*2.8. An Epoxy Ring-Opening Reaction*

Next, 2,2-dimethylpropanoyl chloride (0.5 mL) was added to an aqueous solution of **6** (12.0 mg) at room temperature, and the solution was stirred for 12 h. After the evaporation of the solvent, the product was dissolved in MeOH and purified by reversed-phase HPLC (Phenomenex Hydro-RP column) eluted with CH3OH-H2O (50:50) to yield compound **4** (3.7 mg) and compound **5** (6.4 mg).

#### *2.9. Cell Lines and Culture Conditions*

The human normal cell lines HEK 293T and MRC-9 cell lines were purchased from ECACC (Salisbury, UK). Human normal cell lines PNT2 and RWPE-1 as well as human cancer cell lines PC-3, DU145, 22Rv1, VCaP and LNCaP, as well as a human normal prostate line were purchased from ATCC (Manassas, VA, USA). Human normal cell line HUVEC (passage 11) was kindly donated by Prof. Sonja Loges (University Medical Center Hamburg-Eppendorf, Hamburg, Germany). The human keratinocytes cell line HaCaT was kindly provided by Prof. N. Fusenig (Cancer Research Centre, Heidelberg, Germany). All the cells had a passage number ≤ 30.

Cells were incubated in humidified 5% CO<sup>2</sup> at 37 ◦C. The cells were continuously kept in a culture for 3 months maximum and regularly checked for mycoplasma infection using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Karlsruhe, Germany) and stable phenotype using light microscopy [17].

The following culture media were used: RPMI medium supplemented with GlutamaxTM-I (Invitrogen, Paisley, UK) with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen) for PC-3, DU145, LNCaP, 22Rv1 and PNT2 cells. DMEM medium supplemented with GlutamaxTM-I (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen) for MRC-9, HEK 293 and VCaP cells. Clonetics® EGMTM-2 SingleQuots® medium (Lonza, Walkersville, MD, USA) containing 10% FBS for RWPE-1 cells. Clonetics® EGMTM-2 SingleQuots® medium (Lonza, Walkersville, MD, USA) containing 10% FBS for HUVEC cells. DMEM medium (BioloT, St. Petersburg, Russia) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen) for HaCaT cells.

#### *2.10. MTT Assay*

Cytotoxicity of the isolated compounds to mammalian cells was evaluated using an MTT assay as previously reported with minor modification [18]. In brief, 6000 cells/well were seeded in 96-well planes in 100 µL/well and were incubated overnight. Then the media were exchanged with fresh media containing tested compounds in different concentrations. Following 72 h of incubation, 10 µL of MTT solution (5 mg/mL, Sigma-Aldrich, Munich, Germany) was added to each well, the cells were incubated for 2–4 h. Then the media was carefully aspirated and the plates were dried for 2 h. Then 50 µL/well of DMSO was added to each well to dissolve formazan crystals and the absorbance was measured using plate reader according to the manufacture's protocol. The data were analyzed and the IC50s values were calculated using GraphPad Prism software v.9.1.1 (GraphPad Software, San Diego, CA, USA).

#### *2.11. Sortase Activity Inhibition Assay*

The enzymatic activity of sortase A from *Staphylococcus aureus* was determined using SensoLyte 520 Sortase A Activity Assay Kit \* Fluorimetric \* (AnaSpec AS-72229, AnaSpec, San Jose, CA, USA) in accordance with the manufacturer's instructions. Substances **1**–**6** were dissolved in DMSO and diluted with reaction buffer to obtain a final concentration of 0.8% DMSO, which did not affect enzyme activity. DMSO at a concentration of 0.8% was used as a control. PCMB (4-(hydroxymercuri)benzoic acid) was used as sortase A enzyme activity inhibitor. Fluorescence was measured with a plate reader PHERAStar FS (BMG Labtech, Offenburg, Germany) for 60 min, with a time interval of 5 min. The data were processed with MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany). The results were presented as relative fluorescent units (RFUs) and percentage of the control data, calculated using STATISTICA 10.0 software [14].

#### *2.12. Antimicrobial Activity*

The antibacterial activity of compounds **1**–**6** was evaluated as described previously [19]. The bacterial culture of *Staphylococcus aureus* ATCC 21027 (Collection of Marine Microorganisms PIBOC FEBRAS) was cultured in a Petri dish at 37 ◦C for 24 h on solid medium Mueller Hinton broth with agar—16.0 g/L.

The assays were performed in 96-well microplates in appropriate Mueller Hinton broth. Each well contained 90 µL of bacterial suspension (10<sup>9</sup> CFU/mL). Then, 10 µL diluted at concentrations from 1.5 µM to 100.0 µM using two-fold dilution was added to compounds **1**–**6** (DMSO concentration < 1%). Culture plates were incubated overnight at 37 ◦C, and the OD<sup>620</sup> was measured using a Multiskan Spectrum spectrophotometer (Thermo Labsystems Inc., Beverly, MA, USA). Gentamicin was used as a positive control in concentration 1 mg/mL; 1% DMSO solution in PBS as a negative.

#### *2.13. Biofilm Formation*

The inhibition of the reducing biofilm formation and growth was assessed using the crystal violet biofilm assay as described [20]. Mueller Hinton broth was inoculated with 10<sup>9</sup> CFU/mL of *S. aureus* overnight cultures. A total of 90 µL of this cell suspension was then dispensed into 96-well microtiter plates containing 10 µL of different concentrations of compounds **1**–**6**. After 24 h growth at 37 ◦C the plates were washed with PBS to remove unbound cells. Next, the wells were stained with 0.1% crystal violet solution for 10 min at 37 ◦C. At the completion of the incubation, plates were washed 3 times with PBS and dried. Then, the crystal violet dye from the biofilm was solubilized with 100 µL of ethanol. A total of 100 µL of this solution was then moved to a new microtiter plate for absorbance measurement at λ = 570 nm. The results were reported as percent inhibition normalized to the wild-type control.

#### *2.14. Co-Cultivation of HaCaT Cells with S. aureus*

Co-cultivation of HaCaT cells with *S. aureus* was carried out as described [21]. HaCaT cells at a concentration of 1.5×10<sup>4</sup> cells per well were seeded in 96-well plates for 24 h. Then, culture medium in each well was changed with *S. aureus* suspension (10<sup>2</sup> CFU/mL) in full DMEM medium. Fresh DMEM medium without *S. aureus* suspension was added in other wells as needed. Compounds **1**–**6** at a concentration of 10 µM were added in wells after 1 h. HaCaT cells and *S. aureus* were cultured at 37 ◦C in a humidified atmosphere with 5% (*v*/*v*) CO<sup>2</sup> for 48 h.

After incubation, the plate was centrifuged at 250× *g* for 10 min and 50 µL of supernatant from each well was transferred into the corresponding wells of an optically clear 96-well plate. An equal volume of the reaction mixture (50 µL) from LDH Cytotoxicity Assay Kit (Abcam, Cambridge, UK) was added to each well and incubated for up to 30 min at room temperature. The absorbance of all samples was measured at λ = 450 nm using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) and expressed in optical units (o.u.).

#### *2.15. Statistical Analysis*

All the experiments were performed in biological triplicates. Statistical analyses were performed using GraphPad Prism v.9.1.1 (GraphPad Software, San Diego, CA, USA) or STATISTICA 10.0 software. The data are reported as mean ± SD (standard deviation). For the analysis of statistical differences between the control and drug-exposed group, a one-way ANOVA test followed by Dunnett's post-hoc test was used. Asterisk (\*) indicates statistically significant difference between the treated group and control group if *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Isolated Compounds from Asteromyces cruciatus*

The fungus *Asteromyces cruciatus* was cultivated on a solid rice medium for 21 days. The ethyl acetate extract of the mycelium was fractionated on silica gel, followed by C18- SiO2-column chromatography, and reversed-phase HPLC to produce compounds **1**–**6** (Figure 1).

#### *3.2. Structural Characterization of New Compounds*

The molecular formula of **1** was determined as C11H15ClO3, based on the analysis of HRESIMS (*m/z* 229.0625 [M – H]– calcd for C11H14ClO3, 229.0637), showing the characteristic isotope pattern with one chlorine atom, and confirmed by NMR data. A close inspection of the <sup>1</sup>H and <sup>13</sup>C NMR data (Tables 1 and 2; Figures S13–S19) of **1** by DEPT and HSQC revealed the presence of one methyl (δ<sup>H</sup> 1.80, δ<sup>C</sup> 23.5), two methylenes (δ<sup>H</sup> 1.65, 1.92, δ<sup>C</sup> 33.1; δ<sup>H</sup> 5.14, 5.18, δ<sup>C</sup> 120.7), and five methines (δ<sup>H</sup> 2.96, δ<sup>C</sup> 28.5; δ<sup>H</sup> 4.00, δ<sup>C</sup> 65.7; δ<sup>H</sup> 3.92, δ<sup>C</sup> 68.2; δ<sup>H</sup> 3.57, δ<sup>C</sup> 70.2 and δ<sup>H</sup> 3.81, δ<sup>C</sup> 71.9), including three oxygen-bearing methines, one *sp<sup>2</sup>* quaternary carbon and one triple bond (δ<sup>C</sup> 81.7 and 91.1). The <sup>1</sup>H–1H COSY correlations of H-1(OH)/H-2(OH)/H-3/H-4(OH)/H2-5/H-6/H-1 together with the <sup>1</sup>H-13C HMBC correlations (Figure 2) OH-1 (δ<sup>H</sup> 4.89)/C-6 (δ<sup>C</sup> 28.5); OH-2 (δ<sup>H</sup> 4.92)/C-1 (δ<sup>C</sup> 71.9), C-2 (δ<sup>C</sup> 70.2), C-3 (δ<sup>C</sup> 65.7) and OH-4 (δ<sup>H</sup> 5.06)/C-4 (δ<sup>C</sup> 68.2), C-5 (δ<sup>C</sup> 33.1) indicated the presence of a penta-substituted cyclohexane ring and the location of the hydroxy groups at C-1, C-2 and C-4 in **1**. These data, as well as the chemical shifts of CH-3 (δ<sup>H</sup> 4.00, δ<sup>C</sup> 65.7), indicated the location of the chlorine atom at C-3.

**Figure 1.** Chemical structures of **1**–**6**.

**Figure 2.** Key <sup>1</sup>H–1H COSY, <sup>1</sup>H–13C HMBC (**a**) and ROESY (**b**) correlations of **1**.

The HMBC correlations from H-40a (δ<sup>H</sup> 5.14) to C-2<sup>0</sup> (δ<sup>C</sup> 81.7) and C-3<sup>0</sup> (δ<sup>C</sup> 126.8), from H3-5<sup>0</sup> (δ<sup>H</sup> 1.80) to C-2<sup>0</sup> , C-30 and C-40 (δ<sup>C</sup> 120.7) and cross <sup>1</sup>H–1H COSY correlations between H2-40 and H3-50 revealed the presence of a 3-methyl-3-buten-1-ynyl side chain in **1**. The correlations H-6 (δ<sup>H</sup> 3.19)/C-1<sup>0</sup> (δ<sup>C</sup> 87.5) and C-2<sup>0</sup> (δ<sup>C</sup> 84.1) observed in the HMBC spectrum, recorded in CDCl<sup>3</sup> (Figures S20–S22), established the position of the side chain at C-6.

The relative configurations of **1** were assigned based on ROESY correlations (Figure 2) H-6 (δ<sup>H</sup> 2.96)/H-2 (δ<sup>H</sup> 3.57); OH-4 (δ<sup>H</sup> 5.06)/H-5β (δ<sup>H</sup> 1.65) and H-2; H-3 (δ<sup>H</sup> 4.00)/H-2β (δ<sup>H</sup> 1.92), OH-1 (δ<sup>H</sup> 4.89) and <sup>1</sup>H-1H coupling constants (Table 1). For further investigation, we analyzed the stereoconfigurations of diol at C-1 and C-2 and for protection of these groups before MTPA-esters obtaining the acetonide derivative (**1a**) of compound **1** (Figure 3) was prepared. The small coupling constant (*J*1,2 = 4.3 Hz) and dissimilar magnetic environment of acetonide methyls (∆ = 0.13 ppm) (Figure S57) indicate an *erythro* configuration of the diol group at C-1 and C-2 [22]. The absolute configuration of **1** was established by the modified Mosher's method [23]. Esterification of **1a** with (*S*)- and (*R*)- MTPA chloride occurred at the C-4 hydroxy group to yield the (*R*)-and (*S*) MTPA esters **1a-1** and **1a-2**, respectively. The observed chemical shift differences ∆δ(δ*S*-δ*R*) (Figure 3) indicated the 4*R* configuration and, therefore, the absolute configurations of **1** were established as 1*R*,2*R*,3*R*,4*R*,6*S*. Compound **1** was named acrucipentyn A.

**Figure 3.** Chemical structure of **1a** (**a**) and ∆δ(δS-δR) values (in ppm) for MTPA esters of **1a** (**b**).

The HRESIMS of **2** and **3** showed the peaks of [M–H]– at *m/z* 229.0634 and *m/z* 229.0631, respectively. These data, coupled with <sup>13</sup>C NMR spectral data (DEPT), established the molecular formulas of **2** and **3** as C11H15ClO<sup>3</sup> for both. A close inspection of the <sup>1</sup>H and <sup>13</sup>C NMR data (Tables 1 and 2 and Figures S23–S35) of **2** and **3** by DEPT and HSQC revealed the presence of a penta-substituted cyclohexane ring with three hydroxy groups and a 3-methyl-3-buten-1-ynyl side chain.

The main <sup>1</sup>H–1H COSY and HMBC correlations (Figures S26 and S28) indicated that compound **2** has the same planar structure as **1**. The relative configuration of **2** was assigned based on <sup>1</sup>H-1H vicinal coupling constants (Table 1) and ROESY (Figure S29) correlations H-6 (δ<sup>H</sup> 2.87)/H-4 (δ<sup>H</sup> 4.22) and H-5β (δ<sup>H</sup> 1.91)/H-1 (δ<sup>H</sup> 3.92). Due to the small amount of compound **2,** the absolute configuration establishing by Mosher's method was impossible. Compound **2** was named acrucipentyn B.

The <sup>1</sup>H–1H COSY correlations of H-1(OH)/H-2/H-3(OH)/H-4(OH)/H2-5/H-6 together with the <sup>1</sup>H-13C HMBC correlations (Figure S34) OH-1 (δ<sup>H</sup> 4.38)/C-1 (δ<sup>C</sup> 74.2), C-2 (δ<sup>C</sup> 68.9) and C-6 (δ<sup>C</sup> 35.0); OH-3 (δ<sup>H</sup> 4.50)/C-2, C-3 (δ<sup>C</sup> 79.5) and C-4 (δ<sup>C</sup> 71.1), and OH-4 (δ<sup>H</sup> 4.04)/C-3, C-4 and C-5 (δ<sup>C</sup> 34.2) indicated the location of the hydroxy groups at C-1, C-3, C-4 and a chlorine atom at C-2 in a penta-substituted cyclohexane ring of **3**. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in **3** were determined by HMBC correlations (Figure S34), as for acrucipentyn A (**1**).

The relative configurations of the chiral centers in **3** were determined based on <sup>1</sup>H-1H coupling constants (Table 1). Using the Mosher's method to determine absolute configurations of compound **3** was unsuccessful, due to lability in this compound. Compound **3** was named acrucipentyn C.

The HRESIMS of **4** showed the peak of [M – H]– at *m/z* 227.0471. These data, coupled with <sup>13</sup>C NMR spectral data (DEPT), established the molecular formula of **4** as C11H13ClO3. The <sup>1</sup>H and <sup>13</sup>C NMR (Tables 1 and 2 and Figures S36–S42), DEPT and HSQC spectra showed the presence of three hydroxy protons (δ<sup>H</sup> 5.25, 5.17, 5.04), one methyl group (δ<sup>H</sup> 1.86, δ<sup>C</sup> 23.0), one olefinic methylene (δ<sup>H</sup> 5.32, 5.27, δ<sup>C</sup> 122.2) and five methines (δ<sup>H</sup> 4.25, δ<sup>C</sup> 63.3; δ<sup>H</sup> 4.37, δ<sup>C</sup> 65.1; δ<sup>H</sup> 4.06, δ<sup>C</sup> 67.9; δ<sup>H</sup> 3.87, δ<sup>C</sup> 68.6 and δ<sup>H</sup> 5.92, δ<sup>C</sup> 136.0), including three oxygen-bearing methines and one olefinic methine, two *sp<sup>2</sup>* quaternary carbons and one triple bond (δ<sup>C</sup> 88.2 and 89.9).

The HMBC correlations (Figure 4) from H-3 (δ<sup>H</sup> 4.25) to C-1 (δ<sup>C</sup> 67.9), C-2 (δ<sup>C</sup> 68.6), C-4 (δ<sup>C</sup> 65.1) and C-5 (δ<sup>C</sup> 136.0); from H-5 (δ<sup>H</sup> 5.92) to C-1, C-4, C-6 (δ<sup>C</sup> 123.5) and C-1<sup>0</sup> (δ<sup>C</sup> 88.2), and from OH-4 (δ<sup>H</sup> 5.25) to C-4, C-5, together with <sup>1</sup>H–1H COSY correlations of H-1(OH)/H-2(OH)/H-3/H-4(OH)/H-5, indicated the presence of a penta-substituted cyclohexene ring with ∆ 5,6 double bond, the location of the hydroxy groups at C-1, C-2, C-4 and a chlorine atom at C-3 in **4**. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in **4** were determined by HMBC correlations (Figure S41), as for acrucipentyn A (**1**).

**Figure 4.** Key <sup>1</sup>H–1H COSY, <sup>1</sup>H–13C HMBC correlations of **4** (**a**) and chemical structure of acetonide derivatives **4a** (**b**).

The relative configuration of **4** was assigned based on <sup>1</sup>H-1H coupling constants (Table 1) and ROESY correlation (Figure S42) H-2 (δ<sup>H</sup> 3.87)/OH-4 (δ<sup>H</sup> 5.25). The acetonide derivative (**4a**) of compound **4** (Figure 4) was prepared for further investigation of the stereochemistry at the diol position. The small coupling constant (*J*1,2 = 5.7 Hz) and dissimilar magnetic environment of acetonide methyls (∆ = 0.01 ppm) (Figure S63) indicate an *erythro* configuration of the diol group at C-1 and C-2 [21]. Esterification of **4a** with (*S*)- and (*R*)-MTPA-Cl led to destruction of the compound. Etherification of compound **4** with (*S*)- and (*R*)-MTPA chloride resulted in the formation of esters at three hydroxyl groups, which made it impossible to establish the absolute configuration using the modified Mosher's method. Compound **4** was named acrucipentyn D.

The HRESIMS of **5** showed the peak of [M – H]– at *m/z* 227.0468. These data, coupled with <sup>13</sup>C NMR spectral data (DEPT), suggested the molecular formula of **5** as C11H13ClO3. The <sup>1</sup>H and <sup>13</sup>C NMR data (Tables 1 and 2 and Figures S43–S49) of **5** revealed the presence of a penta-substituted cyclohexene ring with three hydroxy groups and a 3-methyl-3-buten-1-ynyl side chain, the same as in **4**. The location of the hydroxy groups at C-1, C-3, C-4, a chlorine atom at C-2 and a 3-methyl-3-buten-1-ynyl side chain at C-6 in **5** were determined by <sup>1</sup>H–1H COSY and HMBC correlations (Figures S46 and S48), as for acrucipentyn C (**3**).

The relative configuration of **5** was assigned based on ROESY correlations (Figure S49) H-2 (δ<sup>H</sup> 3.69)/H-4 (δ<sup>H</sup> 3.97), OH-1 (δ<sup>H</sup> 5.73), OH-3 (δ<sup>H</sup> 5.48); H-3 (δ<sup>H</sup> 3.31)/H-1 (δ<sup>H</sup> 4.05), OH-4 (δ<sup>H</sup> 5.31) and <sup>1</sup>H-1H coupling constants (Table 1). The attempts to obtain an acetonide of compound **5** were unsuccessful. Etherification of compound **5** with (*S*)- and (*R*)-MTPA-Cl resulted in the formation of esters at three hydroxyl groups, which made it impossible to establish the absolute configuration by the modified Mosher's method. Compound **5** was named acrucipentyn E.

The molecular formula of compound **6** was determined as C11H12O3, based on the analysis of HRESIMS (*m/z* 191.0705 [M – H]−, calcd for C11H11O<sup>3</sup> 191.0714) and NMR data (Tables 1 and 2; Figures S50–S56). The <sup>1</sup>H and <sup>13</sup>C NMR data for this compound were similar to those obtained for (+)-asperpentyn [24,25], with the exception of the CH-2 and CH-3 proton and carbon signals. These data, as well as the biogenetic relationship of compound **6** with acrucipentyns D (**4**) and E (**5**), led us to suggest a configuration of asymmetric centers for it, different from the known asperpentyns.

The ROESY spectrum data and <sup>1</sup>H-1H coupling constants were useless to establish the relative stereochemistry of **6** unambiguously. Therefore, an epoxy ring-opening reaction was carried out in **6**. The reaction of compound **6** with 2,2-dimethylpropanoyl chloride in an aqueous medium (Figure 5) yielded two products, the spectra of which (1H, <sup>13</sup>C NMR, HRESIMS and CD) were identical to acrucipentyns D (**4**) and E (**5**). The presence of only two reaction products confirmed the SN2 mechanism of the epoxy ring opening, which corresponded to the literature data [26]. The orientation of the hydroxyl groups in the reaction products corresponds with the configuration of the epoxy ring in the initial compound. These data made it possible to establish the relative configuration of **6**. Compound **6** was named acrucipentyn F.

**Figure 5.** Scheme of an epoxy ring reaction in compound **6**.

It should be noted that acrucipentyn F is a stereoisomer of well-known fungal metabolites (−)-asperpentyn [27,28] and (+)-asperpentyn [24]. To the best of our knowledge, acrucipentyns are the first chlorine-containing asperpentyn-like compounds. However, it should be noted that several other related groups of 3-methylbutenynyl cyclohexanols, e.g., truncateols from the marine-derived fungi *Truncatella angustata* [29,30] and oxirapentyns from the marine-derived fungi *Beauveria felina* KMM 4639 [31], also have chloro-containing members.

#### *3.3. Biological Activity*

We evaluated the safety and toxicity of compounds **1**–**6** in various human cells. As such, we examined cytotoxicity in ten different human cell lines, including human prostate cells PNT2 and RWPE-1, human embryonic kidney cells HEK 293T, human fibroblast cells MRC-9 and human umbilical vascular endothelial cell line HUVEC, human keratinocytes HaCaT, as well as human prostate cancer cells PC-3, DU145, 22Rv1, VCaP and LNCaP using MTT assay. Indeed, none of the investigated compounds exhibited any significant cytotoxicity at concentrations up to 100 µM, following 72 h of treatment (IC<sup>50</sup> > 100 µM, Figure S65). Additionally, no morphological changes of the cells exposed to the isolated compounds (100 µM for 72 h) could be detected (Figure S66). Therefore, the isolated acrucipentyns A–F were assumed to be nontoxic to mammalian (human) cells.

The inhibitory effect of compounds **1**–**6** on sortase A enzyme from *Staphylococcus aureus* activity was investigated to detect their antibacterial potential (Figure 6).

**Figure 6.** The effect of compounds **1**–**6** on sortase A enzymatic activity. (**a**) The effect of compounds **1**–**6** on sortase A enzymatic activity measured after 10 min of incubation with the substrate. (**b**) The time-dependent effect of compound **3** (50 µM) on sortase A enzymatic activity. Data presented as relative fluorescent units (RFU). DMSO (0.8%) did not show any inhibition activity in comparison with sortase A assay buffer and was used as a control. The sortase inhibitor—4-(hydroxymercuri)benzoic acid (PCMB) in DMSO 0.8% was used as a positive control. All experiments were performed in three independent replicates and the data presented as a mean ± standard error mean (SEM). \* indicates the significant differences with *p* ≤ 0.05.

Compounds **1**, **3**, and **5,** at a concentration of 50 µM, significantly decreased sortase A activity by 18%, 30%, and 21%, respectively (Figure 6a). Compounds **4** and **6** showed less significant effects on sortase A enzymatic activity and compound **2** was inactive in this test. It was observed that an increase in concentrations of **1**–**6** up to 80 µM resulted in some decrease in their sortase A inhibitory activity, which was sometimes detected [32]. The inhibitory effect of compound **3** on sortase A activity was detected throughout the entire period of data acquisition (Figure 6b) and the effects of other studied compounds were similar.

To detect the antibacterial activity of isolated acrucipentyns A–E (**1**–**6**), their inhibitory effects on bacterial growth and biofilm formation of *Staphylococcus aureus* were investigated (Figure 7).

**Figure 7.** Effect of acrucipentyns A–F (**1**–**6**) on growth and biofilm formation of *Staphylococcus aureus*. All experiments were performed in three independent replicates and data were presented as a mean ± SEM.

Compounds **1** and **6** have shown the most pronounced antimicrobial effects against Gram-positive bacterium *S. aureus*. Compound **6** almost completely inhibited the growth of *S. aureus* at a concentration of 100 µM; a two-fold decrease in the concentration of the substance also halved the antimicrobial activity. Compound **1** at a concentration of 100 µM reduced the bacterial growth by 60%. A decrease in concentration to 12.5 µM reduced antimicrobial activity up to 50%. Compound **3** at 100 µM inhibited *S. aureus* growth by 50%. A decrease in the concentration of compound **3** led to a two-fold decrease in activity. The antimicrobial effects of **2**, **4,** and **5**, even at the highest used concentration of 100 µM, do not exceed 50% inhibition of bacterial growth.

When studying the effect of compounds **1**–**6** on the ability to inhibit the biofilm formation by Gram-positive bacteria *S. aureus*, it was noted that compounds **3**, **4**, and **6** have the most pronounced inhibitory activity at a concentration of 100 µM, in the case of which the formation of biofilms is almost absent. A high level of inhibition of biofilm formation in the case of substances **3** and **6** is kept up to concentrations of 12.5 and 25 µM, respectively. When substance **4** is diluted twice, its effect on biofilm formation is halved. Compound **1** inhibits the biofilm formation at concentrations of 12.5–100 µM by 50–70%, respectively. Compounds **2** and **5** at a concentration of 100 µM inhibited biofilm formation by 30%.

Sortase A is an essential component of *S. aureus* virulence because it is responsible for the covalent anchoring of many virulent factors of Gram-positive bacteria onto the cell wall (Appendix A) and, as a result, sortase A plays a key role in the pathogenic processes of *S. aureus* infection [33]. The decrease in sortase activity leads to the abolition of bacterial adhesion to mammalian cells and, thus, is one of the mechanisms preventing the formation of biofilms, which are the predominant form of bacterial existence [34].

The inhibitory effect of investigated compounds **1**, **3**, **4**, and **6** on the biofilm formation correlated with an ability to affect the activity of sortase A. Compound **2**, which did not show a significant effect on biofilm formation, also did not have any effect on the sortase A activity. Opposite to this, compound **5**, in the same experiments, had a significant effect on the activity of sortase A, but had a weaker inhibitory effect on the biofilm formation, in comparison with compounds **1**, **3**, **4**, and **6**. Thus, substances **1**–**6** can be assumed as anti-Staphylococcal agents.

To further confirm their antibacterial properties in a model of infectious damage to human cells, we investigated their effects on lactate dehydrogenase (LDH) release from human keratinocytes HaCaT, co-cultivated with *S. aureus* (Figure 8).

**Figure 8.** Effect of acrucipentyns A–F (**1**–**6**) on LDH release from human keratinocytes HaCaT cocultivated with *Staphylococcus aureus* (Sa) for 48 h. All compounds were tested at a concentration of 10 µM. All experiments were performed in triplicates and data are presented as a mean ± SEM. The difference between control (without Sa) and HaCaT/Sa co-cultivation was statistically significant with *p* < 0.05 (one-way ANOVA test). Asterisk (\*) indicates significant differences (*p* < 0.05) between HaCaT/Sa without compounds and HaCaT/Sa with compounds variants.

In normal conditions, LDH weakly releases from cells to culture media. *S. aureus* caused a significant increase in the LDH release from keratinocytes during co-cultivation. The addition of compounds **1**–**6** at a concentration of 10 µM reduced the LDH release by 30–50%. The greatest effect was registered for compounds **1**, **2**, **4,** and **5**.

Interestingly, compounds **2** and **4**, which showed significant activity in co-cultivation HaCaT cells with *S. aureus*, did not show high antibacterial and anti-biofilm-forming activity, in contrast to substances **1** and **5**. We assume that the cytoprotective effect of substances **1**–**6** in the in vitro infectious skin lesions could be due not only to their anti-*S. aureus* effects, but also to their anti-inflammatory and other cytoprotective effects. Recently, we observed similar dual effects during an investigation on flavuside B, an inhibitor of sortase A enzymatic activity derived from fungi. Flavuside B was able to inhibit *S. aureus* growth and biofilm formation, as well as protect HaCaT keratinocytes against *S. aureus* infection in a co-culture model via an anti-inflammatory pathway [14].

Our work is the very first detailed investigation of anti-Staphylococcal activity of isoprenylated cyclohexanols. To the best of our knowledge, earlier, only oxirapentyns A and D were found as antimicrobial agents among related compounds [35]. Thus, this group of secondary metabolites of marine fungi is interesting for future study, including their structure–activity relationships.

#### **4. Conclusions**

Six new isoprenylated cyclohexanols acrucipentyns A–F (**1**–**6**) were isolated from the alga-derived fungus *Asteromyces cruciatus* KMM 4696. The absolute configuration of acrucipentyn A was assigned by the modified Mosher's method and ROESY data. Acrucipentyns A–E are the very first members of chlorine-contained monocyclic cyclohexanols containing a 3-methylbutenynyl unit. The compounds have shown inhibitory activity against sortase A from *Staphylococcus aureus,* as well as inhibition of *S. aureus* growth and biofilm formation, while no cytotoxicty to mammalian cells was observed. Moreover, acrucipentyns A–F (**1**–**6**) protected human keratinocytes HaCaT from *S. aureus* toxicity in skin infection in an in vitro model. Thus, the isolated compounds hold a good potential as antimicrobal agents and should be further investigated.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jof8050454/s1, Figures S1–S12: CD and UV spectra of compound **1**–**6**, Figures S13–S22, S23–S28, S29–S35, S36–S43, S44–S49 and S50–S56: NMR spectra of compounds **1**–**6**, Figures S57–S58, S59–S60, S61–S62 and S63–S64: NMR spectra of compounds **1a**, **1a-1**, **1a-2**, **4a**, Figure S65: Viability of the various cells exposed to the tested compounds, Figure S66: Microphotographs of human cells exposed to the tested compounds.

**Author Contributions:** Conceptualization, O.I.Z.; Investigation, G.K.O., A.S.A., N.N.K., D.N.P., A.B.R., A.S.M., R.S.P., N.Y.K., A.R.C., E.A.C., O.O.V. and S.A.D.; Methodology, E.A.Y., D.N.P. and A.N.Y.; Project administration, O.I.Z.; Resources, O.I.Z., I.V.G., G.v.A. and A.N.Y.; Supervision, G.v.A. and A.N.Y.; Validation, G.v.A. and A.N.Y.; Visualization, E.A.C.; Writing—Original draft, O.I.Z.; Writing—Review and editing, G.v.A., S.A.D. and A.N.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation, grant number 19-74-10014.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** The study was carried out on equipment at the Collective Facilities Center "The Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS". The study was carried out using the Collective Facilities Center "Collection of Marine Microorganisms PIBOC FEB RAS".

**Conflicts of Interest:** The authors declare no conflict of interest.
