-5 ) chiral center of compound 2 suggests its β-configuration; however, the authors of the primary publication did not provide any spectral data or other proofs supporting this speculation [23]. In the other reports the relative configuration of the C-5 chiral center of 5 -hydroxyasperentin has not been established [22,[24][25][26][27]. The ROESY spectrum of 2 did not contain any cross-peaks, which can be useful to establish the relative stereochemistry of 2. However, the thorough analysis of the vicinal coupling constants for H-15 (δ H 3.96, J = 6.6, 4.2 Hz) and H-14 (δ H 3.70, J = 9.0, 4.3 Hz) together with biogenetic considerations of cladosporin biosynthesis and their derivatives [23,26] suggested a β-configuration of the methyl group at C-15 and a β-configuration of the hydroxy group at C-14. Esterification of 2 with (R)-and (S)-MTPA chloride occurred at the C-14 hydroxy group to give the (S)and (R)-MTPA esters 2a and 2b, respectively. The observed chemical shift differences ∆δ (δS − δR) ( Figure 2) indicated the 14S configuration. Taken together, these data indicate the absolute stereostructure of 2 to be 9R,11R,14S,15S.
The molecular formula of 1 was established to be C 16 Figure 3). Compound 1 was named 14-hydroxyasperentin B (the numeration of atoms was assigned as in the original papers [13,14]). Similar to compound 2, the ROESY spectrum of 1 could not be used to determine the stereoconfigurations of 1. However, based on the obvious biogenetic similarity, the identity of the chemical shift values of compounds 1 and 2 (Table 1) and the specific optical rotation values of [α] D 20 -15.5 (for 1) and [α] D 20 -14.1 (for 2) were found, and the absolute configurations of the stereogenic centers of 1 were assigned as 9R,11R,14S,15S.
The molecular formula of 6 was established to be C 18  were close to those in 5 with the exception of the C-7 (δ C 145.4), C-8 (δ C 39.9), C-9 (δ C 74.2), C-10 (δ C 27.2), C-11 (δ C 18.7), C-12 (δ C 33.3), C-13 (δ C 67.8) and C-14 (δ C 21.3) carbon signals ( Table 2). 1 H-1 H COSY and HSQC spectra of 6 revealed the partial connectivity sequence of the protons as CH 2 (8)−CH(9)−CH 2 (10)−CH 2 (11)−CH 2 (12)−CH(13)−CH 3 (14) and (Figure 3). These data showed the molecular formula corresponded to six degrees of unsaturation, the deshielding of the signal of H-9 to δ H 4.03 and of the corresponding carbon signal (C-9) to δ C 74.2 as well as HMBC correlations from H-9 (δ H 4.03) to C-7 and C-13, which allowed us to suppose the presence of the ring B at C-8. HMBC correlations from the hydroxyl groups from (δ H 5.00) to C-2 and C-3 ; from (δ H 3.99) to C-2 , C-3 and C-4 ; and from (δ H 5.00) to C-4 indicated their locations at C-2 , C-3 and C-4 , respectively. The observed ROESY correlations from H-13 (δ H 3.91) to H 2 -8 (δ H 3.45, δ H 3.14) and H a -11 (δ H 1.85) and from H b -8 (δ H 3.14) to H a -11 ( Figure S63) indicated axial location of H a -11 and H-13 and substituent at C-9 ( Figure 4). This information revealed the β-configuration of H 3 -14 and H-9.   Previously, RALs biosynthetic pathways were investigated in detail in several studies [15,18]. The formation polyketide precursor, which is catalyzed by polyketide synthase, is followed by the further cyclization to β-resorcylic acid [15]. Ultimately this results in the formation of a hybrid structure consisting of 2,4-dihydroxybenzoic acid moiety and a macrolide ring [15]. Usually, the β-resorcylic acid residue has an aliphatic side chain that is attached at C-7 and esterified with the C-1 carboxylic acid, therefore forming a macrolactone ring [15]. However, in the isolated compounds 3−6, the glycerol and butane-1,2,3,4-tetraol were esterified with the C-1 carboxylic acid, therefore forming non-lactonizes β-resorcylic acid derivatives. Thus, our study is (i) the very first report on nonlactonizes RALs having polyol esterified at C-1 and (ii) the second report on β-resorcylic acid harboring a long unesterified aliphatic side chain [16]. Based on this, for the isolated The planar structure of compound 6, named β-resoantarctine C, was also confirmed by the obtaining its pentaacetate derivative 6a ( Figure 5) via acylation reaction. The molecular formula of 6a was established as C 28 H 36 O 13 from a HRESIMS peak at m/z 603.2051 [M + Na] + and was supported by the analysis of 13 C NMR data ( Figure S45).   Previously, RALs biosynthetic pathways were investigated in detail in several studies [15,18]. The formation polyketide precursor, which is catalyzed by polyketide synthase, is followed by the further cyclization to β-resorcylic acid [15]. Ultimately this results in the formation of a hybrid structure consisting of 2,4-dihydroxybenzoic acid moiety and a macrolide ring [15]. Usually, the β-resorcylic acid residue has an aliphatic side chain that is attached at C-7 and esterified with the C-1 carboxylic acid, therefore forming a macrolactone ring [15]. However, in the isolated compounds 3−6, the glycerol and butane- Previously, RALs biosynthetic pathways were investigated in detail in several studies [15,18]. The formation polyketide precursor, which is catalyzed by polyketide synthase, is followed by the further cyclization to β-resorcylic acid [15]. Ultimately this results in the formation of a hybrid structure consisting of 2,4-dihydroxybenzoic acid moiety and a macrolide ring [15]. Usually, the β-resorcylic acid residue has an aliphatic side chain that is attached at C-7 and esterified with the C-1 carboxylic acid, therefore forming a macrolactone ring [15]. However, in the isolated compounds 3-6, the glycerol and butane-1,2,3,4-tetraol were esterified with the C-1 carboxylic acid, therefore forming non-lactonizes β-resorcylic acid derivatives. Thus, our study is (i) the very first report on non-lactonizes RALs having polyol esterified at C-1 and (ii) the second report on β-resorcylic acid harboring a long unesterified aliphatic side chain [16]. Based on this, for the isolated compounds 3−6 we suggest a biosynthetic pathway that includes the same steps as previously reported fungal RALs, but without a macrolide ring formation ( Figure 6).

Activity and Selectivity in Prostate Cancer Cells
Previously, RALs have been reported to exhibit cytotoxic activity in human cancer cell lines in vitro [17]. This effect has been stipulated by Hsp90, protein kinase (PK) and NF-kB inhibition. Moreover, the 14-membered RALs were the most potent among the investigated molecules regarding anticancer activity [17]. Therefore, we examined cytotoxic activity of the three new isolated RALs (3-6) using human prostate cancer cells bearing various levels of drug resistance. Hence, we used PC-3 and DU145 cells, which do not express the full-length androgen receptor (AR) and therefore are non-sensitive to hormonal therapy; docetaxel-and hormonal therapy-resistant PC3-DR cells; 22Rv1 and VCaP cells, which are also insensitive to hormonal therapy due to expression of the AR splice variant V7 (AR-V7) resulting in a ligand-independent AR pathway activation; and hor-

Activity and Selectivity in Prostate Cancer Cells
Previously, RALs have been reported to exhibit cytotoxic activity in human cancer cell lines in vitro [17]. This effect has been stipulated by Hsp90, protein kinase (PK) and NF-kB inhibition. Moreover, the 14-membered RALs were the most potent among the investigated molecules regarding anticancer activity [17]. Therefore, we examined cytotoxic activity of the three new isolated RALs (3-6) using human prostate cancer cells bearing various levels of drug resistance. Hence, we used PC-3 and DU145 cells, which do not express the full-length androgen receptor (AR) and therefore are non-sensitive to hormonal therapy; docetaxel-and hormonal therapy-resistant PC3-DR cells; 22Rv1 and VCaP cells, which are also insensitive to hormonal therapy due to expression of the AR splice variant V7 (AR-V7) resulting in a ligand-independent AR pathway activation; and hormone-sensitive LNCaP cells, which express the wild-type AR (full-length AR).
Overall, compounds 3, 4 and 5 exhibited moderate cytotoxic activity in LNCaP, DU145 and 22Rv1 cells, whereas they were inactive in PC3 and VCaP cells up to 100 µM (Table 4, Figure 7).     Interestingly, compound 3 was active in docetaxel-resistant PC3-DR cells, whereas no activity in PC3 cells was observed (Table 4).
We further examined the effect of the isolated compounds on the activity of pglycoprotein (p-gp). Overexpression of p-gp has been identified to be a major mechanism of drug resistance in cancer cells. It mediates the excretion of various chemotherapeutic agents, including taxanes, out of the cancer cell. To evaluate the effect of the isolated compounds on p-gp activity, we utilized the calcein exclusion assay and used p-gp overexpressing docetaxel-resistant PC3-DR cells as a model [28,29]. Calcein-AM is a non-fluorescent small molecule that can passively enter the cells via diffusion. In the cytoplasm, calcein-AM is hydrolyzed to release green fluorescent-free calcein, which can be then detected. Calcein and calcein-AM are known substrates for p-gp. Therefore, in cells overexpressing p-gp, non-hydrolyzed and therefore non-fluorescent calcein-AM is quickly evacuated back into the extracellular space, and no fluorescence can be observed. The inhibitors of p-gp, such as tariquidar, can block the p-gp-mediated drug excretion and therefore promote the therapeutic effects of the chemotherapeutics. Remarkably, we examined the p-gp inhibitory activity of compounds 3-5. For compounds 3 and 4, moderate p-gp inhibitory activity was observed only at high concentrations of~100 µM, whereas for carbonyl-containing compound 5, significant inhibitory activity was detected at the low concentrations of 5 µM (Figure 8).
Mar. Drugs 2023, 21, x 11 of 18 inhibitory activity was observed only at high concentrations of ~100 µM, whereas for carbonyl-containing compound 5, significant inhibitory activity was detected at the low concentrations of 5 µM (Figure 8). Accumulation of calcein in the cells results from p-gp inhibition, which may be either due to the p-gp blockade by its true inhibitor or by a concurrent substrate molecule. In order to identify whether the tested compounds are p-gp substrates (which ultimately lead to concurrent inhibition of calcein excretion from the cells), we examined the effect of well-established p-gp inhibitor tariquidar (TQD) on cytotoxic activity of the compounds in p-gp-overexpressing PC3-DR cells. Thus, co-treatment with 50 nM TQD did not result in any significant alteration of cytotoxic activity of the isolated compounds (Figure 9), while the activity of docetaxel (a clinically approved chemotherapeutic drug that is known to be a p-gp substrate) was dramatically increased [28]. Therefore, we concluded that unlike to docetaxel, the isolated compounds 3-5 are not p-gp substrates, but rather the true p-gp inhibitors. A high tolerance of the cells to this chemotherapeutic agent is due to p-gp-mediated excretion of the drug [28,29]. Hence, inhibition of p-gp activity should result in the accumulation of docetaxel within the cells, leading to the more pronounced cytotoxic activity of this drug. Therefore, we co-treated PC3-DR cells with docetaxel plus the isolated compounds 3-5. Further measurement of cellular viability followed by data analysis with the Accumulation of calcein in the cells results from p-gp inhibition, which may be either due to the p-gp blockade by its true inhibitor or by a concurrent substrate molecule. In order to identify whether the tested compounds are p-gp substrates (which ultimately lead to concurrent inhibition of calcein excretion from the cells), we examined the effect of well-established p-gp inhibitor tariquidar (TQD) on cytotoxic activity of the compounds in p-gp-overexpressing PC3-DR cells. Thus, co-treatment with 50 nM TQD did not result in any significant alteration of cytotoxic activity of the isolated compounds (Figure 9), while the activity of docetaxel (a clinically approved chemotherapeutic drug that is known to be a p-gp substrate) was dramatically increased [28]. Therefore, we concluded that unlike to docetaxel, the isolated compounds 3-5 are not p-gp substrates, but rather the true p-gp inhibitors.
Mar. Drugs 2023, 21, x 11 inhibitory activity was observed only at high concentrations of ~100 µM, whereas for bonyl-containing compound 5, significant inhibitory activity was detected at the low centrations of 5 µM (Figure 8). Accumulation of calcein in the cells results from p-gp inhibition, which may be ei due to the p-gp blockade by its true inhibitor or by a concurrent substrate molecul order to identify whether the tested compounds are p-gp substrates (which ultima lead to concurrent inhibition of calcein excretion from the cells), we examined the e of well-established p-gp inhibitor tariquidar (TQD) on cytotoxic activity of the c pounds in p-gp-overexpressing PC3-DR cells. Thus, co-treatment with 50 nM TQD not result in any significant alteration of cytotoxic activity of the isolated compounds ( ure 9), while the activity of docetaxel (a clinically approved chemotherapeutic drug is known to be a p-gp substrate) was dramatically increased [28]. Therefore, we conclu that unlike to docetaxel, the isolated compounds 3-5 are not p-gp substrates, but ra the true p-gp inhibitors. Figure 9. Effect of tariquidar on cytotoxicity of the isolated compounds in PC3-DR cells. The were pretreated with tariquidar (TQD, 50 nM) for 1 h and then co-treated with the tested pounds for 48 h. The viability was measured using MTT assay.
A high tolerance of the cells to this chemotherapeutic agent is due to p-gp-medi excretion of the drug [28,29]. Hence, inhibition of p-gp activity should result in the a mulation of docetaxel within the cells, leading to the more pronounced cytotoxic acti of this drug. Therefore, we co-treated PC3-DR cells with docetaxel plus the isolated c pounds 3-5. Further measurement of cellular viability followed by data analysis with SynergyFinder 2.0 software using the ZIP model indicated a synergistic effect of the c binational treatment, especially at a high concentration of both agents (Figure 10). T A high tolerance of the cells to this chemotherapeutic agent is due to p-gp-mediated excretion of the drug [28,29]. Hence, inhibition of p-gp activity should result in the accumulation of docetaxel within the cells, leading to the more pronounced cytotoxic activity of this drug. Therefore, we co-treated PC3-DR cells with docetaxel plus the isolated compounds 3-5. Further measurement of cellular viability followed by data analysis with the SynergyFinder 2.0 software using the ZIP model indicated a synergistic effect of the combinational treatment, especially at a high concentration of both agents ( Figure 10). These findings are in line with the above-reported p-gp inhibitory activity of the isolated compounds 3-5 ( Figure 10A,B).
Mar. Drugs 2023, 21, x 12 of 18 findings are in line with the above-reported p-gp inhibitory activity of the isolated compounds 3-5 ( Figure 10A,B).

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA) in MeOH. UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in MeOH. ECD spectra were measured using a Chirascan-Plus CD Spectrometer (Leatherhead, UK) in MeOH. 1 H and 13 C NMR spectra were recorded in aceton-d 6 on Bruker Avance-500, Avance III-700 and Bruker DPX-300 spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 500 MHz and 125 MHz, 700 and 176 MHz and 300 and 75 MHz, respectively, using TMS as an internal standard. HRESIMS spectra were obtained using Bruker maXis Impact II mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany).

Fungal Strain
The strain of the fungus Penicillium antarcticum KMM 4685 was isolated from superficial mycobiota of the marine brown alga Sargassum miyabei (Sea of Japan) (GenBank sequence dataset and registered under accession number MW527122) [21]. The strain is stored at the Collection of Marine Microorganisms (KMM) of G.B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia).

Cultivation of P. thomii
The fungus was grown stationary at 22 • C for 21 days in 100 Erlenmeyer flasks (500 mL), each containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH 2 PO 4 and 40 mL of natural sea water (Marine Experimental Station of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Troitsa (Trinity) Bay, Sea of Japan).

Extraction and Isolation
At the end of the incubation period, the mycelia and medium were homogenized and extracted with EtOAc (1 L). The obtained extract was dried in vacuo. The residue was dissolved in H 2 O−EtOH (4:1) (300 mL) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3). After evaporation of the EtOAc layer, the residual material (5 g) was passed over a silica column (6 × 15 cm), which was eluted followed by a step gradient from 10:1 to 6:1 EtOH in CHCl 3 (total volume 5 L). Fractions of 250 mL were collected and combined based on TLC results (Si gel, toluene-isopropanol 6:1 and 3:1, v/v). As a result, two fractions were obtained: SM-1327-3 (1.87 g) and SM-1327-4 (640 mg).

Cell Lines and Culture Conditions
The human prostate cancer cells 22Rv1, LNCaP, VCaP and PC-3, DU145 were purchased from ATCC (Manassas, VA, USA). Docetaxel-resistant human prostate cancer cells PC3-DR were generated by the long-term incubation of PC3 cells in the sub-lethal concentrations of docetaxel [30]. The cells were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany). The cells were cultured as previously described [31].

MTT Assay
Effect of the drugs on viability of the cells was evaluated using MTT assay, as previously described [32]. In brief, 6 × 10 3 cells per well in 100 µL per well were plated in 96-well plates and treated with the tested compounds at the indicated concentrations. After 48 h of incubation, the MTT reagent was added, and the viability was measured following 2 h of incubation. The vehicle-treated cells were used as a control. IC50s were calculated using GraphPad Prism v.9.1.1 (GraphPad Software, San Diego, CA, USA).

Drug Combination Studies
The PC3-DR cells were treated with the single drugs at the indicated concentrations and their combinations with docetaxel for 48 h in 100 µL/well as described for MTT assay. The viability was measured using MTT assay, and the generated data were further analyzed using SynergyFinder 2.0 software (https://synergyfinder.fimm.fi [33] (accessed on 9 September 2022)) as previously reported [31]. The difference between the expected and observed drug combination effects was evaluated using Zero Interaction Potency (ZIP) reference model [34]. Synergism was identified as areas with positive δ-values (marker with red), whereas antagonism refers to the areas with negative δ-values (marker with green).

P-Glycoprotein Activity Assay
PC3-DR cells were seeded in a 96-well plate (6 × 10 3 cells/well in 100 µL/well) in the drug-and docetaxel-free medium, incubated overnight and then treated with the tested drugs for 30 min in PBS. Then calcein-AM solution was added to each well, and the green fluorescence was measured using the plate reader following 15 min of incubation according to the manufacturer's protocol. The fluorescence was normalized to the background autofluorescence of the drug solutions as well as to the cellular viability, which was evaluated by MTT assay at the same experimental conditions.

Statistical Analysis
The experiments were performed in biological triplicates. Statistical analyses were performed using GraphPad Prism v.9.1.1 (GraphPad Software, San Diego, CA, USA) and the data are represented as mean ± SD (standard deviation). For the analysis of the statistical significance of the difference between the drug-exposed group and the control group, the one-way ANOVA test and Dunnett's post-hoc test were used; for the difference between two groups, the Student's t-test was used. The statistically significant difference is indicated as (*) if p < 0.05 in either statistical test.

Conclusions
In conclusion, we report isolation and structure elucidation of five new β-resorcylic acid derivatives-β-resoantarctines A-C (3, 5, 6), 8-dehydro-β-resoantarctine A (4) and 14-hydroxyasperentin B (1)-as well as one previously known 14-hydroxyasperentin (2) from the alga-derived fungus Penicillium antarcticum KMM 4685. For the very first time, an absolute configuration of 14-hydroxyasperentin (2) was established using the modified Mosher's method. The configuration of a new compound 1 was determined based on a comparison of its spectral data with those of 2 as well as using biosynthetical considerations. The new isolated compounds 3-6 were structurally and biosynthetically related to resorcylic acid lactones (RAls), but had non-lactonizes structures, possessing an opened macrolide cycle. Compounds 3, 4 and 5 exhibited a moderate cytotoxic activity in LNCaP, DU145 and 22Rv1 human prostate cancer cells. Remarkably, they inhibited an activity of p-glycoprotein at their noncytotoxic concentrations and consequently synergized with docetaxel in p-glycoprotein-overexpressing drug-resistant cells.