3.1. Isolation and Structural Characterization of Compounds
The eight meroterpenoids (1–8) isolated may be sub-grouped into three sub-chemical classes, namely, the memnobotrin-like pentacyclic lactams (1–3), the prenylated isobenzofuranones (4, 5), and the cyclic isobenzofuranone analogues (6, 7, 8). The structural elucidation of each compound is described below, starting with the memnobotrin-like pentacyclic lactams.
Memnobotrin C (
1) was isolated as a yellowish amorphous solid. Positive mode electrospray ionization time-of-flight mass spectrometric analysis ((+)-ESI-TOF MS) of the compound identified a protonated adduct at
m/z 428.2440, which corresponded to the molecular formula C
25H
33NO
5 (calcd. for C
25H
34NO
5, 428.2431). The carbon-13 nuclear magnetic resonance (
13C NMR) spectrum of the compound (
Table 1) showed twenty-five carbon signals in total, including two carbonyl carbons at δ
C 219.7(C3), 171.7 (C7′) and six
sp2 aromatic carbons at δ
C 115.4, 157.9, 100.8, 122.2, 132.6, and 150.0 (C1′–C6′ with oxygenation at C2′ and C6′), which accounted for five degrees of unsaturation in the molecule. These data, in combination with the molecular formula of C
25H
33NO
5, which indicates a total of ten degrees of unsaturation, showed compound
1 to be pentacyclic. Additionally, the NMR data of
1 showed eight
sp3 methylene groups, one of them oxygenated (C10′ δ
C 61.4, δ
H 3.78) and another attached to a nitrogen atom (C9′ δ
C 46.4, δ
H 3.68). There were also two
sp3 methine groups, three
sp3 quaternary carbons with one of them oxygenated (C8 δ
C 78.2), and four singlet methyl groups identified. The following intense heteronuclear multiple bond correlations (HMBC) of the four methyl singlets, i.e., δ
H 1.27 (H12) with δ
C 41.5 (C7), 78.2 (C8), and 52.3 (C9); δ
H 1.09 (H13) with δ
C 219.7 (C3), 48.6 (C4), 56.1 (C5), and 27.1 (C14); δ
H 1.14 (H14) with δ
C 219.7 (C3), 48.6 (C4), 56.1 (C5), and 21.9 (C13); and finally δ
H 1.09 (H15) with δ
C 39.0 (C1), 52.3 (C9), and 37.8 (C10), were consistent with the presence of the drimane-like substructure X in compound
1 (
Figure 2). The low-field chemical shifts of the two hydrogens at δ
H 2.66, 2.48 placed this
sp3 methylene at C2, contiguous to the ketone at δ
C 219.7 (C3). The correlation spectroscopy (COSY) between the hydrogen pairs H1/H2 and H6/H7, coupled with HMBC correlations of H2 (δ
H 2.66) with C1 and C3, corroborated the drimane-like substructure X of compound
1 (
Figure 2). Further, the two downfield hydrogens of the nitrogen-attached methylene at δ
H 3.68 (H9′) gave HMBC cross-peaks with the carbons at δ
C 171.7 (C7′), 50.2 (C8′), and 61.4 (C10′). The two oxygenated methylene hydrogens at δ
H 3.78 (H10′) only gave HMBC cross-peaks with carbon C9′ (δ
C 46.4). These correlations, together with those observed from the singlet aromatic hydrogen at δ
H 6.73 (H3′) to carbons δ
C 115.4 (C1′), 157.9 (C2′), 122.2 (C4′), and 171.7 (C7′), and also the ones from the two methylene hydrogens at δ
H 4.36 (H8′) to the carbons at δ
C 122.2 (C4′), 132.6 (C5′), 150.0 (C6′) and 171.7 (C7′), led to the identification of the phthalimidine-like substructure Y in compound
1 (
Figure 2). The HMBC cross-peaks from the two low-field
sp3 methylene hydrogens at δ
H 2.79 and 2.47 (H11) to the aromatic carbons C1′, C2′, and C6′, to the methylene carbon C9 and also to the oxygenated carbon C8 established the pyran ring that connects the phthalimidine-like substructure Y to the drimane-like substructure X and completes the planar structure of compound
1. This planar structure of compound
1 was confirmed to correspond to a keto-derivative of memnobotrin B previously isolated from
Memnoniella echinata [
17], in which the acetate group attached to C3 in the latter is replaced by a ketone in
1. Although the overlapping proton (
1H) NMR signals for the axial hydrogens H5, H7ax and H9 complicated the determination of the relative configuration in
1, it was assigned using the key NOESY correlations H5/H13 and H9/H11eq, which placed the axial hydrogens H5 and H9, together with the methyl group C13 on the same face of the molecule (
Figure 2). On the other hand, H6ax/H15 and H11ax/H15 NOESY correlations oriented the axial methyl C15 on the opposite face of the molecule (
Figure 2). Intense NOESY correlations were observed from H12 to H7eq and H11ax, thus establishing a β orientation for methyl C12 and confirming an
R configuration at C8, opposite to what was previously reported for memnobotrin B [
17]. Given the similarity in the NMR data of compound
1 and memnobotrin B and the fact that both compounds have been isolated from the same fungal genus,
Memnoniella, suggesting analogous biosynthetic routes, the absolute configuration of compound
1 is predicted to be the same as previously determined by X-ray crystallography for memnobotrin A (and by extension, B) [
17], excluding the configuration at C8, as already explained above.
(+)-ESI-TOF MS analysis was used to assign a molecular formula of C
25H
35NO
5 to memnobotrin D (
2) based on the presence of a protonated adduct at
m/z 430.2595 (calcd. for C
25H
36NO
5, 430.2588). The NMR data of compounds
2 and
1 (
Table 1) were very similar, with the main difference being the replacement of the carbonyl ketone at δ
C 219.7 in compound
1 with a hydroxylated methine group in compound
2 (δ
H 3.21
, δ
C 79.5), giving rise to the two additional hydrogens in the molecular formula of compound
2 (i.e., reduction of the C3 ketone in compound
1 to a hydroxy methine in compound
2). The placement of this new hydroxylated
sp3 methine signal at C3 was confirmed by the HMBC cross-peaks of the hydrogen at δ
H 3.21 (H3) with carbons C4 (δ
C 40.0), C13 (δ
C 28.8) and C14 (δ
C 16.3). Further, HMBC cross-peaks were observed from hydrogens H13 and H14 to the oxygenated carbon at C3 (δ
C 79.5), thus arriving at the planar structure of compound
2 (
Figure 2). Since very similar NOESY correlations were observed in both compounds
1 and
2 (
Figure 2), the relative and absolute configurations of both compounds are proposed to be identical. It is worth mentioning that in compound
2, the dispersion of the signals for hydrogens H5, H7ax and H9 allowed the confirmation of their axial orientation via the key NOESY correlations from H5 to H7ax and H9. An axial orientation was also established for the new hydrogen H3 based on the existence of a large axial/axial coupling constant (11.2 Hz) to H2ax and key NOESY correlations between H3 and H2eq, H1ax, H5 and H13 (
Figure 2).
Memnobotrin E (
3) was easily identified due to its striking similarity to the known memnobotrin B [
17]. The molecular formula of this compound, C
27H
37NO
6, determined by (+)-ESI-TOF mass spectrometry, was reported for a total of 14 other compounds (including memnobotrin B) in the Dictionary of Natural Products [
30]. The proton, carbon-13 and two-dimensional (
1H,
13C and 2D) NMR of compound
3 (
Table 2) confirmed its close structural similarity to memnobotrin B except for one difference. As described above for compounds
1 and
2 (memnobotrins C and D), intense NOESY cross-peaks were also observed between the methyl hydrogens H12 and the methylene hydrogen H7eq and also between the methylene hydrogen H11ax and methyl hydrogens H12 and H15, establishing a β orientation of methyl C12 and therefore an
R configuration at C8 for compound
3. Memnobotrin E (
3) was therefore confirmed to be 8-
epi-memnobotrin B.
With respect to the two prenylated isobenzofuranones isolated (
4,
5), compound
4 appeared as a yellowish amorphous solid with a molecular formula of C
29H
42O
10 established on the basis of its (+)-ESI-TOF MS analysis, which showed the presence of an ammonium adduct at m/z 568.3131 (calcd. for C
29H
46NO
10, 568.3116). The NMR data (
Table 3) of this compound showed a total of twenty-nine carbons, including one carbonyl carbon at δ
C 174.4 (C3), six
sp2 aromatic carbons at δ
C 103.9 (C3a), 165.0 (C4 oxygenated), 116.9 (C5), 156.6 (C6 oxygenated), 101.6 (C7), and 148.3 (C7a), and four olefinic carbons at δ
C 123.5 (C2′), 135.9 (C3′), 125.5 (C6′), and 136.1 (C7′). These carbons account for six degrees of unsaturation which, when compared to the molecular formula of C
29H
42O
10, indicates that the compound has three ring systems, one of them being the aromatic ring. A pyran-sugar ring moiety (the second ring) was also readily identified in the molecule by the chemical shifts of the five oxygenated
sp3 methine groups (C1′′–C5′′) and an
sp3 oxymethylene group (C6′′) in the region of 3.18–4.68 ppm, which were, respectively, placed using the multiplicity of the protons in combination with the COSY correlations observed between the following pairs of hydrogens: H1′′/H2′′, H2′′/H3′′, H3′′/H4′′, H4′′/H5′′and H5′′/H6′′. Considering the coupling constants measured for most of the hydrogens in the sugar moiety and the key NOESY correlations from H1′′ to H2′′, H3′′and H5′′, together with the absence of a NOESY correlation between H4′′ and any of the aforementioned hydrogens, the pyran-sugar moiety in compound
4 was identified as β-mannopyranoside. Due to the scarcity of sample, the absolute configuration of the sugar was tentatively proposed as D. An intense HMBC cross-peak between the hydrogen at δ
H 4.68 (H1′′) and the carbon at δ
C 79.1 (C11′) connected the anomeric carbon (C1′′) of the β-D-mannopyranosyl moiety to the C11′ prenyl part of the molecule via an oxygen bridge. The remaining part of the prenyl chain was established using the intense HMBC cross-peaks of the four singlet methyl hydrogens as follows; δ
H 1.21 (H12′) with carbons δ
C 42.3 (C10′), 79.1 (C11′), and 27.2 (C13′); δ
H 1.22 (H13′) with carbons δ
C 42.3 (C10′), 79.1 (C11′), and 26.6 (C12′); δ
H 1.55 (H14′) with carbons δ
C 125.5 (C6′), 136.1 (C7′), and 41.1 (C8′); and δ
H 1.78 (H15′) with carbons δ
C 123.5 (C2′), 135.9 (C3′), and 41.0 (C4′). The key COSY correlations shown in
Figure 3 supported the structural determination of this part of the molecule. HMBC cross-peaks of the hydrogen at δ
H 3.35 (H1′) with carbons C2′, C3′, C4, C5, and C6 linked the prenyl and aromatic parts of the molecule via the C1′-C5 bond. Additionally, HMBC from the singlet aromatic hydrogen at δ
H 6.47 (H7) to carbons δ
C 103.9 (C3a), C4, C5, and C6 defined the presence of a penta-substituted diol aromatic ring and its connection to the oxygenated methylene carbon at δ
C 71.3 (C1). The low-field singlet
sp3 methylene hydrogens at δ
H 5.20 (H1) showed HMBC correlations with carbons C3, C3a, and C7 (
Figure 3), thus fusing the aromatic ring to the furanone ring. The positions of the oxygenated methylene (C1) and carbonyl (C3) carbons of the furanone ring were confirmed by the intense NOESY and COSY correlations between the hydrogens at H1 and H7. All the above data confirmed compound
4 as a 10′-dehydroxy-11′-β-D-mannopyranosyl C3-carbonyl derivative (rather than C1 carbonyl) of memnoconol [
17].
In the case of compound
5, its (+)-ESI-TOF MS analysis showed the presence of a [M+H]
+ ion at
m/z 403.2118 (calcd. for C
23H
31O
6, 403.2115), thus establishing a molecular formula of C
23H
30O
6. There were many similarities between the NMR data of compounds
5 and
4 (
Table 3); however, one readily noticeable difference was the absence of the β-D-mannopyranosyl moiety in compound
5. The
13C NMR data of this compound showed a total of twenty-three carbons. These included eleven
sp2 carbons, one carbonyl at δ
C 172.4 (C3), six aromatic carbons at δ
C 102.2 (C3a), 166.1 (C4 oxygenated), 111.3 (C5), 155.6 (C6 oxygenated), 103.1 (C7), and 151.5 (C7a), and four olefinic carbons at δ
C 119.0 (C1′), 127.2 (C2′), 125.7 (C6′), and 136.3 (C7′). These eleven unsaturated
sp2 carbons accounted for six degrees of unsaturation in the molecule, which, when compared to its formula of C
23H
30O
6, indicates that compound
5 is tricyclic. HMBC cross-peaks observed from the singlet aromatic hydrogen at δ
H 6.31 (H7) to the carbons at δ
C 70.3 (C1), C3a and C5, together with the HMBC cross-peak of the singlet low-field
sp3 methylene hydrogens at δ
H 5.11 (H1) to carbons C3, C3a, C7, and C7a, established a substructure of fused aromatic and furanone rings. NOESY and COSY correlations observed between the hydrogens at H1 and H7 confirmed the placement of carbons C1 and C3 in the same positions as previously observed in compound
4. Apart from the absence of the β-D-mannopyranosyl moiety, a second difference was identified in the prenyl part of compound
5, where the singlet methyl group at C15′ gave chemical shifts of δ
H 1.45/δ
C 27.1 in comparison to the previous observance of this methyl group at δ
H 1.78/ δ
C 16.4 in compound
4. Additionally, the hydrogens of this methyl group(H15′) gave HMBC cross-peaks with the carbons at δ
C 42.2 (C4′), 81.0 (C3′), and C2′, suggesting the placement of this C15′ methyl group at the oxygenated carbon C3′, resulting in the presence of an ether bridge between C3′ and the aromatic carbon C6 (i.e., C3′-O-C6 cyclization). The C3′-O-C6 cyclization caused the C3′-C2′ double bond previously observed in compound
4 to shift to a new position between C1′ and C2′ in compound
5, giving rise to a substructure similar to what was previously reported for salfredin B
11 [
31]. This substructure was confirmed by the fact that the NMR chemical shifts reported for salfredin B
11 were very similar to those recorded for this substructural part of compound
5 [
31]. The introduction of a new pyran ring, together with the shifting of the position of the double bond in compound
5, were further confirmed by two HMBC correlations, i.e., (a) the one from the olefinic hydrogen at δ
H 6.72 (H1′) to carbons C3′, C4 and C6, and (b) the other from the olefinic hydrogen at δ
H 5.52 (H2′) to carbons C3′ and C5. The last difference between compounds
4 and
5 was at C10′, where a hydroxylation is observed in compound
5 (CH-OH, δ
C 79.1 δ
H 3.21) as opposed to the CH
2 group present in compound
4 (δ
C 42.3 δ
H 1.44). The structure of compound
5 (
Figure 3) was therefore determined as a new pyran derivative of a C3-carbonyl analogue of memnoconol [
17]. The scarcity of the sample prevented the determination of the absolute configuration at chiral centers C3′ and C10′.
With respect to the third compound class (cyclic isobenzofuranone analogues
6,
7, and
8), the molecular formula of compound
6 was established as C
22H
32O
3 based on the presence of a protonated adduct (i.e., a [M+H]
+ ion) at
m/z 345.2426 in its (+)-ESI-TOF MS data (calcd. for C
22H
33O
3, 345.2424). The NMR data of this compound showed a total of twenty-two carbons (
Table 4), six of them being
sp2 aromatics at δ
C 108.2, 156.5, 108.3, 138.0, 109.8, and 154.8 (C1′–C6′), which together with the ring accounted for four of the degrees of unsaturation. When compared to the molecular formula of C
22H
32O
3, this suggests the compound has three more rings. The intense HMBC cross-peaks observed for the four singlet methyl hydrogens at δ
H 1.15 (H12), 1.01 (H13), 0.81 (H14), 0.94 (H15), together with the HMBC cross-peaks from the two low-field
sp3 methylene hydrogens at δ
H 2.57, 2.27 (H11) to the aromatic carbons C1′, C2′, C6′, the methine carbon C9 and also to the oxygenated quaternary carbon C8, were used to establish the structure of compound
6 as a drimane-like substructure connected to an aromatic ring through a pyran ring (same as in compound
2). There was a fifth singlet methyl hydrogen at δ
H 2.14 (H7′) in the NMR data, which gave intense HMBC cross-peaks with carbons C3′, C4′ and C5′, hence defining the tetrasubstituted aromatic ring and establishing the planar structure of compound
6 as a tetracyclic benzopyrane (
Figure 4). HMBC correlations from hydrogen H3′ to carbons C1′, C2′, C5′ and C7′, and from hydrogen H5′ to carbons C1′, C3′, C6′ and C7′ confirmed this structural proposal. NOESY correlations similar to those observed in compound
2 established the same stereochemistry in both compounds.
In the case of compound
7, the presence of a protonated adduct ([M+H]
+ ion) at
m/z 385.2014 in its (+)-ESI-TOF MS data (calcd. for C
23H
29O
5, 385.2010) agreed with a molecular formula of C
23H
28O
5 for the compound. The NMR data of this compound showed a total of twenty-three carbons with many similarities to compound
6 (
Table 4), including six
sp2 aromatic carbons at δ
C 117.0, 165.6, 101.9, 103.3, 148.2, and 156.6 (C1′–C6′). However, two additional olefinic carbons at δ
C 149.5 (C8) and 108.4 (C12) and two carbonyl carbons at δ
C 219.0 (C3) and 174.7 (C8′) were identified in the NMR data of compound
7. In all, these accounted for seven degrees of unsaturation, which, when compared to the molecular formula of C
23H
28O
5, suggests the presence of three cycles in addition to the aromatic ring. Three singlet methyl hydrogen signals were present in this compound, and from these, the following HMBC cross-peaks were observed: δ
H 1.04 (H13) to carbons δ
C 26.5 (C14), 48.8 (C4), 56.9 (C5), and C3 (ketone); δ
H 1.07 (H14) to carbons δ
C 22.2 (C13), C4, C5, and C3; and δ
H 1.01 (H15) to carbons δ
C 38.4 (C1), 41.0 (C10), C5, and 54.4 (C9). These HMBCs defined a drimane-like substructure (with a C3 ketone) for compound
7. Further, HMBC cross-peaks from the two low-field olefinic hydrogens at δ
H 5.11 and 4.73 (H12) to the carbons at δ
C 39.2 (C7) and C9, together with HMBC cross-peaks from hydrogens H7, H9 and H11 all to carbon C8, placed a double bond between carbons C8 and C12, thus breaking the C8-O-C6′ bridge and opening up the pyran ring previously observed at this position in compound
6 (
Figure 4). Additionally, HMBC cross-peaks similar to those previously described in compounds
4 and
5 established the same substructure for the isobenzofuranone part of compound
7 (C1′ to C8′). The NOESY and COSY correlations observed between hydrogens H3′ and H7′ corroborated the carbonyl position at C8′, thus completing the proposed structure of compound
7 (
Figure 4). NOESY correlations around the drimane substructure confirmed the same stereochemistry in both compounds
6 and
7.
The structure of compound
8 was easily elucidated due to its close similarity to phomoarcherin A [
32]. The molecular formula of compound
8, determined as C
23H
30O
5 by (+)-ESI-TOF MS analysis, matched that of phomoarcherin A [
32]. The
1H,
13C and 2D NMR data of
8 (
Table 5) confirmed it to be very similar to phomoarcherin A with two main exceptions: (a) HMBC cross-peaks similar to those found in compound
7 established the same substructure for the isobenzofuranone part (carbonyl at C8′) of compound
8, and (b) NOESY correlations (around the drimane substructure) similar to those observed in compound
2 established the same stereochemistry in both compounds
2 and
8. Thus, as shown in
Figure 5, the structure of compound
8 was established as the 8-
epi, C8′-carbonyl analogue of phomoarcherin A [
32].
All the eight new compounds (
1–
8) isolated from the
M. dichroa (CF-080171) extract belong to the tetraketide-terpenoid class of compounds, which are known to be the largest class of meroterpenoids isolated from fungi [
9]. A plausible biosynthetic pathway (
Scheme 1) may involve an initial condensation step between orsellinic acid and farsenyl pyrophosphate to form a linear meroterpenoid intermediate, which may later undergo subsequent derivatizations to produce the various compounds isolated [
9]. For example, the glycosylation of the linear intermediate at C11′ and cyclization between the unstable aldehyde and a pre-oxidized methyl substituent of the orsellinic acid residue results in the creation of the isobenzofuranone ring and the eventual production of compound
4 [
33,
34,
35]. Alternatively, hydroxylations at C10′, C11′ and cyclization between C3′ and the para hydroxyl group of the orsellinic acid residue produce compound
5 [
36,
37]. The cyclic derivatives,
6–
8, are formed by total cyclization of the prenyl substructure of the linear intermediate (or partial cyclization in the case of compound
7) and either lactonization of the orsellinic residue in the case of compounds
7 and
8 or the decarboxylation of orsellinic residue in the case of compound
6, with different degrees of oxidation in the resulting drimane residue as in each case [
38]. The cyclic compounds
1–
3 may also be formed by total cyclization of the prenyl part of the intermediate followed by lactamization (instead of lactonization) of the orsellinic acid residue with further derivatizations [
39]. The resulting drimane residue could also undergo further derivatization, as in each case [
9].
3.2. Biological Activity
For early drug discovery purposes, the standard
P. falciparum 3D7 lactase dehydrogenase and transgenic
T. cruzi β-D-galactosidase whole antiparasitic assays were utilized in the bioassay-guided process to purify the active components of the
M. dichroa CF-080171 extract, which had been previously identified as active against both parasites. All the isolated compounds (
1–
8) were tested in their pure forms against the
P. falciparum 3D7 and
T. cruzi Tulahuen C4 parasitic strains, and their EC
50 values were determined (
Table 6). Of the different compound classes isolated, memnobotrins C (
1) and D (
2) showed the most potent antiparasitic activity with EC
50 values of 0.040 and 0.201 µM, respectively, against
P. falciparum 3D7, and 0.226 and 1.37 µM respectively, against
T. cruzi Tulahuen C4. The presence of a ketone at C3 in compound
1 improves its biological activity 5-fold against both
P. falciparum 3D7 and
T. cruzi Tulahuen C4 when compared to the presence of a hydroxyl at this same position (C3) in compound
2. However, memnobotrin E (
3), which has an acetate group attachment at this same position (C3), showed 179-fold lower potency against
P. falciparum 3D7 and 157-fold lower potency against
T. cruzi Tulahuen C4 in comparison to compound
1 (
Table 6).
In the case of compounds
4 and
5, which had some structural similarities to memnoconol [
17], compound
4 exhibited an interesting potency with EC
50 values of 0.243 and 0.934 µM against
P. falciparum 3D7 and
T. cruzi Tulahuen C4 respectively, whereas compound
5 showed moderate potency against
P. falciparum 3D7 (EC
50 of 8 µM) and was inactive against
T. cruzi Tulahuen C4 at 50 µM. The presence of a sugar moiety in compound
4 may be implicated in the significantly higher bioactivity observed in this compound. With respect to compounds
6–
8, only
6 showed moderate activity in both parasites with EC
50 values of 4.0 and 7.0 µM against
P. falciparum 3D7 and
T. cruzi Tulahuen C4, respectively. Compound
7 was inactive in both parasites at 100 and 50 µM, respectively, whereas compound
8 showed slight activity (EC
50 of 17.8 µM) against
P. falciparum 3D7 only.
In order to determine their general cytotoxic effect, the EC
50 values of the isolated compounds were also determined in vitro against two different cell lines, i.e., liver carcinoma HepG2 cells and L6 rat skeletal muscle cells. Although the crude
M. dichroa CF-080171 extract from which the compounds were isolated had previously been cleared as non-cytotoxic against the HepG2 cells at the primary screening stage, it was essential to confirm the potential cytotoxic effect of the isolated pure compounds since factors such as purity and failure to reach the effective inhibitory concentration may have masked the proper effects of the respective compounds in the crude extract prior to their purification. The L6 rat skeletal muscle cells were used here as a second line of cytotoxic screen to ensure robustness since the intracellular
T. cruzi β-D-galactosidase antiparasitic assay is performed in this same host cell line. As seen in
Table 6, the three new compounds which exhibited the most interesting antiparasitic activity, i.e., compounds
1,
2 and
4, also exhibited low micromolar EC
50 values of 1.20, 4.53, and 4.84 µM, respectively, when tested for their cytotoxicity against hepatocytic carcinoma Hep G2 cells by means of a cell viability MTT assay. Although the three compounds (
1,
2 and
4) demonstrated some level of selectivity towards the
P. falciparum 3D7 (selectivity indices of 30, 22.5 and 19.9, respectively) as compared to their cytotoxicity against the Hep G2 cells, the fact that the EC
50 values recorded were in the low micromolar range was a hint of general cytotoxicity. To confirm this, the two most potent compounds,
1 and
2, were further tested against L6 rat skeletal muscle cells, and both proved to be cytotoxic with EC
50 values of ˂0.098 µM for compound
1 and 1.39 µM for compound
2. The L6 host cells are routinely used in evaluating cytotoxicity of potential antimalarial compounds [
40,
41,
42]; thus, the cytotoxic nature of compounds
1 and
2 in this cell line demonstrates their inherent cytotoxic nature. As would be expected, compound
6, which showed moderate antiparasitic activity in both parasites, also exhibited some cytotoxicity against L6 cells (EC
50 value of 2.0 µM), whereas the
T. cruzi-inactive compounds
5 and
7 were also inactive at 50 µM in L6 host cells (
Table 6).
Although the literature reports many triprenyl phenolic compounds isolated from
Stachybotrys and
Memnoniella fungi, only two compounds belonging to the memnobotrin subclass have ever been previously isolated, i.e., memnobotrins A and B. Memnobotrin B was reported to show some cytotoxic activity at 100 µM against three different cell lines, with inhibition percentages in the range of 80–90% [
17]. Considering these observations, the newly discovered antiparasitic memnobotrins C and D (compounds
1 and
2) provide very interesting biologically relevant analogues to the memnobotrin subclass despite their inherent cytotoxicity. Additionally, the superior biological activity of memnobotrins C and D in comparison to memnobotrins A, B and E, shows how the C3-substituent affects the overall potency of this compound class, thus providing some insight into their structure-activity relationship (SAR) with respect to possible chemotherapeutic developments using medicinal chemistry methods. Some of these medicinal chemistry methods may include (a) the full or partial direct replacement of the C3 substituent to generate several classes of analogues, (b) the reduction of the electronic densities around certain strategic parts of the molecule, and (c) the introduction of a structural element of metabolic interest [
43]. The various analogues generated from such medicinal chemistry methods could be tested in a SAR study to ascertain which of them would exhibit the best selectivity against only the parasites for further development. It would also be interesting to further investigate the biological effect of this compound class in (a) cell life cycle/morphological perturbations and (b) target deconvolution studies in both
P. falciparum and
T. cruzi parasites [
44].