2.1. Chemistry
The CH
2Cl
2-MeOH extract of the lyophilized sponge
Haliclona sp. was first subjected to a normal-phase silica gel column chromatography to yield 12 fractions. Fraction 9 was subjected to repetitive reverse-phase semi-preparative and analytical HPLC to yield six pure compounds (
1–
6) (
Figure 2). Among them, three are known and were identified as osirisynes A (
4), B (
5), and E (
6) by comparison with published spectroscopic data; the other three are new and were named osirisynes G-I (
1–
3). Their elucidation is described below.
Osirisyne G (
1) was obtained as a white amorphous solid. The molecular formula, C
47H
72O
12, was established from a HRESIMS molecular ion peak at
m/
z 827.4950 [M − H]
−, indicating 12 degrees of unsaturation (
Figure S1). Analysis of the 1D and 2D
1H and
13C NMR data for
1 (CD
3OD,
Table 1,
Figures S2–S5) revealed resonances and correlations consistent with those of a long-chain highly oxygenated polyacetylene, like osirisynes A–F [
12] or fulvynes A–I [
11]. The
1H NMR spectrum of
1 recorded in CD
3OD showed the presence of four olefinic protons (δ
H 5.88 (1H, ddd,
J = 15.4, 6.2, 1.3 Hz), δ
H 5.76 (1H, ddd,
J = 15.4, 5.7, 1.1 Hz), δ
H 5.62 (1H, dtd,
J = 15.3, 6.5, 0.8 Hz), and δ
H 5.43 (1H, ddt,
J = 15.3, 7.1, 1.4 Hz), an acetylenic proton [δ
H 2.92 (1H, d,
J = 2.2 Hz), nine oxygenated methines (δ
H 5.11 (1H, m), δ
H 4.82 (1H, dm,
J = 5.7 Hz), δ
H 4.60 (1H, d,
J = 4.2 Hz), δ
H 4.33 (1H, td,
J = 6.7, 1.6 Hz), δ
H 4.09 (1H, q,
J = 6.0 Hz), δ
H 3.97 (1H, m), δ
H 3.69 (1H, tt,
J = 10.9, 6.3 Hz), δ
H 3.61 (1H, td,
J = 8.6, 2.5 Hz), and δ
H 3.43 (1H, dd,
J = 8.1, 4.3 Hz) and a series of methylene groups in the range δ
H 2.50–1.30. The
13C NMR spectrum of
1 showed the presence of a ketone C-19 (δ
C 214.5), a carboxylic acid C-1 (δ
C 161.3), eight sp carbons due to four triple bonds C-2, C-3, C-32, C-33, C-35, C-36, C-46, and C-47 (δ
C 79.9, 83.4, 85.0, 83.8, 80.9, 81.9, 84.7, 74.9), four sp
2 carbons due to two double bonds C-25, C-26, C-43, and C-44 (δ
C 132.3, 134.3, 136.0, 130.3), nine oxymethines C-4, C-5, C-6, C-27, C-31, C-34, C-38, C-42, and C-45 (δ
C 65.0, 78.5, 72.7, 73.5, 62.5, 52.3, 70.7, 72.2, 62.5), and several methylene groups were also present.
All proton-bearing carbons were assigned by HSQC experiment. Analysis of the COSY and HMBC correlations aided in recognizing the partial structures a–d (
Figure 3) of the long alkyl chain of compound
1. The COSY correlations revealed the presence of the spin system C-4—C-5—C-6—C-7 for the partial structure a and the HMBC correlations between H-4, C-2, and C-3 and between H-5 and C-2 indicated the carbon resonances of the triple bond in fragment a. For the partial structure b, the HMBC correlations between H-18 and C-19, H-20 and C-19, C-21 and C-22, H-21 and C-19, C-22 and C-23, in addition to the COSY correlations between H-17 and H-18 as well as H-20 and H-21 indicated the presence of the ketone C-19 in the spin system C-17—C-18—C-19—C-20—C-21—C-22. The COSY correlations between H-24 and H-25, H-25 and H-26, H-26 and H-27, as well as H-27 and H-28 revealed the presence of an oxymethine C-27 linked to a sp
2 carbon C-26 in the spin systems C-24—C-25—C-26—C-27—C-28. The two spin systems mentioned above can be linked by analysis of HMBC correlations between H-22, C-23, and C-24; H-23, C-24, and C-25; H-24, C-25, and C-26; H-25, C-23, H-26, and C-24. HMBC correlations between H-31, C-32, and C-33; H-34 and C-32; C-33, C-35, and C-36; and H-37, C-34, C-3,5 and C-36 indicated the carbon resonances of the two triple bonds in fragment c, thus, by using the COSY correlations, the spin system C-30—C-31—C-32—C-33—C-34—C-35—C-36—C-37—C-38—C-39 was revealed. The partial structure d was revealed by COSY correlations between H-41 and H-42; H-42 and H-43; H-43 and H-44; H-44 and H-45; and between H-45 and H-47 and by HMBC correlations between H-45 and C-43, C-44, C-46, and C-47 and between H-44 and C-42 and C-46. The geometries of the two double bonds were easily assigned as 25
E, 43
E by the coupling constant analysis of the olefinic protons (
J = 15.3 and 15.4 Hz, respectively). The relative configuration of compound
1 remained unassigned. Any attempts to obtain suitable derivatives (Mosher’s esters) for a stereochemical analysis were unsuccessful due to the small amount of product and the high number of chiral carbons. The connectivities between partial structures a–d for
1 as well as the number of the linking methylene groups were established on the basis of the molecular formula and ESI–MS/MS data. The combinations of partial structures a + b + c + d represented 674 m.u. whereas the molecular structure weight was 828 m.u. The difference corresponding to 11 methylene groups determined the length of the alkyl chains between the different partial structures. For the ESI-MS/MS analysis, Cu
II was used for compound ionization so the species sought were detected as [(M − H) + Cu
II]
+. Indeed, a reduction of Cu
II copper to Cu
I can occur during the electrospray ionization process [
21]. This reduction is accompanied by the formation of radical species, which can be at the origin of specific fragmentations during the experiments of collision-induced dissociation (CID) and cannot be obtained from the dissociation of protonated species [M + H]
+ for this type of compound [
22]. Indeed, the CID of these protonated species is generally accompanied by the loss of non-specific molecules such as H
2O molecules. The ESI–MS/MS spectra (
Figure 4) showed different fragment ions that indicated the presence of nine methylenes between a and b, one methylene between b and c, and one methylene between c and d. The fragmentation of the molecule was explained by different dissociation mechanisms (
Figure 5). In ESI
+–MS/MS, the peak at
m/
z 846.4 corresponded to the loss of a CO
2 molecule and confirmed the presence of the carboxylic acid C-1. The presence of this carboxylic acid was also confirmed in ESI
−–MS/MS by the presence of the ion [M − H]
− at
m/
z 827.5 and the peak at
m/
z 783.5 corresponded to the loss of a CO
2 molecule. In ESI
+–MS/MS, the peak at
m/
z 761.4 can be explained by a rearrangement between the three hydroxyls in C-4, C-5, and C-6, yielding to a loss of a H
2O molecule and a 129.0 Da fragment corresponding to the C
5H
3O
3 formula. This mechanism confirmed the sequence C-1 to C-6. The peak at
m/
z 542.2 can be explained by a fragmentation between C-27 and C-28. This fragment confirmed the link between the partial structure b and c by one methylene. The peak at
m/
z 708.3 can be explained by a fragmentation between C-37 and C-38. The loss of a 182.1 Da fragment corresponding to a C
10H
14O
3 confirmed the link between the partial structure c and d by one methylene. In ESI
−–MS/MS, the peak at
m/
z 601.4 was also due to a fragmentation between C-37 and C-38 and confirmed this link. These fragments (
m/
z 542.2 and 708.3 in ESI
+–MS/MS and
m/
z 601.4 in ESI
−–MS/MS) established the link between partial structures b, c, and d. In order to correspond to the molecular formula C
47H
72O
12, the aliphatic chain between the partial structures a and b had to possess 12 methylenes.
Osirisyne H (
2) was obtained as a white amorphous solid. The molecular formula, C
47H
72O
11, was established from a HRESIMS molecular ion peak at
m/
z 811.5002 [M − H]
−, indicating 12 degrees of unsaturation (
Figure S6). Analysis of the 1D and 2D
1H and
13C NMR data for
2 (
Table 2,
Figures S9–S13) revealed resonances and correlations consistent with those of a long-chain highly oxygenated polyacetylene, like osirisyne G (
1). The
1H NMR spectrum of
2 recorded in CD
3OD showed the presence of four olefinic protons (δ
H 5.88 (1H, ddd,
J = 15.4, 6.2, 1.3 Hz), δ
H 5.76 (1H, ddd,
J = 15.4, 5.7, 1.1 Hz), δ
H 5.61 (1H, dtd,
J = 15.3, 6.7, 0.6 Hz), and δ
H 5.42 (1H, ddt,
J = 15.3, 7.1, 1.3 Hz), an acetylenic proton (δ
H 2.92 (1H, d,
J = 2.2 Hz), eight oxygenated methines (δ
H 5.04 (1H, quint,
J = 1.8 Hz), δ
H 4.82 (1H, dm,
J = 5.7 Hz), δ
H 4.60 (1H, d,
J = 4.3 Hz), δ
H 4.09 (1H, q,
J = 6.1 Hz), δ
H 3.97 (1H, q,
J = 6.3 Hz), δ
H 3.69 (1H, m), δ
H 3.62 (1H, td,
J = 8.8, 2.3 Hz), and δ
H 3.43 (1H, dd,
J = 8.1, 4.3 Hz), and a series of methylene groups in the range δ
H 2.50–1.30. The
13C NMR spectrum of
2 showed the presence of a ketone C-19 (δ
C 214.5), a carboxylic acid C-1 (δ
C 158.6), eight sp carbons due to four triple bonds C-2, C-3, C-32, C-33, C-35, C-36, C-46, and C-47 (δ
C 80.0, 83.3, 79.3, 84.3, 81.1, 81.5, 84.0, 74.5), four sp
2 carbons due to two double bonds C-25, C-26, C-43, and C-44 (δ
C 132.2, 134.3, 136.0, 130.4), eight oxymethines C-4, C-5, C-6, C-27, C-34, C-38, C-42, and C-45 (δ
C 65.0, 78.5, 72.9, 73.4, 52.4, 70.6, 72.2, 62.4), and several methylene groups were also present. Osirisyne H (
2) was different from
1 by the presence of the methylene C-31 (δ
H 2.23 (2H, td,
J = 6.9, 2.0 Hz); δ
C 19.1) instead of an oxygenated methine. The molecular formula determined by HRESIMS, C
47H
72O
11 for osirisyne H (
2) and C
47H
72O
12 for osirisyne G (
1), in addition to the HSQC correlation between H-31 and C-31 and the HMBC correlations between H-31, C-32, and C-33, confirmed this difference. The connectivities between partial structures a, b, c’, and d for
2 as well as the number of the linking methylene groups were established on the basis of the molecular formula and ESI–MS/MS data. As osirisyne G (
1), the ESI–MS/MS spectra showed different fragment ions that indicated the presence of nine methylenes between a and b, one methylene between b and c’, and one methylene between c’ and d (
Supplementary Materials, Figures S7 and S8).
Osirisyne I (
3) was obtained as a white amorphous solid. The molecular formula, C
47H
72O
11, was established from a HRESIMS molecular ion peak at
m/
z 811.4998 [M − H]
−, indicating 12 degrees of unsaturation (
Figure S14). Analysis of the 1D and 2D
1H and
13C NMR data for
3 (
Table 2,
Figures S18–S24) revealed resonances and correlations consistent with those of a long-chain highly oxygenated polyacetylene, like osirisyne G (
1). The
1H NMR spectrum of
3 recorded in CD
3OD showed the presence of six olefinic protons (δ
H 5.88 (1H, ddd,
J = 15.3, 6.2, 1.2 Hz), δ
H 5.76 (1H, ddd,
J = 15.4, 5.7, 1.1 Hz), δ
H 5.62 (1H, dq,
J = 14.5, 7.0 Hz), δ
H 5.62 (1H, dq,
J = 14.5, 7.0 Hz), δ
H 5.43 (1H, ddt, 15.3, 7.1, 1.3), and δ
H 5.43 (1H, ddt, 15.3, 7.1, 1.3), an acetylenic proton (δ
H 2.91 (1H, d,
J = 2.2 Hz), nine oxygenated methines (δ
H 5.11 (1H, q,
J = 1.7 Hz), δ
H 4.82 (1H, dm,
J = 5.7 Hz), δ
H 4.33 (1H, td,
J = 6.7, 1.6 Hz), δ
H 4.19 (1H, d,
J = 5.2 Hz), δ
H 4.09 (1H, q,
J = 6.1 Hz), δ
H 3.98 (1H, q, 6.3 Hz), δ
H 3.97 (1H, q, 6.3 Hz), δ
H 3.69 (1H, m), and δ
H 3.56 (1H, ddd,
J = 8.6, 5.0, 3.4 Hz), and a series of methylene groups in the range δ
H 2.50–1.30. The
13C NMR spectrum of
3 showed the presence of a carboxylic acid C-1 (δ
C 161.5), eight sp carbon due to four triple bonds C-2, C-3, C-32, C-33, C-35, C-36, C-46, and C-47 (δ
C 80.7, 83.3, 85.5, 84.4, 80.7, 82.1, 84.4, 74.9), six sp
2 carbons due to two double bonds C-19, C-20, C-25, C-26, C-43, and C-44 (δ
C 132.5, 134.5, 132.3, 134.4 136.2, 130.5), nine oxymethines C-4, C-5, C-21, C-27, C-31, C-34, C-38, C-42, and C-45 (δ
C 67.2, 75.5, 73.6, 73.6, 62.6, 52.5, 70.9, 72.4, 62.6), and several methylene groups were also present. Osirisyne I (
3) is similar to osirisyne G (
1), however, the
13C NMR spectrum of
3 indicated the disappearance of the carbonyl group C-19. Instead, signals for a new double bond, C-19, C-20, and a hydroxyl-bearing methine, C-21, appeared. This partial structure was confirmed by COSY correlations between H-18 and H-19; H-19 and H-20; H-20 and H-21; and between H-21 and H-22. Osirisyne I (
3) was also different from osirisyne G (
1) due to the presence of the methylene C-6 (δ
H 1.47 (1H, m), 1.67 (1H, m); δ
C 33.3) instead of an oxygenated methine. The molecular formula determined by HRESIMS, C
47H
72O
11 for osirisyne I (
3) and C
47H
72O
12 for osirisyne G (
1), in addition to the HSQC correlation between H-6 and C-6 and the HMBC correlations between H-6, C-4, and C-5, confirmed this difference. The connectivities between partial structures a’, b’, c, and d for
3 as well as the number of the linking methylene groups were established on the basis of the molecular formula and ESI–MS/MS data. Regarding osirisyne G (
1), the ESI–MS/MS spectra showed different fragment ions that indicated the presence of nine methylenes between a’ and b’, one methylene between b’ and c, and one methylene between c and d (
Supplementary Materials, Figures S15 and S16).
2.2. Biological Activity
While the CH
2Cl
2-MeOH extract from
Haliclona sp. sponge presented significant anti-tyrosinase activity (31.1%) (
Figure 6), one of these molecules, osirisyne E (
6), was already described as an enzyme inhibitor. This molecule, as well as osyrisines C and F, had shown Na
+/K
+ ATPase and reverse transcriptase (RT) inhibitory activities [
13]. The six isolated osirisynes (
1–
6) were submitted to a biological evaluation against seven different targets involved in aging or age-related diseases. These targets include biological assays on catalase and sirtuin 1 activation and on CDK7, Fyn kinase, tyrosinase, elastase, and proteasome inhibition.
Catalase is a common enzyme located at the peroxisome that prevents cell oxidative damage (oxidized proteins, lipids, and DNA) by converting hydrogen peroxide (H
2O
2) into water (H
2O) and dioxygen (O
2). This antioxidant enzyme prevents the accumulation of hydrogen peroxide, which is continuously produced by metabolic reactions and belongs to the reactive oxygen species (ROS), in cellular organelles and tissues. Indeed, ROS are associated to the pathogenesis of numerous diseases including age-related diseases [
23,
24,
25]. Finding natural products that are catalase activators can increased the intracellular antioxidant defense system capacity and can be useful in preventing these diseases.
Sirtuin 1 is a member of the sirtuin family of proteins, a group of very promising targets for anti-aging approaches [
26] with activities linked to crucial biological processes like regulating ribosomal DNA recombination, gene silencing, DNA repair, chromosomal stability, and longevity [
27].
CDK7 is one of the cyclin-dependent kinases (CDKs), known for their critical roles in cell cycle regulation but also involved in other physiological process like DNA repair and transcription [
28]. This kinase has been reported in recent studies to be crucial for the pathogenesis of certain cancer types driven by transcription of a key set of genes and has been validated as a therapeutic target for cancers [
29,
30,
31].
Fyn is a member of the Src family of protein tyrosine kinases (PTKs), an important class of molecules in human biology. Fyn’s biological functions are diverse, and include signaling via the T cell receptor, regulation of brain function, as well as adhesion mediated signaling [
32]. Recent studies highlight the involvement of this kinase in different age-related diseases such as cancers [
33] or Alzheimer’s disease [
34]. Fyn interacts with both protein Tau and amyloid β-peptide, two key players responsible for the major pathologic hallmarks of Alzheimer’s disease [
35,
36] and inhibitors of this kinase seem to be a promising novel approach therapy of this disease [
18].
Tyrosinase, a copper-containing metalloenzyme, is a key enzyme involved in melanogenic processes [
37]. In humans, melanin helps defend skin from the damage caused by UV light, however, excess levels of melanin can cause various dermatological disorders including hyperpigmentations, melisma, freckles, and age spots. Many tyrosinase inhibitors have been used in cosmetics and pharmaceutical products for the prevention of overproduction of melanin in the epidermis, however, side effects may occur following the chronic exposure to these compounds, so research of new tyrosinase inhibitors is still important for the development of new cosmeceuticals agents [
38].
Elastase is a proteinase enzyme capable of degrading elastin, the main component of the elastic fibers responsible for the mechanical properties of connective tissue [
39,
40]. In the skin, the elastic fibers, together with the collagenous fibers, form a network under the epidermis and are responsible for skin elasticity. Therefore, elastase, by breaking down elastin, decreases the skin elasticity and increases skin aging, resulting in visible skin changes like wrinkles. So, elastase inhibitors are important cosmeceuticals agents by preventing loss of skin elasticity.
Proteasomes are protein complexes containing a common core, referred to as the 20S proteasome, that degrade unneeded or damaged proteins by proteolysis, this process is often called the ubiquitin-proteasome pathway [
41]. This ubiquitin-proteasome pathway may be critical in cell cycle regulation, and due to these multiple functions, proteasome malfunctions are involved in a certain number of pathologies, in particular those linked to aging such as cancers and neurodegenerative diseases. Therefore, inhibitors of proteasome are highly sought for the treatment of these diseases [
42].
Five compounds (
1;
3–
6) inhibited proteasome activity and two compounds (
5–
6) inhibited CDK7 and Fyn kinase (
Table 3). Osirisyne B (
5) was the most active compound with IC
50 on FynB kinase, CDK7 kinase, and proteasome inhibition of 18.44 µM, 9.13 µM, and 0.26 µM, respectively.
The activity of these compounds seems to be related to the number of oxygen atoms. Osirisynes B (5) and E (6) are the two compounds with the lowest number of oxygens and presented inhibition activities on three enzymes, FYN kinase, proteasome, and CDK7. Furthermore, comparison between osirisyne B (5) and osirisyne E (6) highlighted the increase of the inhibition activity with the presence of a ketone instead of a hydroxyl function. In our continuing search for bioactive metabolites, studies on this family of compounds from sponges are still going on and a structure–activity relationship (SAR) study will be made when more similar compounds are isolated and tested.