**2. Results and Discussion**

Compound **1** was an orange amorphous powder which was analyzed to have the molecular formula of C32H26O13, with 20 unsaturation degrees, by HRFABMS analysis ([M + H]<sup>+</sup> *m*/*z* 619.1449, calcd C32H27O13, 619.1446). The 13C NMR data of this compound showed signals of four carbonyl (δc 190.5, 188.7, 185.7, 183.5) and twenty deshielded methine and quaternary carbons (δc 166.9–104.6). Aided by the HSQC data, the chemical shifts of corresponding methine protons at δ<sup>H</sup> 7.65, 7.51, 6.81, and 6.78 in the 1H NMR data revealed the presence of aromatic moieties (Table 1). Since these NMR features were accounted for 14 unsaturation degrees, **1** must be a hexacyclic compound.


**Table 1.** 13C and 1H NMR data of compounds **1**–**3** in CD3OD.

*<sup>a</sup>*–*<sup>c</sup>* Measured at 150/600, 100/400, and 200/800 MHz for 13C/ 1H NMR, respectively. *<sup>d</sup>* Assigned by HMBC data. *<sup>e</sup>* Not detected.

A combination of 13C and 1H NMR and HSQC data diagnosed the remaining NMR signals as an oxy-quaternary carbon (δc 74.6) and three oxymethine (δC/δ<sup>H</sup> 75.2/3.79, 70.6/4.73, and 70.1/4.26), two oxymethyl (δC/δ<sup>H</sup> 57.0/3.70 and 56.9/3.69), and two methyl (δC/δ<sup>H</sup> 22.3/1.33 and 16.6/2.23) groups. Aided by the literature study, the overall spectroscopic features suggested **1** to be a bianthraquinone consisting of each one unit of anthraquinone and tetrahydroanthraquinone.

Having the information above, the structure determination of **1** was mostly accomplished by extensive H–C long-range analyses for this proton-deficient compound (Figure 2). Firstly, long-range correlations of two aromatic protons at δ<sup>H</sup> 7.51 (H-1) and 7.65 (H-4) and a benzylic methyl proton at δ<sup>H</sup> 2.23 (H3-11) with neighboring carbons not only constructed a 2-hydroxy-3-methylbenzene moiety (C-1-C-4, C-4a, and C-9a) but also placed two carbonyls at δ<sup>C</sup> 188.7 (C-9) and 183.5 (C-10) *ortho*-substituted to the benzene. Similarly, combined HMBC and LR-HSQMBC correlations of the protons at δ<sup>H</sup> 6.78 (H-7) and 3.69 (H3-12) with neighboring carbons revealed the presence of an 8-hydroxy-5-methoxybenzene moiety (C-5-C-8, C-8a, and C-10a). The linkage between C-8a and C-9 was also secured by crucial H-7/C-9 correlation. Although it was not evidenced by HMBC data, the carbon chemical shifts of C-10 (δ<sup>C</sup> 183.5) and C-10a (δ<sup>C</sup> 133.4), in conjunction with the MS-derived unsaturation degree, directly linked these carbons, thus, constructing an anthraquinone moiety for **1**.

**Figure 2.** Key correlations of COSY (bold), HMBC (arrow), and LR-HSQMBC (*J*CH = 2 Hz, dashed arrow) experiments for compounds **1**, **3**, and **4**.

Meanwhile, 1H–1H COSY data showed direct spin coupling (*J* = 7.5 Hz) between oxymethine protons at δ<sup>H</sup> 4.73 (H-1- ) and 3.79 (H-2- ) (Figure 2). Subsequently, HMBC correlations of these protons and an isolated oxymethine and a methyl proton at δ<sup>H</sup> 4.26 (H-4- ) and 1.33 (H-11- ), respectively, with the olefinic and hydroxyl-bearing carbons, defined a 3-methyl-2,3,4,5-tetrahydroxy cyclohexene-type moiety (C-1- –C-4- , C-4a', and C-9a'). The attachment of two carbonyl carbons at δ<sup>C</sup> 190.5 (C-9- ) and 185.7 (C-10- ) at the cyclohexene unit was also accomplished by their HMBC correlations with H-4 and H-1- , respectively.

The HMBC correlations of an aromatic and a methoxy proton at δ<sup>H</sup> 6.81 (H-7- ) and 3.70 (H3-12- ), respectively, with aromatic carbons, revealed an 8- -hydroxy-5- -methoxybenzene (C-5- –C-8- , C-8a', and C-10a'), analogous to the C-5–C-10a unit. Subsequently, further connection to the diketo-bearing cyclohexene unit constructing a tetrahydroanthraquinone moiety was also accomplished by the HMBC

and LR-HSQMBC correlations between these. Since the anthraquinone and tetrahydroanthraquinone moieties had open ends at C-6 and C-6- , respectively, C–C linkage between these carbons was anticipated. The crucial evidence was provided by the HMBC data, in which long-range correlations were found at H-7/C-6 and H-7- /C-6. Thus, the planar structure of **1** was determined as a bianthraquinone analogous to alterporriols [3]. Although significant numbers of bianthraquinones were reported from diverse fungi, those having a 6-6- C–C linkage are rather rare. To the best of our knowledge, alterporriol K–M are the only previous examples having the same type of C–C linkage [21].

The structure of **1**, designated as alterporriol Z1, possessed four stereogenic centers at C-1- –C-4 at the aliphatic ring and a chiral axis at C-6/C-6- , requiring configurational determination. Firstly, relative configurations at the aliphatic ring were assigned by proton–proton coupling constants and NOESY analyses (Figure 3). The large coupling (*J* = 7.5 Hz) between H-1 and H-2 oriented both protons axial to the cyclohexene ring that was supported by the NOESY cross peak at H-2- /1- -OH. Then, the neighboring 11- -CH3 group was equatorially oriented by the cross peak H-2- /H3-11- . Although both H-4 and 4- -OH protons showed spatial proximity with H3-11- , the NOESY cross peak at H-2- /H-4 was crucial to the axial orientation of H-4- (Supplementary Materials, Figure S9 and Table S2). Thus, the overall relative configurations were assigned as 1- *S*\*, 2- *R*\*, 3- *S*\*, and 4- *S*\*. The absolute configurations at these centers were approached by computational methods and are described later.

**Figure 3.** Key correlations of NOESY (arrow) experiments for compounds **1**, **3**, and **7**.

The configuration at the C-6/C-6 chiral axis was assigned by ECD measurements. As shown in Figure 4, the measured ECD profile of **1** showed significant Cotton effects at 269 (Δε 35.79) and 285 (Δε −36.06) nm, assigning an a*R* configuration at the C-6/C-6 axis. Although this interpretation was supported by the same configuration at structurally related alterporriols A and L [21,22], a question would arise from the possible contribution of ECD by the C-1- –C-4 stereogenic centers. That is, the absolute configurations at these centers would remarkably influence overall ECD profiles. Conversely, depending on the results, it would be also possible to deduce the absolute configurations of stereogenic centers at the cyclohexene moiety. To clarify this, ECD data were calculated for the a*R* atropisomeric contribution with two possible absolute configurations at the stereogenic centers. The results were that these profiles were very similar to each other at both wavelengths and intensity of Cotton effects, regardless of the configurations at the cyclohexene moiety (Figure 5). Thus, the a*R* configuration was unambiguously assigned for the chiral axis.

**Figure 4.** Experimental ECD spectra of compounds **1**–**4**.

**Figure 5.** Calculated ECD spectra of compounds **1** and **2**.

Having fixed axial chirality, the absolute configurations at C-1- –C-4 were subsequently approached by DP4 calculations. However, two models derived from the opposite configurations expected very similar 13C and 1H NMR data from each other (Supplementary Materials, Figure S52 and Table S1), which was consistent with the results of ECD analysis. Thus, the absolute configurations of the cyclohexene moiety remained unassigned [23]. Overall, the structure of alterporriol Z1 (**1**) was determined to be a bianthraquinone of the alterporriol class.

The molecular formula of alterporriol Z2 (**2**) was established to be C32H26O13, identical to **1**, by HRFABMS analysis ([M + H]<sup>+</sup> *m*/*z* 619.1454, calcd for C32H27O13, 619.1446). A comparison of the 13C and 1H NMR data of this compound with those of **1** revealed a very close structural similarity between them (Table 1). Subsequently, the extensive 1D and 2D NMR analyses of **2** deduced the same planar structure and relative configurations of the aliphatic ring as those of 1. Accordingly, the structural difference was traced to the chiral axis at C-6–C-6- , in which the measured ECD spectrum of **2** showed a quasi-mirror profile from **1** (Figure 4). The noticeably reduced intensities of Cotton effects would be attributed from the stereogenic center-bearing cyclohexene moiety. However, the calculated ECD data of **2** were virtually identical to each other, regardless of absolute configurations at the cyclohexene stereogenic centers, eradicating the possibility of the reversal of measured ECD by these factors (Figure 5). Thus, the structure of alterporriol Z2 (**2**) was defined as an atropisomer of alterporriol Z1 (1).

A minor constituent of alterporriol Z3 (**3**) was isolated as a dark red amorphous solid that was analyzed for the molecular formula of C33H28O13 by HRFABMS analysis ([M + Na]<sup>+</sup> *m*/*z* 655.1430, calcd for C33H28O13Na, 655.1422). The 13C and 1H NMR data of this compound were similar to those of **1** and **2**, indicating the same bianthraquinone nature (Table 1). An extensive examination of these data, in conjunction with a literature and database search, revealed that **3** had close structural similarity with a congener alterporriol N (**6**) [23]. The most conspicuous difference in NMR data was the presence of an additional methoxy group at δC/δ<sup>H</sup> 62.8/3.77 in **3**.

Given this information, the planar structure of **3** was determined by HMBC analyses. Despite the limited amount of obtained materials, almost all carbons and protons except for unprotonated C-10a' were adequately assigned by HSQC and HMBC correlations (Figure 2). In this way, **3** was defined as consisting of an anthraquinone and a tetrahydroanthraquinone moiety, as with other compounds. The newly appeared methoxy group (C-13- ) was also attached at C-1 by COSY correlation at H-1- /H-2- and HMBC correlation at H3-13- /C-1- . Due to the lack of neighboring protons, the C–C linkage was not directly evidenced by HMBC data. However, the diagnostic chemical shifts of the unprotonated C-1 and C-5 carbons at δ<sup>C</sup> 129.0 and 130.7, respectively, were indicative of the linkage between the anthraquinone and tetrahydroanthraquinone moiety at these carbons.

The configurations at cyclohexene and C-1/C-6 chiral axis of **3** were assigned using the same methods as in **1** and **2**. Firstly, the small coupling constants (*J* = 4.7 Hz) between the vicinal H-1- and H-2 indicated the orientations of these to be either axial–equatorial or equatorial–equatorial to the cyclohexene ring. Then, the NOESY data showed mutual cross peaks among H-2- , H-4- , and H3-11- , placing these at the same phase of the cyclohexene ring (Figure 3). Therefore, the former two oxymethine protons were axially oriented, while the C-11 methyl group was equatorially oriented to the ring system. Aided with the additional cross peak with H-2- , the H-1 oxymethine proton was also equatorially oriented. Thus, the overall relative configurations were assigned as 1- *R*\*, 2- *R*\*, 3- *S*\*, and 4- *S*\*. The absolute configuration of the C-1/C-6 chiral axis of **3** was also assigned by ECD measurement. As shown in Figure 4, the ECD data of this compound were similar to 1, assigning the same a*R* configuration. Thus, the structure of alteroporriol Z3 (**3**) was defined as a new bianthraquinone of the alterporriol class.

In addition to **1**–**3**, the crude extract of *Stemphylium* sp. contained three known bianthraquinones (**4**–**6**). Based upon the results of combined spectroscopic analyses, these were identified as alterporriols F (**4**) [24], G (**5**) [25], and N (**6**) [23], respectively. The spectroscopic data of these compounds were in good accordance with those in the literature. Among these, the configuration of C-5/C-5 chiral axis of **4**, previously unassigned, was determined to be a*R* by the ECD measurement in this work (Figure 4).

In addition to bianthraquinones, the culture broth of *Stemphylium* sp. contained three new meroterpenoids. The molecular formula of compound **7**, a brown amorphous solid, was deduced to be C21H30O6 with seven unsaturation degrees, by HRFABMS analysis ([M + H]<sup>+</sup> *m*/*z* 379.2118, calcd for C21H31O6, 379.2115). The 13C NMR data of this compound showed signals of three significantly deshielded carbons (δ<sup>C</sup> 200.4, 180.9, and 170.4) and three olefinic carbons (δ<sup>C</sup> 139.7, 127.5, and 107.5). The odd numbers of the latter carbons were indicative of a highly differentiated double bond possibly consisting of a deshielded carbon (δ<sup>C</sup> 170.4) and a shielded carbon (δ<sup>C</sup> 107.5). Between two carbonyl carbons, the conspicuous one (δ<sup>C</sup> 200.4) was readily assigned as a ketone, while the other one (δ<sup>C</sup> 180.9) was thought to be either a carboxylic acid or an ester group by a strong absorption band at 1755 cm−<sup>1</sup> in the IR data. Aided by the 1H NMR and HSQC data, the remaining 15 carbons in the 13C NMR data were diagnosed to be one oxy-quaternary (δ<sup>C</sup> 85.8), three oxymethine (δ<sup>C</sup> 78.1, 74.5, and 66.8), one methine (δ<sup>C</sup> 40.8), seven methylene (δ<sup>C</sup> 46.8, 40.2, 34.5, 33.6, 30.5, 26.3, and 19.8), and three methyl (δ<sup>C</sup> 20.1, 17.7, and 16.4) carbons (Table 2). Overall, preliminary examination of the spectroscopic data suggested **7** to be a tricyclic compound possessing two carbonyl groups and two double bonds.


**Table 2.** 13C and 1H NMR Data of compounds **7**–**9** in CD3OD.

*<sup>a</sup>*, *<sup>b</sup>* Measured at 100/800 and 100/500 MHz for 13C/ 1H, respectively.

The planar structure of **7** was determined by combined 1H–1H COSY and HMBC analyses (Figure 2). That is, COSY data revealed a linear assembly of an oxymethine and two methylene groups (δC/δ<sup>H</sup> 33.6/2.62 and 2.32, 30.5/2.18 and 1.97, and 66.8/4.30, C-2–C-4). Then, the HMBC correlations of these protons with neighboring carbons not only placed a ketone (δ<sup>C</sup> 200.4, C-1) and a tetra-substituted double bond (δ<sup>C</sup> 170.4 and 107.5, C-6 and C-5) at the adjacent locations, but also constructed a 4-oxygenated cyclohexanone moiety (C-1–C-6, ring A): H-4/C-2, C-3, C-5, and C-6, H2-3/C-1, and H2-2/C-1 and C-6. The COSY data also found a direct linkage between methylene and oxymethine (δC/δ<sup>H</sup> 19.8/2.59 and 2.22 and 78.1/3.95, C-7 and C-8). Subsequently, their attachment at C-6 of ring A was secured by the HMBC of H2-7 with ring carbons: H2-7/C-1, C-5, and C-6. Similarly, a quaternary carbon (δ<sup>C</sup> 85.8, C-9) and a methyl group (δC/δ<sup>H</sup> 20.1/1.35, C-19) were linearly connected at C-8 by a number of key HMBC correlations: H-8/C-9 and H3-19/C-8 and C-9.

The COSY data revealed the presence of another linear spin system consisting of each one of methylene, oxymethine, and olefinic methine (δC/δ<sup>H</sup> 46.8/2.42 and 2.07, 74.5/4.80, and 127.5/5.17, C-10–C-12; Figure 2). The accomplishment of a full olefin (with δ<sup>C</sup> 139.7, C-13) as well as the attachment of a vinyl methyl group (δC/δ<sup>H</sup> 16.4/1.65, C-20) was made by HMBC correlations: H-11/C-13 and H3-20/C-12 and C-13. The linkage of this moiety at the C-9 quaternary carbon was also secured by HMBC data: H3-19/C-10 and H2-10/C-8 and C-9.

Based on the COSY data, the remaining proton signals were found to form a linear assembly of three methylenes, a methine, and a methyl group (δC/δ<sup>H</sup> 40.2/1.99 (2H); 26.3/1.42 (2H); 34.5/1.59 and 1.38, 40.8/2.37; and 17.7/1.11, C-14-C-17, and C-21). Subsequently, the attachments of this moiety at C-13 and a carbonyl group (δ<sup>C</sup> 180.9, C-18) were secured by a series of HMBC correlations: H3-20/C-14; H2-14/C-12; and C-13, H-17/C-18, and H3-21/C-18. Thus, the framework of **7** was constructed as a C21 meroterpenoid of the tricycloalterfurene class.

The 2D NMR-based structure elucidation of **7** assigned, in addition to the C-1 ketone, six oxygenated positions at C-4, C-5, C-8, C-9, C-11, and C-18, accounting for two rings inherent from the mass data. A literature study suggested cyclic ether linkages at C-5/C-9 and C-8/C-11 constructing a dihydropyran (ring B) and a tetrahydrofuran (ring C), respectively, while the remaining carbons were functionalized as the 4-hydroxy and 21-carboxylic acid group. The comparison of 13C and 1H NMR data of **7** were in good accordance with tricycloalterfurene A [15], supporting this interpretation. Crucial evidence was provided in the process for relative and absolute configurations and described later.

The configurations at the stereogenic centers of **7** were initially approached by NOESY data (Figure 3). The 13*E* configuration was assigned by cross peaks at H-11/H3-20 and H-12/H2-14 as well as the diagnostic carbon chemical shifts of C-20 (δ<sup>C</sup> 16.4) and C-14 (δ<sup>C</sup> 40.2). The NOESY data also placed H-7β (δ<sup>H</sup> 2.22), H-8, H3-19, H-10β (δ<sup>H</sup> 2.42), and H-11 at one phase of the B/C ring plane by a series of cross peaks: H-7β/H3-19, H-8/H3-19, H-8/H-11, H3-19/H-10β, and H-10β/H-11. To have these cross peaks, the B/C ring juncture must have a *cis* orientation. Overall, relative configurations at the stereogenic centers were assigned as 8*R*\*, 9*R*\*, and 11*R*\*.

The configurations at the remotely placed C-4 and C-17 stereogenic centers were independently assigned by modified Mosher's and phenylglycine methyl ester (PGME) methods, respectively. That is, the treatments of **7** with (*R*)- and (*S*)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride (MTPA-Cl) produced corresponding (*S*)- and (*R*)-MTPA esters, **7**-4*S* and **7**-4*R*, respectively. The resulting Δδ (δ*<sup>S</sup>* − δ*R*) values between these esters assigned the 4*R* absolute configuration (Figure 6). Similarly, **7** was also converted to (*S*)- and (*R*)-PGME amides, **7**-18*S* and **7**-18*R* by treatments with (*S*)- and (*R*)-PGME, respectively. Subsequently, the Δδ (δ*<sup>S</sup>* − δ*R*) values between the amides clearly assigned the 17*S* configuration (Figure 7). As described earlier, the productions of 4-esters and 17-amides by these reactions unambiguously confirmed the presence of 4-hydroxy and 18-carboxylic acid groups.

**Figure 6.** Δδ values [Δδ = δ*<sup>S</sup>* − δ*R*] obtained for (*S*)- and (*R*)-MTPA esters of **7** and **8**.

**Figure 7.** Δδ values [Δδ = δ*<sup>S</sup>* − δ*R*] obtained for (*S*)- and (*R*)-PGME amide derivatives of **7** and **8**.

Finally, given the absolute configurations at the remote stereogenic centers, those at rings B and C were determined by computational methods. As shown in Figure 7, the measured ECD profile of **7** matched well with the calculated one, with 8*R*, 9*R*, and 11*R* configurations in both wavelength and intensity of the absorption maximum at 258 nm. Overall, the absolute configurations of **7** were determined to be 4*R*, 8*R*, 9*R*, 11*R*, and 17*S* by combined NOESY, chemical derivatization, and computational methods. Thus, the structure of **7**, designated as tricycloalterfurene E, was determined to be a tricyclic meroterpenoid.

The molecular formula of compound **8** was deduced to be C21H30O6, identical to **7**, by HRFABMS analysis ([M + H]<sup>+</sup> *m*/*z* 379.2120, calcd for C21H31O6, 379.2115). The spectroscopic data of this compound were also very similar to those of **7**, suggesting the same tricyclic meroterpenoid nature. However, detailed examination of its 13C and 1H NMR and HSQC data revealed noticeable differences in the signals of carbons and protons at the hydroxyl-bearing cyclohexanone moiety (ring A) (Table 2). Although 1H-1H COSY data showed the presence of the same linear assembly of a hydroxyl-methine and two methylene protons as **7**, the HMBC data revealed grossly different proton−carbon correlations. That is, the C-1 ketone (δ<sup>C</sup> 199.9) was correlated with hydroxyl-methine (δ<sup>H</sup> 4.07) and methylene (δ<sup>H</sup> 2.24 and 1.83) protons. The latter methylene protons were additionally correlated to the C-6 (δ<sup>C</sup> 105.6) and C-5 (δ<sup>C</sup> 170.8) olefinic carbons. Therefore, **8** was structurally defined to be a regioisomer of **7** bearing a hydroxyl group at C-2.

The relative and absolute configurations of **8** were pursued stepwise as for **7**. First, the NOESY data of this compound showed identical proton–proton spatial proximities to **7**, revealing the same relative configurations at rings B and C. Then, the absolute configuration at C-2 was also assigned as *R* by modified Mosher's method (Figure 6). Strikingly, however, the 1H NMR spectra of PGME-amides of **8** from treatments with (*S*)- and (*R*)-PGME were virtually identical to each other and consisted of pairing proton signals. A detailed examination indeed revealed that **5** was a mixture of two 17-epimers with a ratio of 1:0.57. Based upon the proton intensity, the PGME analysis also concluded the 17*S* and 17*R* configuration for the major and minor constituents, respectively. The analytical and spectroscopic behaviors of 8 as not an epimeric mixture but a single compound would be attributed from the far remote location of C-17 from other stereogenic centers. Having this, the absolute configurations of **8** were also assigned by ECD measurements. As shown in Figure 8, the measured ECD profile of **8** was very similar to that of **7**, defining the same absolute configurations for the ring portion between these compounds. This interpretation was firmly supported by the calculated ECD data of **8** with fixed absolute configurations and epimeric ratio (1:0.57), which were almost identical to the measured one. Thus, the structure of **8**, designated as tricycloalterfurene F, was determined to be an epimeric mixture of tricyclic meroterpene carboxylic acids.

**Figure 8.** Experimental and calculated ECD spectra of compounds **7**–**9**.

The molecular formula of tricycloalterfurene G (**9**) was established as C22H32O6 by HRFABMS analysis ([M + Na]<sup>+</sup> *m*/*z* 415.2104, calcd for C22H32O6Na, 415.2091). The 13C and 1H NMR data of this compound were very similar to those of **7**, with appearance of a methoxy group (δC/δ<sup>H</sup> 52.1/3.65) as the most noticeable difference (Table 2). This methyl group was placed at the C-18 carboxylic group by combined 2D NMR data, including crucial HMBC correlation between the methoxy proton and carboxylic carbon (δ<sup>C</sup> 178.9). After deducing the same NOESY-based relative configurations as **7**, the experimental ECD profiles of **9** were also very similar to those of **7,** assigning the same absolute

configurations between these (Figure 8). Thus, the structure of tricycloalterfurene G (**9**) was defined to be an ester derivative of tricycloalterfurene E (**7**).

The obtained bianthraquinones (**1**–**6**) and meroterpenoids (**7**–**9**) were assayed for their cytotoxicity. However, all of these were inactive (IC50 » 20 μM) against a number of human cancer cell-lines: A549 (lung cancer), HCT116 (colon cancer), MDA-MB-231 (breast cancer), PC3 (prostate cancer), SK-Hep1 (liver cancer), and SNU638 (stomach cancer). In addition, these compounds were inactive (MIC > 128 μg/mL) against diverse human pathogenic bacterial strains *Enterococcus faecalis*(ATCC19433), *Enterococcus faecium* (ATCC19434), *Klebsiella pneumoniae* (ATCC10031), *Proteus hauseri* (NBRC3851), *Salmonella enterica* (ATCC14028), and *Staphylococcus arueus* (ATCC6538p). These compounds were also inactive (IC50 > 145 μM) against microbial key enzymes sortase A (SrtA) and isocitrate lyase (ICL).

In our further assay using bianthraquinones, the anti-inflammatory activities were indirectly evaluated by ability to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 mouse macrophages. As shown in Figure 9, compounds **1**, **2**, and **4**–**6** showed moderate anti-inflammatory activity with IC50 values of 11.6 ± 0.7, 16.1 ± 1.1, 9.6 ± 1.6, 8.4 ± 0.4, and 10.7 ± 0.6 μM, respectively, while 3 was inactive. No measurable cytotoxicity was observed against mouse macrophages at these concentrations (Supplementary Materials, Figure S55). To further elucidate the inhibitory mechanism on NO production, the effect of compounds on the protein expression levels of iNOS and COX-2, the key inflammatory mediators, was evaluated. As shown in Figure 10, protein levels of iNOS and COX-2 in RAW 264.7 cells incubated with LPS for 18 h increased in comparison to quiescent cells, but this was significantly down-regulated by treatment of the compounds (20 μM) 30 min prior to LPS exposure. These findings showed that the bianthraquinones would be potential candidates for the anti-inflammatory related study.

**Figure 9.** Concentration-dependent effect of **1**, **2**, and **4**–**6** on lipopolysaccharide (LPS)-stimulated NO production in RAW 264.7 cells. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 indicate significant differences relative to the vehicle-treated control group.

**Figure 10.** Effect of compounds **1**, **2**, and **4**–**6** on LPS-induced protein expressions of iNOS and COX-2 in RAW 264.7 cells. The cells were treated with 20 μM of compounds for 30 min prior to LPS (1 μg/mL) treatment and incubated for 18 h. Protein expression levels were determined by Western blotting analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

Optical rotations were measured on a JASCO P1020 polarimeter (Jasco, Tokyo, Japan) using a 1 cm cell. UV spectra were acquired with a Hitachi U-3010 spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). ECD spectra were recorded on an Applied Photophysics Chirascan plus CD spectrometer. IR spectra were recorded on a JASCO 4200 FT-IR spectrometer (Jasco, Tokyo, Japan) using a ZnSe cell. 1H and 13C NMR spectra were measured in CD3OD and THF-*d8* solutions on Bruker Avance -500, -800 instruments (Billerica, MA, USA) and JEOL -400, -600 instruments (Peabody, MA, USA). High resolution FAB mass spectrometric data were obtained at the Korea Basic Science Institute (Daegu, Korea), and were acquired using a JEOL JMS 700 mass spectrometer (Jeol, Tokyo, Japan) with *meta*-nitrobenzyl alcohol (NBA) as the matrix. Semi-preparative HPLC separations were performed on a Spectrasystem p2000 equipped with a Spectrasystem RI-150 refractive index detector. All solvents used were spectroscopic grade or distilled from glass prior to use.
