Chloro-Furanocembranolides from Leptogorgia sp. Improve Pancreatic Beta-Cell Proliferation

Two new chloro-furanocembranolides (1, 2) and two new 1,4-diketo cembranolides (3, 4) were isolated from the crude extract of Leptogorgia sp. together with a new seco-furanocembranolide (5) and the known Z-deoxypukalide (6), rubifolide (7), scabrolide D (8) and epoxylophodione (9). Their structures were determined based on spectroscopic evidence. Four compounds: 1, 2, 7 and 8 were found to activate the proliferation of pancreatic insulin-producing (beta) cells.


Results
Compound 1 was obtained as an oil whose EIMS spectrum showed peaks at m/z [M − 1] + 409/411, with relative intensities suggesting one chlorine atom. These peaks correspond to the molecular formula C 20 H 23 ClO 7  Connectivity information obtained from COSY, HSQC and HMBC experiments unambiguously determined the planar structure of compound 1 as a furanocembranolide, containing a C-18 oxidized to aldehyde, a vicinal diol at C-7-C-8, a C-10-C-20 α,β-epoxy-γ-lactone moiety and a chloroisopropenyl group at C-1.  The HMBC correlations H 2 -16/C-17, C-15, C-1 and H 2 -17/C-16, C-15, C-1  locate a chloroisopropenyl group at C-1 of fragment I, whereas the correlations H 3 -19/C-7, C-8 and C-9 allowed us lengthen fragment II by adding a vicinal dihydroxyl moiety bonded to C-9. The north end of both fragments II and I are connected together by insertion of a furane ring, in agreement with the HMBC correlations H-5/C-3, C-4, C-6 and H 2 -2 with C-4. An epoxy lactone binds the southern ends of fragments I and II between C-13 and C-10, as deduced from the HMBC correlations H-10/C-11, C-12, C-20; H 2 -13/C-20, C-11, C-12 and H 2 -14 with C-12. Thus, the structure of 1, with nine degrees of unsaturation, has been established.    Compound 2 was obtained as an oil whose EIMS spectrum showed a molecular ion at m/z [M] + 394/396, with an isotopic pattern for a chlorine atom in the molecular formula C 20 H 23 ClO 6 (HREIMS) (m/z 394.1181 [M] + , calcd. for C 20 H 23 35 ClO 6 394.1183). The 13 C NMR spectrum and correlations in the HSQC spectrum indicated seven quaternary carbons, six methines, six methylenes and one methyl ( Table 1). Absorptions for a hydroxyl group at 3558 cm −1 and carbonyl group at 1747 cm −1 were observed in its IR spectrum. 1 H and 13 C NMR data resemble those of 1. The principal differences lie in the chemical shifts of H-7, H 2 -9, C-9 and C-19, which were: δ H-7 4.59 (1H, s), δ H-9 1.89 (1H, m); 2.59 (1H, dd); δ C-9 43.0 and δ C-19 19.6, compared with those of 1 (Table 1). These shift values suggested that compounds 1 and 2 differ in the configuration of C-7 and C-8. Also, their molecular formulas diverge by 16 amu of oxygen. This suggests that the γ-lactone ring system is devoid of the epoxide ring on 2. This absence is confirmed by the new signals observed in the 1 H and 13 C NMR spectra for a proton (δ H-11 5.86 (1H, s)) of a disubstituted olefin (δ C-11 148.6; δ C-12 136.2). Thus, the structure of 2 with nine degrees of unsaturation was established as shown in Figure 1 by COSY, HSQC and HMBC experiments. The relative configurations of compounds 1 and 2 were ascertained by NOESY experiments, molecular mechanics [11], chemical shift studies and comparison of their spectroscopic data with those of previously described cembranoids, leptodiol [1], lophodiol A [12] and sinumaximol B [13] (Figure 3). HSQC spectrum indicated seven quaternary carbons, six methines, six methylenes and one methyl (Table 1). Absorptions for a hydroxyl group at 3558 cm −1 and carbonyl group at 1747 cm −1 were observed in its IR spectrum. 1 H and 13 C NMR data resemble those of 1. The principal differences lie in the chemical shifts of H-7, H2-9, C-9 and C-19, which were: δH-7 4.59 (1H, s), δH-9 1.89 (1H, m); 2.59 (1H, dd); δC-9 43.0 and δC-19 19.6, compared with those of 1 (Table 1). These shift values suggested that compounds 1 and 2 differ in the configuration of C-7 and C-8. Also, their molecular formulas diverge by 16 amu of oxygen. This suggests that the γ-lactone ring system is devoid of the epoxide ring on 2. This absence is confirmed by the new signals observed in the 1 H and 13 C NMR spectra for a proton (δH-11 5.86 (1H, s)) of a disubstituted olefin (δC-11 148.6; δC-12 136.2). Thus, the structure of 2 with nine degrees of unsaturation was established as shown in Figure 1 by COSY, HSQC and HMBC experiments.
The relative configurations of compounds 1 and 2 were ascertained by NOESY experiments, molecular mechanics [11], chemical shift studies and comparison of their spectroscopic data with those of previously described cembranoids, leptodiol [1], lophodiol A [12] and sinumaximol B [13] ( Figure 3). In compound 1, the observed NOEs of H-5 with H-7 and H3-19, together with those of H-7 with H3-19 suggested that the adjacent hydroxyl groups at C-7-C-8 should be in a cis relationship. In compound 2, the observed NOEs of H3-19 with H-5 and H-10 indicate that these protons and Me-19 must be on same side of the molecule, whereas the NOEs of H3-19 with H-9a and of H-7 with H-9b indicates that H-7 and Me-19, so the vicinal diols on C-7-C-8, have a trans-relationship. Therefore, the relative configuration of C-8 is opposite to that on compound 1.
The configurations of C-7-C-8 vicinal diols were corroborated by comparison of the 1 H and 13 C NMR chemical shifts around the diol moiety C-7-C-8 of compounds 1 and 2 with those of the related diols leptodiol, lophodiol A and sinumaximol B, shown in Table 2. The chemical shifts of C-19 and H-7 of compounds 1 and 2 present strong differences (ΔδC-19 = 2.8 ppm and ΔδH-7 = 0.7). The chemical shifts of C-19 and H-7 of leptodiol (δC-19 22.7; δH-7 5.12) and lophodiol A (δC-19 22.7; δH-7 5.24), both with the C-7-C-8 diols in an α-cis-relationship, are very similar to those of compound 1. Whereas the chemical shifts of C-19 and H-7 of sinumaximol B (δC-19 19.8; δH-7 4.52), whose C-7-C-8 diols show a trans-relationship, present strong differences (upfield ΔδC-19 ≈ 2.6 ppm and downfield ΔδH-7 ≈ 0.8) which are very similar to those presented by compound 2 in comparison to compound 1. In compound 1, the observed NOEs of H-5 with H-7 and H 3 -19, together with those of H-7 with H 3 -19 suggested that the adjacent hydroxyl groups at C-7-C-8 should be in a cis relationship. In compound 2, the observed NOEs of H 3 -19 with H-5 and H-10 indicate that these protons and Me-19 must be on same side of the molecule, whereas the NOEs of H 3 -19 with H-9a and of H-7 with H-9b indicates that H-7 and Me-19, so the vicinal diols on C-7-C-8, have a trans-relationship. Therefore, the relative configuration of C-8 is opposite to that on compound 1.
In compound 1, a striking 1 H NMR signal is the singlet observed for H-11 (δ 3.73, s) due to the roughly 90 • dihedral angle formed between H-10 and H-11. This results in a small J H-10, H-11 that confirms the relative configuration of C-10 and C-11 as represented in the energetically favourable conformation shown in Figure 3.
Finally, in compound 1, the relative configuration of C-1 was secured by the NOESY correlation of H-13b with H-1 and H-13a with H-11, as shown in the 3D model in Figure 3. In compound 2, the observed NOEs of H-1 with H-2a and H-14a, as well as of H-14a with H-11 and of H-2b with H-14b, indicate that the isopropenyl group of 2 is situated on the alpha side of the molecule. Therefore, both compounds belong to the furanocembranolide of the α-series and their relative configurations are: 1R*, 7S*, 8S*, 10S*, 11S* and 12S* for compound 1 and 1R*, 7S*, 8R* and 10S* for compound 2. In compound 1, a striking 1 H NMR signal is the singlet observed for H-11 (δ 3.73, s) due to the roughly 90° dihedral angle formed between H-10 and H-11. This results in a small JH-10, H-11 that confirms the relative configuration of C-10 and C-11 as represented in the energetically favourable conformation shown in Figure 3.
Finally, in compound 1, the relative configuration of C-1 was secured by the NOESY correlation of H-13b with H-1 and H-13a with H-11, as shown in the 3D model in Figure 3. In compound 2, the observed NOEs of H-1 with H-2a and H-14a, as well as of H-14a with H-11 and of H-2b with H-14b, indicate that the isopropenyl group of 2 is situated on the alpha side of the molecule. Therefore, both compounds belong to the furanocembranolide of the α-series and their relative configurations are: 1R*, 7S*, 8S*, 10S*, 11S* and 12S* for compound 1 and 1R*, 7S*, 8R* and 10S* for compound 2. Compound 3 was obtained as an oil whose EIMS spectrum showed a peak at m/z 362 [M] + , which corresponds to the molecular formula C20H26O6 (HREIMS) (m/z 362.1737 [M] + , calcd. for C20H26O6 362.1729). These data are in agreement with the 13 C NMR spectrum, which displayed correlations in the HSQC spectrum indicative of seven quaternary carbons, four methines, six methylenes and three methyls. Absorptions for a hydroxyl group at 3475 cm −1 and carbonyl groups at 1751, 1721 and 1701 cm -1 were observed in their IR spectrum. According to the degree of unsaturation given by the 13 C NMR data, 3 must be a tricyclic compound.
In the 1 H and 13 C NMR experiments (Table 3) (Figure 4). The HMBC correlations of H2-16/C-1, C-17 and H3-17/C-16, C-15, C-1 locates the isopropenyl group at C-1 in fragment I, whereas the HMBC correlations of H2-2 and H3-18 with C-3 connects fragments I and II through C-3. The HMBC correlations H3-19/ C-7, C-8, C-9, along with those of H-5 and H2-7 with C-6, allowed us to extend fragment II by connecting it with substructure III through C-6. Both ends of the fragment I and substructure III (C-6-C-9) are connected by inserting an α,β-unsaturated γ-lactone bonded to C-13 and C-9, respectively, due to the HMBC correlations H2-13/C-20, C-11, C-12 and those of H-11/C-10 and H2-9/C-10, C-11. The quaternary feature of C-10 comes from the oxygen linkage between C-5 and C-10 supported by an HMBC correlation of H-5 with C-10. Therefore, the tricyclic structure of 3 has been established as depicted in Figure 4. In compound 1, a striking 1 H NMR signal is the singlet observed for H-11 (δ 3.73, s) due to the roughly 90° dihedral angle formed between H-10 and H-11. This results in a small JH-10, H-11 that confirms the relative configuration of C-10 and C-11 as represented in the energetically favourable conformation shown in Figure 3.
Finally, in compound 1, the relative configuration of C-1 was secured by the NOESY correlation of H-13b with H-1 and H-13a with H-11, as shown in the 3D model in Figure 3. In compound 2, the observed NOEs of H-1 with H-2a and H-14a, as well as of H-14a with H-11 and of H-2b with H-14b, indicate that the isopropenyl group of 2 is situated on the alpha side of the molecule. Therefore, both compounds belong to the furanocembranolide of the α-series and their relative configurations are: 1R*, 7S*, 8S*, 10S*, 11S* and 12S* for compound 1 and 1R*, 7S*, 8R* and 10S* for compound 2. Compound 3 was obtained as an oil whose EIMS spectrum showed a peak at m/z 362 [M] + , which corresponds to the molecular formula C20H26O6 (HREIMS) (m/z 362.1737 [M] + , calcd. for C20H26O6 362.1729). These data are in agreement with the 13 C NMR spectrum, which displayed correlations in the HSQC spectrum indicative of seven quaternary carbons, four methines, six methylenes and three methyls. Absorptions for a hydroxyl group at 3475 cm −1 and carbonyl groups at 1751, 1721 and 1701 cm -1 were observed in their IR spectrum. According to the degree of unsaturation given by the 13 C NMR data, 3 must be a tricyclic compound.
In the 1 H and 13 C NMR experiments (Table 3) (Figure 4). The HMBC correlations of H2-16/C-1, C-17 and H3-17/C-16, C-15, C-1 locates the isopropenyl group at C-1 in fragment I, whereas the HMBC correlations of H2-2 and H3-18 with C-3 connects fragments I and II through C-3. The HMBC correlations H3-19/ C-7, C-8, C-9, along with those of H-5 and H2-7 with C-6, allowed us to extend fragment II by connecting it with substructure III through C-6. Both ends of the fragment I and substructure III (C-6-C-9) are connected by inserting an α,β-unsaturated γ-lactone bonded to C-13 and C-9, respectively, due to the HMBC correlations H2-13/C-20, C-11, C-12 and those of H-11/C-10 and H2-9/C-10, C-11. The quaternary feature of C-10 comes from the oxygen linkage between C-5 and C-10 supported by an HMBC correlation of H-5 with C-10. Therefore, the tricyclic structure of 3 has been established as depicted in Figure 4. . These data are in agreement with the 13 C NMR spectrum, which displayed correlations in the HSQC spectrum indicative of seven quaternary carbons, four methines, six methylenes and three methyls. Absorptions for a hydroxyl group at 3475 cm −1 and carbonyl groups at 1751, 1721 and 1701 cm −1 were observed in their IR spectrum. According to the degree of unsaturation given by the 13 C NMR data, 3 must be a tricyclic compound.
In the 1 H and 13 C NMR experiments (Table 3) (fragment II) (Figure 4). The HMBC correlations of H 2 -16/C-1, C-17 and H 3 -17/C-16, C-15, C-1 locates the isopropenyl group at C-1 in fragment I, whereas the HMBC correlations of H 2 -2 and H 3 -18 with C-3 connects fragments I and II through C-3. The HMBC correlations H 3 -19/ C-7, C-8, C-9, along with those of H-5 and H 2 -7 with C-6, allowed us to extend fragment II by connecting it with substructure III through C-6. Both ends of the fragment I and substructure III (C-6-C-9) are connected by inserting an α,β-unsaturated γ-lactone bonded to C-13 and C-9, respectively, due to the HMBC correlations H 2 -13/C-20, C-11, C-12 and those of H-11/C-10 and H 2 -9/C-10, C-11. The quaternary feature of C-10 comes from the oxygen linkage between C-5 and C-10 supported by an HMBC correlation of H-5 with C-10. Therefore, the tricyclic structure of 3 has been established as depicted in Figure 4.   35 Cl, 396.1340). Considering the HSQC correlations, signals observed in the 13 C NMR spectrum indicate seven quaternary carbons, four methines, seven methylenes and two methyls. Absorptions for a hydroxyl group at 3420 cm −1 and carbonyl groups at 1727, 1690 and 1647 cm -1 were observed in their IR spectrum. 1 H and 13 C NMR data ( Table 3) resemble those of 3. The molecular formula of 4 showed that one proton of 3 is substituted by a chlorine atom in 4. Its corresponding data from both 1 H and 13 C NMR reveal that the substitution is situated on the isopropenyl appendage, where the methyl group of 3 changed to chloromethylene in 4. This substitution was confirmed by the fragment (m/z 321.1335 [M   Table 3) (Table 3). Absorptions for carbonyl groups at 1655, 1650 and 1638 cm −1 were noted in the IR spectrum.
In addition to the 1 H and 13 C NMR data registered for an isopropenyl group, a furan ring and a α,β-unsaturated-γ-lactone, other notable key signals were detected for the following functional groups: aldehyde [δ H- showed that fragments I and II are linked through an α,β-unsaturated-γ-lactone. HMBC correlations of H 3 -19/C-8, C-9 allowed us to place the methyl-ketone. Finally, the aldehyde must be located at C-6, in good agreement with the chemical shift observed for the aldehyde group of seco-bipinnatin J (δ H-7 9.52 (1H, s), δ C-7 177.5), the only seco-derivative [9] isolated from Pseudopterogorgia, which showed the same scission pattern. Compound 5 is the first seco-furanocembranolide isolated from genus Leptogorgia. Z-deoxypukalide [1], also isolated in this work, can be considered a biogenetic precursor of 5 by oxidative cleavage of the corresponding ∆ 7,8 . Since Z-deoxypukalide belongs to the α-cembranolide series, we assign the same relative configurations 1R*, 10S* to 5. NOESY experiments, studies of coupling constants and molecular mechanics calculations suggest that 3 and 4 have the same relative stereochemistry (Figure 1). In both compounds, NOEs were observed for H-5 with H-9a and H-11 and for H3-19 with H-9a and H-9b, defining a relative configuration for C-5 and C-10 and establishing H3-19 on C-8 in a pseudo-equatorial disposition and therefore coplanar to H-5. Also, the large coupling constants of H-5 (J = 10.4 Hz) in 3 and H-5 (J = 10.3 Hz) in 4 indicate that H-5 and H-4 are trans-diaxial, as the observed NOE between H-5 and H3-18 corroborates. Finally, the NOE observed between H-11 and H-1 places the isopropenyl group on the opposite side to the Me-18. Therefore, the overall relative configuration for 3 and 4 should be 1R*, 4R*, 5S*, 8R* and 10S*.
Compound 5 was obtained as an oil with an EIMS spectrum peak at m/z 388 [M] + , which corresponds to the molecular formula C21H24O7 (HREIMS) (m/z 388.1524 [M] + , calcd. for C21H24O7, 388.1522). These data are in agreement with the 13 C NMR spectrum, which displayed correlations in the HSQC spectrum indicating eight quaternary carbons, five methines, five methylenes and three methyls (Table 3). Absorptions for carbonyl groups at 1655, 1650 and 1638 cm −1 were noted in the IR spectrum.
Compound 5 is the first seco-furanocembranolide isolated from genus Leptogorgia. Zdeoxypukalide [1], also isolated in this work, can be considered a biogenetic precursor of 5 by oxidative cleavage of the corresponding Δ 7,8 . Since Z-deoxypukalide belongs to the α-cembranolide series, we assign the same relative configurations 1R*, 10S* to 5  It should be expected that compounds 1-5 belong to the same enantiomeric series as Z-deoxypukalide, (6) whose absolute configuration we have previously determined using an NMR-based method using Pirkle's reagent [1].

Activation of Pancreatic Beta-Cell Proliferation
Several strategies have been proposed to recover functional beta-cell mass loss in diabetes mellitus onset; one of them is to activate beta-cell proliferation [3]. In previous work, we showed that furanocembranolides such as epoxypukalide, pukalide, Z-deoxypukalide and leptolide augment beta-cell proliferation [4][5][6]. In order to acquire detailed knowledge of the proliferation effect induced by furanocembranolides, compounds 1, 2 and rubifolide (7) were used to treat synchronized INS-1 cells and proliferation was then measured. INS-1 cells were preincubated with 0.1 µM of each product and proliferation was measured by BrdU incorporation (Table 4), showing a 2-3-fold increase in proliferation. Although it is difficult to reach a conclusion regarding the functional groups that could modulate this proliferation activity, these results also support chloro-furanocembranolides being potential activators of pancreatic beta-cell proliferation.
Furthermore, synchronized INS-1 cells were treated with the nor-cembranolide, scabrolide D (8), (Table 4), showing a 2.8 ± 0.69-fold change above untreated cells (1.0). We therefore consider it of interest in searching for compounds of the furanocembranolide and nor-1,4-diketocembranolide families, in order to develop a new class of antidiabetic agents. Table 4. Beta-cell proliferation measurement after treating INS-1 beta-cells with each compound.