Kallopterolides A–I, a New Subclass of seco-Diterpenes Isolated from the Southwestern Caribbean Sea Plume Antillogorgia kallos

Kallopterolides A–I (1–9), a family of nine diterpenoids possessing either a cleaved pseudopterane or a severed cembrane skeleton, along with several known compounds were isolated from the Caribbean Sea plume Antillogorgia kallos. The structures and relative configurations of 1–9 were characterized by analysis of HR-MS, IR, UV, and NMR spectroscopic data in addition to computational methods and side-by-side comparisons with published NMR data of related congeners. An investigation was conducted as to the potential of the kallopterolides as plausible in vitro anti-inflammatory, antiprotozoal, and antituberculosis agents.


Introduction
In the field of natural products chemistry cleavage of a ring with the addition of one or more hydrogen atoms at each terminal group thus created is often indicated by the prefix "seco-" [1].One of the first seco-diterpenes to be described in the literature was isolated from the tobacco plant in 1984 [2].Such ∆ 11,12 seco-cembrane stemmed from selective oxidative cleavage of one olefin double bond within the cembrane ring.In most cases, either a C-C double bond or a vicinal diol is ruptured by oxidative cleavage.Several types of seco-cembranes have been reported throughout the literature [3].Within the cembrane family of natural products cleavage usually takes place at the ∆ 4,5 , ∆ 5,6 , or ∆ 11,12 position, with most such open-chain congeners having thus far been isolated from tobacco [4].
The sea plume Pseudopterogorgia bipinnata has been previously reported as being a prolific source of structurally interesting ∆ 2,3 and ∆ 7,8 seco-cembranes endowed with unusual functionality [5].For instance, in 2000 our laboratory reported the isolation of seco-bipinnatin J (Figure 1), the first ∆ 7,8 seco-cembrane containing 4-furanal and α,β-unsaturated-γ-lactone moieties [6].Subsequently, three additional members of the same family, caucanolides D-F (Figure 1), were isolated following a re-collection of the same sea feather [7].The latter metabolites possessed two distinct α,β-unsaturated-γ-lactone moieties arranged in a linear fashion [5].Interestingly, in the preceding 2005 investigation we also described a small subset of ring-opened compounds whose structures could not be rationalized as those stemming from a cembrane precursor but rather from a pseudopteranebased diterpene.Up until now, caucanolides A-C (Figure 1) represented the only family of marine natural products based on a ∆ 2,3 seco-pseudopterane skeleton.Like the ∆ 2,3 seco-cembranes, the ∆ 2,3 seco-pseudopteranes arise presumably from oxidative cleavage of the C-2/C-3 bond.Salient structural features of the latter compounds worth highlighting are the N 1 ,N 1 -dimethyl-N 2 -acylformamidine and α,β-unsaturated-γ-hydroxy-ε-lactone moiety present in caucanolides B and C, respectively.As part of our ongoing investigation of Caribbean marine invertebrates as likely sources of bioactive natural products, we recently investigated the chemical composition of the marine sea plume Antillogorgia kallos (previously described as Pseudopterogorgia kallos) collected in Old Providence Island, Colombia [8].During this investigation, we isolated and characterized two new seco-pseudopterane diterpenes, which we have named kallopterolides A and B (1 and 2), along with ten seco-cembranes of which six are new, namely kallopterolides C-H (3)(4)(5)(6)(7)(8), and four are known (caucanolides C-F, Figure 1).We also isolated and partially characterized a labile compound, which we named kallopterolide I (9).The latter congener is a C14 derivative that seems to arise from the further degradation of either kallopterolide F (6), G (7), or H (8). The chemical structures of all the natural products stemming from this investigation were established from the analysis of 1D and 2D NMR, IR, UV, and HRMS spectral data.A modern computational method was used to validate the assigned stereochemistry.After solving all their molecular structures, samples of pure compounds were screened in vitro for antimicrobial activity against Mycobacterium tuberculosis and Plasmodium falciparum.Some of the compounds isolated were also screened for anti-inflammatory activity.Figure 2 depicts the chemical structures of the title compounds.As part of our ongoing investigation of Caribbean marine invertebrates as likely sources of bioactive natural products, we recently investigated the chemical composition of the marine sea plume Antillogorgia kallos (previously described as Pseudopterogorgia kallos) collected in Old Providence Island, Colombia [8].During this investigation, we isolated and characterized two new seco-pseudopterane diterpenes, which we have named kallopterolides A and B (1 and 2), along with ten seco-cembranes of which six are new, namely kallopterolides C-H (3)(4)(5)(6)(7)(8), and four are known (caucanolides C-F, Figure 1).We also isolated and partially characterized a labile compound, which we named kallopterolide I (9).The latter congener is a C 14 derivative that seems to arise from the further degradation of either kallopterolide F (6), G (7), or H (8). The chemical structures of all the natural products stemming from this investigation were established from the analysis of 1D and 2D NMR, IR, UV, and HRMS spectral data.A modern computational method was used to validate the assigned stereochemistry.After solving all their molecular structures, samples of pure compounds were screened in vitro for antimicrobial activity against Mycobacterium tuberculosis and Plasmodium falciparum.Some of the compounds isolated were also screened for anti-inflammatory activity.Figure 2 depicts the chemical structures of the title compounds.
The relative stereochemistry of 1 was established through a combination of 2D-NO-ESY (Figure 4a) and coupling constant (J) data analyses (Table 1, Figure 3b,c) in tandem with molecular modeling studies.This task was facilitated by the fact that the stereogenic centers within 1 are contiguous.First off, the 1 H NMR spectrum of 1 was re-recorded in CD3OD (see Table S1 in the Supplementary Materials), which enhanced signal splitting and facilitated the J analysis of the pivotal proton H-7.The improved resolution revealed that the conformational flexibility of 1, particularly alongside the C-6-C-7-C-8 bonds, was somewhat restricted, allowing some helpful conclusions to be drawn from these data.In point, the proton resonance ascribed to H-7, which occurs as a doublet of doublet, is coupled with both oxymethines H-6 and H-8.The magnitude of the coupling constant between H-7 and H-8 (JH7-H8 = 10.0 Hz) indicated that rotation along the C-7-C-8 bond is restricted and that these protons adopt an anti-periplanar arrangement (Figure 4a).If we place H-7 above the plane (β-configuration), then H-8 must have the α-configuration (below the plane).The absence of a NOESY cross-peak between these vicinal protons supported this contention.On the other hand, H-7 showed a NOESY correlation with H-6, The relative stereochemistry of 1 was established through a combination of 2D-NOESY (Figure 4a) and coupling constant (J) data analyses (Table 1, Figure 3b,c) in tandem with molecular modeling studies.This task was facilitated by the fact that the stereogenic centers within 1 are contiguous.First off, the 1 H NMR spectrum of 1 was re-recorded in CD 3 OD (see Table S1 in the Supplementary Materials), which enhanced signal splitting and facilitated the J analysis of the pivotal proton H-7.The improved resolution revealed that the conformational flexibility of 1, particularly alongside the C-6-C-7-C-8 bonds, was somewhat restricted, allowing some helpful conclusions to be drawn from these data.In point, the proton resonance ascribed to H-7, which occurs as a doublet of doublet, is coupled with both oxymethines H-6 and H-8.The magnitude of the coupling constant between H-7 and H-8 (J H7-H8 = 10.0 Hz) indicated that rotation along the C-7-C-8 bond is restricted and that these protons adopt an anti-periplanar arrangement (Figure 4a).If we place H-7 above the plane (β-configuration), then H-8 must have the α-configuration (below the plane).The absence of a NOESY cross-peak between these vicinal protons supported this contention.On the other hand, H-7 showed a NOESY correlation with H-6, which established their spatial proximity on the β face of the molecule.
This conclusion was validated by the small axial-equatorial coupling constant between these protons (J H6-H7 = 4.1 Hz).Furthermore, the fixed conformation depicted in Figure 4a was supported by the strong NOESY correlations observed between H-5/Me-16, H-5/H-7, H-7/Me-19, Me-19/H-18β, and H-8/H-18α.Farther along the eastern quadrant, the peak assignment for Me-14 and Me-15 in the 1 H-NMR spectrum of 1 was based on the NOESY cross-peak between H-2 and Me-15.While these methods allowed us to establish the assignments described, we should point out that the relative configuration drawn for 1, namely, 6S*, 7S*, and 8R*, correlates well with the known absolute configuration of other diterpenes co-isolated during this investigation (namely, kallolide A, kallolide A acetate, kallolide C, bipinnapterolide A, and gersemolide).This observation aligns with our contention that, most likely, 1 originates following oxidation/cleavage at C-2/C-3 of a suitable pseudopterane-based precursor (see Figure 1 and Scheme S1 in the Supplementary Materials).This conclusion was validated by the small axial-equatorial coupling constant between these protons (JH6-H7 = 4.1 Hz).Furthermore, the fixed conformation depicted in Figure 4a was supported by the strong NOESY correlations observed between H-5/Me-16, H-5/H-7, H-7/Me-19, Me-19/H-18β, and H-8/H-18α.Farther along the eastern quadrant, the peak assignment for Me-14 and Me-15 in the 1 H-NMR spectrum of 1 was based on the NOESY cross-peak between H-2 and Me-15.While these methods allowed us to establish the assignments described, we should point out that the relative configuration drawn for 1, namely, 6S*, 7S*, and 8R*, correlates well with the known absolute configuration of other diterpenes co-isolated during this investigation (namely, kallolide A, kallolide A acetate, kallolide C, bipinnapterolide A, and gersemolide).This observation aligns with our contention that, most likely, 1 originates following oxidation/cleavage at C-2/C-3 of a suitable pseudopterane-based precursor (see Figure 1 and Scheme S1 in the Supplementary Materials).
Kallopterolide B (2), an optically active yellowish oil, α 20 +5.0 (c 1.0, MeOH), showed a pseudomolecular [M + 1] + ion peak at m/z 345.1696 in the HR-FAB-MS corresponding to a molecular formula of C20H25O5 (calcd 345.1702).The IR and UV spectroscopic data for 2 were very similar to those recorded for kallopterolide A (1).Further examination revealed that their 1 H and 13 C NMR data in CDCl3 were also almost identical, indicating that both compounds possess identical functionality, namely, two α,β-unsaturated-γ-lactones, one α-substituted-β,β-dimethyl-α,β-unsaturated aldehyde, and one isopropenyl group.Therefore, we concluded that these compounds must be diastereomers.A detailed side-by-side comparison of the 1 H NMR spectra of 1 and 2 (Table 1) revealed that the minor differences observed could be explained by inverting the configuration in 2 at C-6.The reversal at C-6 from S* in 1 to R* in 2 was rendered by subtle differences in the 1 H NMR chemical shifts and coupling constants for H-6 [δH 5.34 (ddd, 1H, 3.9, 2.0, 1.9 Hz) in 1 vs. δH 5.05 (dd, 1H, 1.7, 1.6 Hz) in 2] and H-7 [δH 2.21 (dd, 1H, 10.0, 4.3 Hz) in 1 vs. δH 2.60 (dd, 1H, 7.0, 7.0 Hz) in 2] (Table 2).The IR and UV spectroscopic data for 2 were very similar to those recorded for kallopterolide A (1).Further examination revealed that their 1 H and 13 C NMR data in CDCl 3 were also almost identical, indicating that both compounds possess identical functionality, namely, two α,β-unsaturated-γ-lactones, one α-substituted-β,β-dimethyl-α,β-unsaturated aldehyde, and one isopropenyl group.Therefore, we concluded that these compounds must be diastereomers.A detailed sideby-side comparison of the 1 H NMR spectra of 1 and 2 (Table 1) revealed that the minor differences observed could be explained by inverting the configuration in 2 at C-6.The reversal at C-6 from S* in 1 to R* in 2 was rendered by subtle differences in the 1 H NMR chemical shifts and coupling constants for H-6 [δ H 5.34 (ddd, 1H, 3.9, 2.0, 1.9 Hz) in 1 vs. δ  2).Moreover, the coupling constant values between H-6/H-7 (J H6-H7 = 7.0 Hz) and H-7/H-8 (J H7-H8 = 7.0 Hz) [9] (Figure 5a,b), combined with molecular modeling studies and key NOESY correlations between H-6 and H-9 and between H-6 and H-18α (Figure 4b) suggested that these proton pairs lie within spatial proximity toward the α face.Interestingly, the chemical shift of H-6 (δ H 5.05) in 2 appears upfield when compared to that of H-6 (δ H 5.34) in 1.This shielding albeit small can be explained by the proximity of H-6 to ∆ 17 (anisotropic effect).Conversely, when H-6 has the opposite equatorial-like orientation (as in 1) the olefin functionality and the latter proton lie too far away from each other (Figure 4b).Moreover, the coupling constant values between H-6/H-7 (JH6-H7 = 7.0 Hz) and H-7/H-8 (JH7-H8 = 7.0 Hz) [9] (Figure 5a,b), combined with molecular modeling studies and key NO-ESY correlations between H-6 and H-9 and between H-6 and H-18α (Figure 4b) suggested that these proton pairs lie within spatial proximity toward the α face.Interestingly, the chemical shift of H-6 (δH 5.05) in 2 appears upfield when compared to that of H-6 (δH 5.34) in 1.This shielding albeit small can be explained by the proximity of H-6 to Δ 17 (anisotropic effect).Conversely, when H-6 has the opposite equatorial-like orientation (as in 1) the olefin functionality and the latter proton lie too far away from each other (Figure 4b).Given that kallopterolides A (1) and B (2) possess very similar NMR spectra, we sought additional computational support for our stereochemical assignment.For this, we used a machine learning-augmented DFT method, DU8ML, which in the past proved both fast and accurate for natural products of this size [10,11].As shown in Table 3, calculated chemical shifts alone are not sufficient to differentiate between the potential diastereomers.This was not unexpected, given that the actual experimental-experimental RMSD value for 13 C NMR shifts of the two compounds is a mere 0.38 ppm.We, therefore, have included all three parameters, i.e., RMSDs for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and 13 C chemical shifts, presented as triads in Table 3, e.g., {1.73/0.19/1.49}.Analysis of the four diastereomers reveals that the most important differentiating factor is the calculated proton spin-spin coupling constants, with RMSD values for the wrong diastereomers exceeding 1.5 Hz.The two correct diastereomers have shown good matches across all three calculated RMSDs.For details, see Scheme S2 in the Supplementary Materials.Given that kallopterolides A (1) and B (2) possess very similar NMR spectra, we sought additional computational support for our stereochemical assignment.For this, we used a machine learning-augmented DFT method, DU8ML, which in the past proved both fast and accurate for natural products of this size [10,11].As shown in Table 3, calculated chemical shifts alone are not sufficient to differentiate between the potential diastereomers.This was not unexpected, given that the actual experimental-experimental RMSD value for 13 C NMR shifts of the two compounds is a mere 0.38 ppm.We, therefore, have included all three parameters, i.e., RMSDs for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and 13 C chemical shifts, presented as triads in Table 3,    Kallopterolide D (4), [α]  20 -10.0 (c 0.9, CHCl3), was isolated as an optically active yellowish oil.The molecular formula C20H24O6, deduced from HR-FAB-MS analysis of its pseudomolecular ion (m/z [M + H] + 361.1652, calcd for C20H25O6 361.1651), required nine sites of unsaturation.The IR spectrum of 4 indicated the presence of hydroxyl (3461 cm −1 ), aldehyde (2870 cm −1 ), ester (1775, 1752 cm −1 ), and olefin (3090, 1663, 1625 cm −1 ) functionalities.The UV spectrum (MeOH) showed maxima at λmax 210 nm (ε 16,800) and λmax 263 nm (ε 19,800).The 13 C NMR spectrum displayed twenty signals (Table 4), of which eight were olefinic and three were carbonyl carbon resonances, suggesting that compound 4 was also bicyclic.Interpretation of the 1D and 2D NMR spectra revealed the presence in 4 of the following fragments: -CH2-CH2-C(CHO)=C(CH3)2 (C-1, C-2 and C-13 through C-17) (see Figure 6c) as well as a tertiary methyl carbinol linked to a α,β-unsaturated-γ-lactone through a methylene carbon (C-8 through C-12 and C-19 through C-20) (see Figure 6b).The connectivity between the C-9 methylene bridge and the internal γ-butenolide was accomplished from 1   Kallopterolide D (4), [α]  20 -10.0 (c 0.9, CHCl3), was isolated as an optically active yellowish oil.The molecular formula C20H24O6, deduced from HR-FAB-MS analysis of its pseudomolecular ion (m/z [M + H] + 361.1652, calcd for C20H25O6 361.1651), required nine sites of unsaturation.The IR spectrum of 4 indicated the presence of hydroxyl (3461 cm −1 ), aldehyde (2870 cm −1 ), ester (1775, 1752 cm −1 ), and olefin (3090, 1663, 1625 cm −1 ) functionalities.The UV spectrum (MeOH) showed maxima at λmax 210 nm (ε 16,800) and λmax 263 nm (ε 19,800).The 13 C NMR spectrum displayed twenty signals (Table 4), of which eight were olefinic and three were carbonyl carbon resonances, suggesting that compound 4 was also bicyclic.Interpretation of the 1D and 2D NMR spectra revealed the presence in 4 of the following fragments: -CH2-CH2-C(CHO)=C(CH3)2 (C-1, C-2 and C-13 through C-17) (see Figure 6c) as well as a tertiary methyl carbinol linked to a α,β-unsaturated-γ-lactone through a methylene carbon (C-8 through C-12 and C-19 through C-20) (see Figure 6b).The connectivity between the C-9 methylene bridge and the internal γ-butenolide was accomplished from 1   Kallopterolide D (4), [α]  20 -10.0 (c 0.9, CHCl3), was isolated as an optically active yellowish oil.The molecular formula C20H24O6, deduced from HR-FAB-MS analysis of its pseudomolecular ion (m/z [M + H] + 361.1652, calcd for C20H25O6 361.1651), required nine sites of unsaturation.The IR spectrum of 4 indicated the presence of hydroxyl (3461 cm −1 ), aldehyde (2870 cm −1 ), ester (1775, 1752 cm −1 ), and olefin (3090, 1663, 1625 cm −1 ) functionalities.The UV spectrum (MeOH) showed maxima at λmax 210 nm (ε 16,800) and λmax 263 nm (ε 19,800).The 13 C NMR spectrum displayed twenty signals (Table 4), of which eight were olefinic and three were carbonyl carbon resonances, suggesting that compound 4 was also bicyclic.Interpretation of the 1D and 2D NMR spectra revealed the presence in 4 of the following fragments: -CH2-CH2-C(CHO)=C(CH3)2 (C-1, C-2 and C-13 through C-17) (see Figure 6c) as well as a tertiary methyl carbinol linked to a α,β-unsaturated-γ-lactone through a methylene carbon (C-8 through C-12 and C-19 through C-20) (see Figure 6b).The connectivity between the C-9 methylene bridge and the internal γ-butenolide was accomplished from 1   Kallopterolide D (4), [α]  20 -10.0 (c 0.9, CHCl3), was isolated as an optically active yellowish oil.The molecular formula C20H24O6, deduced from HR-FAB-MS analysis of its pseudomolecular ion (m/z [M + H] + 361.1652, calcd for C20H25O6 361.1651), required nine sites of unsaturation.The IR spectrum of 4 indicated the presence of hydroxyl (3461 cm −1 ), aldehyde (2870 cm −1 ), ester (1775, 1752 cm −1 ), and olefin (3090, 1663, 1625 cm −1 ) functionalities.The UV spectrum (MeOH) showed maxima at λmax 210 nm (ε 16,800) and λmax 263 nm (ε 19,800).The 13 C NMR spectrum displayed twenty signals (Table 4), of which eight were olefinic and three were carbonyl carbon resonances, suggesting that compound 4 was also bicyclic.Interpretation of the 1D and 2D NMR spectra revealed the presence in 4 of the following fragments: -CH2-CH2-C(CHO)=C(CH3)2 (C-1, C-2 and C-13 through C-17) (see Figure 6c) as well as a tertiary methyl carbinol linked to a α,β-unsaturated-γ-lactone through a methylene carbon (C-8 through C-12 and C-19 through C-20) (see Figure 6b).The connectivity between the C-9 methylene bridge and the internal γ-butenolide was accomplished from 1   Kallopterolide D (4), [α]  20 -10.0 (c 0.9, CHCl3), was isolated as an optically active yellowish oil.The molecular formula C20H24O6, deduced from HR-FAB-MS analysis of its pseudomolecular ion (m/z [M + H] + 361.1652, calcd for C20H25O6 361.1651), required nine sites of unsaturation.The IR spectrum of 4 indicated the presence of hydroxyl (3461 cm −1 ), aldehyde (2870 cm −1 ), ester (1775, 1752 cm −1 ), and olefin (3090, 1663, 1625 cm −1 ) functionalities.The UV spectrum (MeOH) showed maxima at λmax 210 nm (ε 16,800) and λmax 263 nm (ε 19,800).The 13 C NMR spectrum displayed twenty signals (Table 4), of which eight were olefinic and three were carbonyl carbon resonances, suggesting that compound 4 was also bicyclic.Interpretation of the 1D and 2D NMR spectra revealed the presence in 4 of the following fragments: -CH2-CH2-C(CHO)=C(CH3)2 (C-1, C-2 and C-13 through C-17) (see Figure 6c) as well as a tertiary methyl carbinol linked to a α,β-unsaturated-γ-lactone through a methylene carbon (C-8 through C-12 and C-19 through C-20) (see Figure 6b).The connectivity between the C-9 methylene bridge and the internal γ-butenolide was accomplished from 1     In all, three stereogenic centers are present in kallopterolide D: the two asymmetric carbon atoms at C-8 and C-10 and the Δ 6 trisubstituted double bond.From the outset, we realized that the relative stereochemistry of kallopterolide D was going to be difficult to ascertain given the acyclic nature of its structure as well as the non-adjacency of its two chiral carbons [12].It should, therefore, be noticed that, except for the Z-configuration assigned to Δ 6 (vide infra), the 8R*and 10S* configurations depicted in 4 should be taken as tentative.First off, since the 10S absolute configuration for bipinnatin J (see Scheme S1 in the Supplementary Materials), a likely biogenetic precursor to 4, has been established by asymmetric synthesis, we assigned the S* configuration at C-10 in 4 [13].Interestingly, after conducting a series of molecular modeling studies and 2D-NOESY experiments we envisioned that kallopterolide D has the propensity to adopt the S-shaped conformation shown in Figure 7.In all, three stereogenic centers are present in kallopterolide D: the two asymmetric carbon atoms at C-8 and C-10 and the ∆ 6 trisubstituted double bond.From the outset, we realized that the relative stereochemistry of kallopterolide D was going to be difficult to ascertain given the acyclic nature of its structure as well as the non-adjacency of its two chiral carbons [12].It should, therefore, be noticed that, except for the Z-configuration assigned to ∆ 6 (vide infra), the 8R* and 10S* configurations depicted in 4 should be taken as tentative.First off, since the 10S absolute configuration for bipinnatin J (see Scheme S1 in the Supplementary Materials), a likely biogenetic precursor to 4, has been established by asymmetric synthesis, we assigned the S* configuration at C-10 in 4 [13].Interestingly, after conducting a series of molecular modeling studies and 2D-NOESY experiments we envisioned that kallopterolide D has the propensity to adopt the S-shaped conformation shown in Figure 7.In all, three stereogenic centers are present in kallopterolide D: the two asymmetric carbon atoms at C-8 and C-10 and the Δ 6 trisubstituted double bond.From the outset, we realized that the relative stereochemistry of kallopterolide D was going to be difficult to ascertain given the acyclic nature of its structure as well as the non-adjacency of its two chiral carbons [12].It should, therefore, be noticed that, except for the Z-configuration assigned to Δ 6 (vide infra), the 8R*and 10S* configurations depicted in 4 should be taken as tentative.First off, since the 10S absolute configuration for bipinnatin J (see Scheme S1 in the Supplementary Materials), a likely biogenetic precursor to 4, has been established by asymmetric synthesis, we assigned the S* configuration at C-10 in 4 [13].Interestingly, after conducting a series of molecular modeling studies and 2D-NOESY experiments we envisioned that kallopterolide D has the propensity to adopt the S-shaped conformation shown in Figure 7.   4) and subsequent analysis of 2D NMR data suggested that 5 possessed the same partial structures and identical interconnectivity as those of 4. Thus, we concluded that kallopterolides D ( 4) and E (5) are diastereomers.Following side-by-side comparisons of their 1 H and 13 C NMR spectra, we quickly realized that the minor spectral differences observed were ascribable to a change in relative stereochemistry in 5 at the C-8 position.Specifically, we argue that the change at C-8 from R* in kallopterolide D (4) to S* in kallopterolide E ( 5) could be inferred from subtle differences in the 13  As in 4, molecular modeling analyses in combination with 2D-NOESY experiments indicated that at 20 • C, a solution of kallopterolide E (5) in CDCl 3 does not adopt a linear conformation either.Instead, compound 5 attains a more stable S-shape conformation (Figure 8).As we saw before in 4, strong NOESY correlations were observed in 5 between H-5/H-7 and H-5/H-18, which strongly argued for the Z-geometry of ∆ 6 .This time, however, and contrary to what was observed for kallopterolide D ( 4 4) and 2D-NMR spectroscopic data of 3 with those for stereoisomers 4 and 5 quickly revealed the presence in 3 of the already familiar partial structures a-c (devoid of relative stereochemistry) previously remarked in Figure 6.On the other hand, when the UV spectra in MeOH of these stereoisomers were compared, 3 revealed a subtle hypsochromic effect (λ max 257 nm for 3 vs.263 nm for 4), suggesting a change in the geometry of kallopterolide C (3) about the 5-ethylidenyl-3-methyl-2(5H) furanone functionality [7].Careful comparisons of the 1 H-and 13 C-NMR spectra of these compounds supported this contention [14][15][16][17].For instance, the change in geometry at ∆ 6 from Z in kallopterolide E ( 5) to E in kallopterolide C (3) was clearly implied by the differences in the 13   9).
The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ 13C ) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ 13C ) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations.The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E (5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations.  Kallopterolide F (6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C 20 H 24 O 5 , which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10) revealed the presence of partial structures -CH 2 -CH 2 -C(CHO)=C(CH 3 ) 2 (C-1 to C-2 and C-13 to C-17), α-methyl-α,β-unsaturated-γlactone (C-3 to C-6 and C-18), and α,β-unsaturated-γ-lactone (C-10 to C-12 and C-20) as in kallopterolides C-E (3-5).
Table 5.Comparison of the experimental 13 C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated 13  The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations.
Table 5.Comparison of the experimental 13 C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated 13 C chemical shift RMSDs of possible diastereomers a .

SS-E SS-Z SR-Z SR-E C
1.23 Kallopterolide F ( 6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C20H24O5, which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10 The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations.
Table 5.Comparison of the experimental 13 C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated 13 C chemical shift RMSDs of possible diastereomers a .

SS-E SS-Z SR-Z SR-E C
1.23 Kallopterolide F ( 6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C20H24O5, which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10 The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations.
Table 5.Comparison of the experimental 13 C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated 13 C chemical shift RMSDs of possible diastereomers a .

SS-E SS-Z SR-Z SR-E C
1.23 Kallopterolide F ( 6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C20H24O5, which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10 The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations. Kallopterolide F ( 6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C20H24O5, which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10 The results of DU8ML calculations for the potential candidate structures of kallopterolides C (3), D (4), and E ( 5) are presented in Table 5.The fact that C-8 is quaternary presents an additional challenge of stereochemical assignment, as the 1 H-1 H spin-spin coupling constants are not informative.In this case, the assignment was solely based on 13 C data.Two unambiguous matches were identified: kallopterolide C (3) as the SS-E isomer, RMSD (δ13C) = 1.23 ppm, and kallopterolide E (5) as the SS-Z isomer, RMSD (δ13C) = 1.12 ppm.The stereoconfiguration of kallopterolide D (4) was then confirmed as SR-Z, i.e., as the remaining choice between the SR-Z and SR-E stereoconfigurations. Kallopterolide F ( 6) and kallopterolide G (7) were isolated as optically active yellowish oils with similar optical rotations and spectroscopic data.The HR-MS analysis of each compound suggested the same molecular formula of C20H24O5, which indicated nine degrees of unsaturation.The IR, 1 H, and 13 C NMR spectra indicated the presence of olefin, aldehyde, and ester functionalities.Careful analysis of the 1 H, 13 C (Table 6), DEPT-135, HMQC, 1 H-1 H COSY, and HMBC (Figure 10  The difference of sixteen mass units in the molecular formula of 6 and 7 suggested that the tertiary hydroxy group in kallopterolides C-E (3-5) must have been replaced by a trisubstituted alkene across C-7/C-8.This observation was corroborated by the absence of a broad IR absorption band ascribable to hydroxy functionality in the IR spectra of kallopterolide F (6) and kallopterolide G (7).The presence of a distinct trisubstituted olefin in kallopterolide F (6)   Key HMBC correlations between H-6 with C-8, H-7 with C-5, C-9, in addition to tho H-10 with C-8, C-9 connected the α-methyl-α,β-unsaturated-γ-lactone (C-3 to C-6) at C-7 the α,β-unsaturated-γ-lactone at C-8 (C-10 to C-12 and C-20).Concurrent 1 H-1 H COSY e iments distinctively indicated the coupled proton spin systems across all these substruc Careful evaluation of the overall 2D NMR data recorded for kallopterolide F ( 6) and lopterolide G (7) demonstrated that these compounds shared the same planar structure.The "open-chain" nature of structures 6 and 7 severely hampered our ability to assign their relative stereochemistry.In addition, DU8ML failed to differentiate compounds 6 and 7's relative configurations confidently, thus our assignments should be taken as tentative.As a convenient starting point, we adopted for 6 and 7 the same 10S configuration as that usually found in cembranolides from other sea plume species belonging to the Pseudopterogorgia genus.Moreover, careful analysis of the NOESY spectra of compounds 6 and 7, as well as side-side comparisons of their coupling constant data together with molecular modeling studies, established that both molecules share a similar 3D conformation (Figure 11).In particular, the pivotal H-10 proton, in 7, showed a NOESY cross-peak with the H 3 -19 protons.The latter methyl protons, in turn, showed a NOESY correlation with H-6.If we assume that H-10 lies in the β face, H-6, too, must be assigned to the same face.The anti-periplanar relationship between H-6 and H-7 was deduced from the coupling constant value of 8.6 Hz.The "open-chain" nature of structures 6 and 7 severely hampered our ability to assign their relative stereochemistry.In addition, DU8ML failed to differentiate compounds 6 and 7 s relative configurations confidently, thus our assignments should be taken as tentative.As a convenient starting point, we adopted for 6 and 7 the same 10S configuration as that usually found in cembranolides from other sea plume species belonging to the Pseudopterogorgia genus.Moreover, careful analysis of the NOESY spectra of compounds 6 and 7, as well as sideside comparisons of their coupling constant data together with molecular modeling studies, established that both molecules share a similar 3D conformation (Figure 11).In particular, the pivotal H-10 proton, in 7, showed a NOESY cross-peak with the H3-19 protons.The latter methyl protons, in turn, showed a NOESY correlation with H-6.If we assume that H-10 lies in the β face, H-6, too, must be assigned to the same face.The anti-periplanar relationship between H-6 and H-7 was deduced from the coupling constant value of 8.6 Hz.Interestingly, the NOESY spectrum of kallopterolide G (7) displayed similar NOESY correlations except for the key correlation between H-10 and H3-19 that was not present between these protons in the NOESY spectrum of kallopterolide F (6).These dissimilarities connote that bis-butenolides 6 and 7 are epimers at C-6 (Figure 11).Molecular modeling experiments corroborated these observations and established that the most likely relative stereochemistry for kallopterolides F (6) and G ( 7) is 6R*,10S* and 6S*,10S*, respectively.In both 6 and 7, the E geometry was assigned to the Δ 7 -trisubstituted olefin based Interestingly, the NOESY spectrum of kallopterolide G (7) displayed similar NOESY correlations except for the key correlation between H-10 and H 3 -19 that was not present between these protons in the NOESY spectrum of kallopterolide F (6).These dissimilarities connote that bis-butenolides 6 and 7 are epimers at C-6 (Figure 11).Molecular modeling experiments corroborated these observations and established that the most likely relative stereochemistry for kallopterolides F (6) and G ( 7) is 6R*,10S* and 6S*,10S*, respectively.In both 6 and 7, the E geometry was assigned to the ∆ 7 -trisubstituted olefin based on the shielded methyl carbon resonance at δ C 18.1 in kallopterollide F (6) and δ C 17.2 in kallopterolide G (7), respectively.The respective absence of a NOESY cross-peak between H-7 and H 3 -19 in the spectrum of each compound confirmed the proposed geometry.
Kallopterolide H (8) was isolated as a yellowish oil, [α] 20 D +56.9 (c 1.0, acetone).The HR-ESI-MS exhibited a pseudomolecular ion [M + H] + at m/z 361.1654 (calcd 361.1651,C 20 H 25 O 6 ), appropriate for a molecular formula of C 20 H 24 O 6 .The latter required nine degrees of unsaturation, which was supported by 13 C NMR and DEPT NMR data (Table 6).A difference of sixteen mass units in the molecular formula of kallopterolide G (7) in relation to kallopterolide H (8) revealed the presence of an extra oxygen atom in compound 8.The 1 H and 13 C NMR data for kallopterolide H (8) were very similar to those for kallopterolide G ( 7), but they did not show the characteristic aldehyde resonances at δ H 10.10 (s, 1H, H-2) or δ C 190.7 (C, C-2).On the other hand, the appearance of a shielded carbonyl resonance at δ C 172.3 (C, C-2), in addition to a broad IR absorption band at 3446 cm −1 , corroborated the presence of a carboxylic acid functionality.This information suggested that kallopterolide H ( 8) is the C-2 carboxylic acid derivative of kallopterolide G (7).Because similar NOEs and 1D NMR data ( 1 H and 13 C NMR) were observed for each compound, it was concluded that most likely they possess identical stereochemistry.Unfortunately, the DU8ML method failed to assign the relative configuration for 8 confidently due to intra-or inter-molecular H-bonds in the conformational equilibrium.
Lastly, kallopterolide I ( 9) was isolated as a homogeneous yellowish oil.Unfortunately, ensuing decomposition of this compound after purification made it impossible for us to obtain IR, [α] D , UV, or HR-MS data.The planar structure of this compound was however elucidated using 2D NMR data collected prior to its decomposition.The overall 1D NMR data (Table 1) for compound 9 showed fourteen carbon resonances corresponding to two carbon-carbon and three carbon-oxygen double bonds indicating six sites of unsaturation.Careful analysis of the 1 H, 13 C DEPT-135, HMQC, 1 H-1 H COSY, and HMBC quickly revealed the presence of the -CH 2 -CH 2 -C(CHO)=C(CH 3 ) 2 and α,β-unsaturated-γ-lactone substructures.The appearance of two carbon resonances at δ C 204.5 (C, C-8) and 30.5 (CH 3 , C-19), combined with the proton signal at δ H 2.21 (s, 3H, H 3 -19) swiftly led us to identify a methyl ketone functionality.The presence of the latter functionality was confirmed by HMBC correlations of C-8 with H 2 -9 and H 3 -19.The lactone moiety was connected with the C-9 methylene from HMBC correlations of H 2 -9 to C-10 and C-11.This compound must likely stem from the C-7/C-8 oxidative cleavage of kallopterolides F (6), G (7) or H (8). The 10S stereochemistry depicted in 9, although as likely as not to be correct, is implied and thus subject to confirmation.

Biogenesis
The co-occurrence of kallopterolides A-I with pseudopterane and cembrane diterpenes within the same organism suggests that compounds 1-9 could arise from successional oxidation-ring cleavage of the latter metabolites.As a matter of convenience, we could envision the title compounds as belonging to one of the following three subclasses, respectively, the ∆ 2,3 seco-pseudopteranes [kallopterolides A (1) and B (2)], the ∆ 2,3 secocembranes [kallopterolides C-H (3-8)], and the ∆ 2,3 , ∆ 7,8 bis-seco-cembrane [kallopterolide I (9)].Hereafter, a hypothetical biogenetic proposal has been put forward (see Scheme S1 in the Supplementary Materials) linking the kallopterolides to either the furanocembranolides or the furanopseudopteranolides, related diterpenes concomitant with this sea plume as well as other gorgonian species of the same order [18][19][20][21][22][23].In so far as the absolute structures of some of these plausible precursors have been established, Scheme S1 in the Supporting Materials justifies our bias in choosing the stereochemistry depicted in structures 1-9.

Biological Activity
Marine natural products are important sources of biologically active agents, and a plethora of bioactive compounds have been extracted from marine organisms like tunicates, sponges, soft corals, and molluscs [24].These biologically active compounds have been reported to modulate various biological activities and have anti-inflammatory, antifungal, and anticancer effects [25].Despite our best efforts to detect meaningful bioactivity, the kallopterolides demonstrated marginal or no activity at all as potential antiinflammatory, antiprotozoal, or antituberculosis agents.Thus, kallopterolides A-E (1-5) demonstrated minimal effects on the release of TXB 2, O 2 − , or lactate dehydrogenase (LDH) (a marker for cell cytotoxicity) from E. coli lipopolysaccharide-activated rat neonatal microglia in vitro [26].On the other hand, kallopterolides D (4) and E (5) showed no activity against chloroquine-resistant Plasmodium falciparum W2 (IC 50 values ≥ 50 µg/mL) [27].Kallopterolide E (5) was tested for in vitro antituberculosis activity against Mycobacterium tuberculosis H 37 Rv, but it was found to exhibit only marginal mycobacterial growth inhibition (23%) at a concentration of 6.25 µg/mL.Likewise, kallopterolide A (1) showed 0% growth inhibition at the same concentration [28].It should be remarked here that kallopterolides F-I (6-9) could not be tested in the anti-inflammatory, antiplasmodial, or antituberculosis assays due to either scarcity or untimely decomposition of the natural products.

General Experimental Procedures
1D-and 2D-NMR spectra were recorded with a Bruker DPX-300 or DRX-500 FT-NMR spectrometers (Bruker Corporation, Billerica, MA, USA).Infrared and UV spectra were obtained with a Nicolet Magna FT-IR 750 spectrometer (Nicolet Instrument Corporation, Madison, WI, USA) and a Shimadzu UV-2401 PC UV-Visible (Shimadzu Corporation, Columbia, MD, USA) recording spectrophotometers, respectively.Optical rotations were obtained with an Autopol IV (Rudolph Research Analytical, Hackettstown, NJ, USA) automatic polarimeter.HR-EI-MS, HR-FAB-MS, HR-ESI-MS, and LR-EI-MS analyses were generated at the Mass Spectrometry Laboratory of the University of Illinois at Urbana-Champaign.Routine molecular modeling studies were performed with MacSpartan Pro and/or Spartan 04' Programs (Wavefunction, Irvine, CA, USA).Column chromatography was performed using silica gel (35-75 mesh) (Analtech, Newark, DE, USA) and TLC analyses were carried out using glass precoated silica gel plates.HPLC was performed using either an Ultrasphere polar-bonded Cyano semi-preparative (Avantor, Radnor, PA, USA) column (5 µ, 10 mm × 25 cm) or an Ultrasphere normal-phase Si gel semi-preparative column (5 µ, 10 mm × 25 cm).All HPLC separations were carried out using a flow rate = 2 mL/min with isocratic elution of the mobile phase with the UV detector set at λ = 220 nm.All solvents used were either spectral grade or were distilled from glass before use.The percentage yield of each compound is based on the weight of the dry gorgonian specimen.

Collection and Extraction of Antillogorgia kallos
Fresh specimens of the sea plume Antillogorgia kallos (Bielschowsky, 1918) [8] were collected by hand using SCUBA at depths of 83-91 ft in Old Providence Island, Colombia, on 15-16 March 2002.A voucher specimen is stored in the Chemistry Department of the University of Puerto Rico-Río Piedras Campus.The organism was partially air-dried, frozen, and lyophilized prior to extraction.The dry specimens (1.07 kg) were blended using a mixture of CH 2 Cl 2 /MeOH (1:1) (20 × 1 L).After filtration, the crude extract was concentrated and stored under vacuum to yield a greenish gum (166 g).The crude extract was suspended in water (2 L) and extracted with n-hexane (3 × 2 L), CHCl 3 (3 × 2 L), and EtOAc (2 × 2 L).Each extract was concentrated under reduced pressure to yield 71.9 g of the n-hexane extract, 39.3 g of the CHCl 3 extract, and 1.47 g of the EtOAc extract.

Computational Method
For the calculations and details regarding the computational method used, see Scheme S2 in the Supplementary Materials.

Anti-Inflammation Bioassay
The anti-inflammation assays were performed at the Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois by members of Professor Alejandro M. S. Mayer's Research Group.Rat neonatal microglia (2 × 10 5 cells) were seeded into each well of 24-well flat-bottom culture clusters and stimulated with bacterial lipopolysaccharide (LPS) (0.3 ng/mL) in Dulbecco's modified Eagle medium + 10% fetal bovine serum + penicillin + streptomycin for 17 h in a humidified 5% CO 2 incubator at 35.9 • C. Media were then removed, and microglia were washed with warm (37 • C) Hanks' balanced salt solution (HBSS) and then incubated with the title compounds (0.01-10 µM) or vehicle (DMSO) for 15 min prior to stimulation with phorbol 12-myristate 13-acetate (PMA) (1 µM).All experimental treatments were run in triplicate and in a final volume of 1 mL.Seventy minutes after PMA stimulation, HBSS was aspirated from each well and O 2 -, TXB 2 , and LDH release were determined as described elsewhere [26].

Antiplasmodial Bioassay
The antiplasmodial activity of some of the isolated compounds was evaluated against a chloroquine-resistant (Indochina W2) strain of Plasmodium falciparum using a novel DNAbased microfluorimetric method.This method was developed, and all the antiplasmodial bioassays were performed, at the Instituto de Investigaciones Científicas Avanzadas y Servicios de Alta Tecnología, Ciudad del Saber, Clayton, Panama.Detailed description of the experimental method used for this assay is given elsewhere [27].In this bioassay compounds displaying an IC 50 value > 10 µg/mL are considered inactive and those with an IC 50 value ≤ 10 µg/mL are considered active.

Antimycobacterial Bioassay
The antitubercular activity of some of the isolated compounds was evaluated against the laboratory strain Mycobacterium tuberculosis H 37 Rv.A detailed description of the experimental method used for this antitubercular assay has been previously described [28].All the antimycobacterial assays were performed in the Institute for Tuberculosis Research, University of Illinois at Chicago, College of Pharmacy, 833 S. Wood Street (M/C 964) Chicago, IL by members of Professor Scott G. Franzblau's Research Group.In this bioassay compounds displaying inhibitory growth percentage ≥ of 90% at 6.25 µg/mL are considered active.

Figure 4 .
Figure 4. Computer-generated perspective views for the lowest energy conformers for (a) kallopterolide A (1) and (b) kallopterolide B (2).Some hydrogen atoms have been omitted for clarity.

Figure 6 .
Figure 6.Top: Key HMBC correlations required for the assembly of partial units (a-c).Bottom: 1 H-1 H COSY and HMBC correlations required to interconnect units (a-c), thus yielding the complete planar structure (d) for kallopterolide D (4).

Figure 7 .
Figure 7. Computer-generated perspective view for the lowest energy conformer of kallopterolide D (4) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.Strong NOESY correlations between H-7 with both H-5 and H-10 quickly established the Z geometry about the Δ 6 olefin and vouched for our assignment for the 10S* relative stereochemistry in 4. Furthermore, the conspicuous absence of NOEs between H-10 and Me-19 and between H-7 and H2-9αβ, combined with strong NOEs between Me-19 and H2-9αβ, all argued for the 8R* configuration.Additional validation for the proposed S-shape conformation of 4 stems from the multiplicity and coupling constant values for H-10 (ddd, JH-9α/H-10 = 4.2 Hz, JH-9β/H-10 = 8.2 Hz, and JH-10/H-11 = 2.6 Hz) as well as the supplementary

Figure 6 .
Figure 6.Top: Key HMBC correlations required for the assembly of partial units (a-c).Bottom: 1 H-1 H COSY and HMBC correlations required to interconnect units (a-c), thus yielding the complete planar structure (d) for kallopterolide D (4).

Molecules 2024 , 21 Figure 6 .
Figure 6.Top: Key HMBC correlations required for the assembly of partial units (a-c).Bottom: 1 H-1 H COSY and HMBC correlations required to interconnect units (a-c), thus yielding the complete planar structure (d) for kallopterolide D (4).

Figure 7 .
Figure 7. Computer-generated perspective view for the lowest energy conformer of kallopterolide D (4) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.Strong NOESY correlations between H-7 with both H-5 and H-10 quickly established the Z geometry about the Δ 6 olefin and vouched for our assignment for the 10S* relative stereochemistry in 4. Furthermore, the conspicuous absence of NOEs between H-10 and Me-19 and between H-7 and H2-9αβ, combined with strong NOEs between Me-19 and H2-9αβ, all argued for the 8R* configuration.Additional validation for the proposed S-shape conformation of 4 stems from the multiplicity and coupling constant values for H-10 (ddd, JH-9α/H-10 = 4.2 Hz, JH-9β/H-10 = 8.2 Hz, and JH-10/H-11 = 2.6 Hz) as well as the supplementary

Figure 8 .
Figure 8. Computer-generated perspective view of the lowest energy conformer for kallopterolide E (5) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.Kallopterolide C (3) was isolated as an optically active yellowish oil, α 20 +12.5 (c 0.4, MeOH).The LR-EI-MS of 3 exhibited its molecular ion [M] +• at m/z 360, appropriate for a molecular formula of C20H24O6.However, attempts to measure the exact mass of 3 using HR-MS techniques (HR-EI-MS, HR-FAB-MS and HR-ESI-MS) failed to secure this information.Interestingly, the IR spectrum of 3 was quite similar to those of kallopterolide D (4) and kallopterolide E (5).Side-by-side comparisons of the 1D-NMR (Table4) and 2D-NMR spectroscopic data of 3 with those for stereoisomers 4 and 5 quickly revealed the presence in 3 of the already familiar partial structures a-c (devoid of relative stereochem-

Figure 9 .
Figure 9. Computer-generated perspective view of the lowest energy conformer for kallopterolide C (3) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.

Figure 9 .
Figure 9. Computer-generated perspective view of the lowest energy conformer for kallopterolide C (3) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.
C chemical shift RMSDs of possible diastereomers a .C (3) showing important NOESY correlations.Several hydrogen atoms have been omitted for clarity.

Figure 10 .
Figure 10.Selected 1 H-1 H COSY and HMBC correlations for kallopterolide F (6).Figure 10.Selected 1 H-1 H COSY and HMBC correlations for kallopterolide F (6). Key HMBC correlations between H-6 with C-8, H-7 with C-5, C-9, in addition to those of H-10 with C-8, C-9 connected the α-methyl-α,β-unsaturated-γ-lactone (C-3 to C-6) at C-7, and the α,β-unsaturated-γ-lactone at C-8 (C-10 to C-12 and C-20).Concurrent 1 H-1 H COSY experiments distinctively indicated the coupled proton spin systems across all these substructures.Careful evaluation of the overall 2D NMR data recorded for kallopterolide F (6) and kallopterolide G (7) demonstrated that these compounds shared the same planar structure.The "open-chain" nature of structures 6 and 7 severely hampered our ability to assign their relative stereochemistry.In addition, DU8ML failed to differentiate compounds 6 and 7's relative configurations confidently, thus our assignments should be taken as tentative.As a convenient starting point, we adopted for 6 and 7 the same 10S configuration as that usually found in cembranolides from other sea plume species belonging to the Pseudopterogorgia genus.Moreover, careful analysis of the NOESY spectra of compounds 6 and 7, as well as side-side comparisons of their coupling constant data together with molecular modeling studies, established that both molecules share a similar 3D conformation (Figure11).In particular, the pivotal H-10 proton, in 7, showed a NOESY cross-peak with the H 3 -19 protons.The latter methyl protons, in turn, showed a NOESY correlation with H-6.If we assume that H-10 lies in the β face, H-6, too, must be assigned to the same face.The anti-periplanar relationship between H-6 and H-7 was deduced from the coupling constant value of 8.6 Hz.

Figure 11 .
Figure 11.Computer-generated perspective views for the lowest energy conformers for (a) kallopterolide F (6) and (b) kallopterolide G(7).Some hydrogen atoms have been omitted for clarity.

Figure 11 .
Figure 11.Computer-generated perspective views for the lowest energy conformers for (a) kallopterolide F (6) and (b) kallopterolide G(7).Some hydrogen atoms have been omitted for clarity.
a NMR spectra were recorded in CDCl 3 at 25 • C; 1 H and 13 C NMR chemical shift values are in ppm and referenced to the residual CHCl 3 (δ = 7.26) or CDCl 3 (δ = 77.0)ppm signals.b 13 C NMR multiplicities were deduced from a DEPT NMR experiment.c Chemical shifts and 1 H-1 H coupling constant values are approximated due to second-order effects.

Table 2 .
Key differences observed between the 1 H and13C NMR spectra of kallopterolides A (1) and B (2) in CDCl 3 at 25 • C.

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .
a Root Mean Square Deviations (RMSDs) for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and13C chemical shifts, presented as triads in curly brackets.Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Red bold font highlights bad (i.e.too high) RMSD numbers for J's indicating a mismatch.

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .
a Root Mean Square Deviations (RMSDs) for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and13C chemical shifts, presented as triads in curly brackets.Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Red bold font highlights bad (i.e.too high) RMSD numbers for J's indicating a mismatch.

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .
a Root Mean Square Deviations (RMSDs) for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and13C chemical shifts, presented as triads in curly brackets.Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Red bold font highlights bad (i.e.too high) RMSD numbers for J's indicating a mismatch.

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .
a Root Mean Square Deviations (RMSDs) for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and13C chemical shifts, presented as triads in curly brackets.Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Red bold font highlights bad (i.e.too high) RMSD numbers for J's indicating a mismatch.

Table 3 .
Comparison of the experimental NMR spectroscopic data of kallopterolides A (1) and B (2) with DU8ML-calculated NMR parameter of possible diastereomers a .
a Root Mean Square Deviations (RMSDs) for the 1 H-1 H spin-spin coupling constants, 1 H chemical shifts, and13C chemical shifts, presented as triads in curly brackets.Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Red bold font highlights bad (i.e.too high) RMSD numbers for J's indicating a mismatch.

Table 5 .
Comparison of the experimental13C-NMR spectroscopic data of kallopterolides C (3), D (4), Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Values highlighted represent unambiguous matches. a

Table 5 .
Comparison of the experimental13C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated13C chemical shift RMSDs of possible diastereomers a .Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Values highlighted represent unambiguous matches. a

Table 5 .
Comparison of the experimental13C-NMR spectroscopic data of kallopterolides C (3), D (4), and E (5) with calculated13C chemical shift RMSDs of possible diastereomers a .Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Values highlighted represent unambiguous matches. a Blue circles (and alphanumeric characters) denote the S configuration.Red circles (and alphanumeric characters) denote the R configuration.Values highlighted represent unambiguous matches. a