Insights into Cytotoxic Behavior of Lepadins and Structure Elucidation of the New Alkaloid Lepadin L from the Mediterranean Ascidian Clavelina lepadiformis

The chemical investigation of the Mediterranean ascidian Clavelina lepadiformis has led to the isolation of a new lepadin, named lepadin L, and two known metabolites belonging to the same family, lepadins A and B. The planar structure and relative configuration of the decahydroquinoline ring of lepadin L were established both by means of HR-ESIMS and by a detailed as extensive analysis of 1D and 2D NMR spectra. Moreover, microscale derivatization of the new alkaloid lepadin L was performed to assess the relative configuration of the functionalized alkyl side chain. Lepadins A, B, and L were tested for their cytotoxic activity on a panel of cancer cell lines (human melanoma [A375], human breast [MDA-MB-468], human colon adenocarcinoma [HT29], human colorectal carcinoma [HCT116], and mouse myoblast [C2C12]). Interestingly, a deeper investigation into the mechanism of action of the most cytotoxic metabolite, lepadin A, on the A375 cells has highlighted its ability to induce a strongly inhibition of cell migration, G2/M phase cell cycle arrest and a dose-dependent decrease of cell clonogenity, suggesting that it is able to impair self-renewing capacity of A375 cells.


Introduction
The marine environment represents an extraordinary source of new and structurally complex bioactive metabolites characterized by a wide and incredible array of chemical structures showing antiallergic, anti-atherosclerotic, antibacterial, anticancer, anticoagulant, and antidiabetic to antifungal, antihyperlipidemic, antihypertensive, anti-inflammatory, and antioxidant to antiviral, cardioprotective, immunoadjuvant, and hypocholesterolemic activity [1,2]. Ascidians represent highly preferential drug resources in the ocean, being prolific producers of bioactive marine natural products, due to the strong immune defensive system and their associated symbiotic microbes [3][4][5]. In particular, the unique molecules isolated from ascidians have contributed with their considerable structural diversity, that remain a key inspirational factor in the search for new therapeutics, to a wealth of active compounds. Typically, most ascidian compounds contain one or two heteroatoms, most frequently nitrogen and/or oxygen and occasionally sulfur, that are presumably derived from amino acids and many of these are biologically active [6,7]. The lepadin family, comprising, until now, eleven decahydroquinoline members, was identified from ascidians belonging to the genera Clavelina, Didemnum, and Aplidium as well as their predators [8][9][10][11][12]. These secondary metabolites are characterized by 5-alkyl-3-hydroxydehydroquinoline nucleus with a diversified array of relative stereochemical relationships at C-2, C-3, C-4a, C-5, and  (Table 1) showed typical resonances of a decahydroquino The 13 C NMR spectrum (Table 1) displayed signals for all 20 carbons, including carbonyl carbon at δC 171.8. The general features of the proton spectrum of lepa clearly resembled those of other lepadins, as illustrated by (i) the large band of al methylene protons at δH 1.30, (ii) the diagnostic resonances of perhydroquinoline tem (H-2, H-3, H-4a, H-8a), (iii) the typical oxymethine proton at δH 5.18, and ( ondary aliphatic methyl group (δH 1.33, d, J = 6.0 Hz). The analysis of COSY, HS HMBC 2D NMR spectra of 3 led to the assignment of all protonic and carbon re through the key recorded homonuclear and heteronuclear correlations (Table 1 NMR spectra allowed to highlight a close correlation between the known lepa and the novel lepadin L (3). Indeed, compounds 1 and 3 showed similar featur the presence of the same decahydroquinoline core and the 2-hydroxyacetat linked at C3. The most significant differences in the 13 C NMR spectra of lepadin were confined to the signals of the substituent at C-5 (C1′-C8′). Similarly, the spectra of these two compounds mainly differed for the resonances of two oxy of an acyclic 1,2 diol (δH 3.45 and δH 3.85). These data together with information by analysis of 2D NMR data allowed the sequential assignment of all the proto alkyl chain at C-5; indeed, 1 H-1 H spin system could be further extended to the s by following the chain of proton couplings from H-5 through to H-8′. The COSY In the frame of our research project aimed at the detailed chemical characterization of marine secondary metabolites from invertebrates [6,18,19], herein, we describe the results provided by the analysis of the metabolic content of the Mediterranean ascidian Clavelina lepadiformis. This analysis led to the isolation of the already known lepadins A (1) and B (2) [9,10] along with a new alkaloid belonging to the same family, named lepadin L (3, Figure 1). We have then explored their potential effects on viability and proliferation on different cancer cell lines, namely A375, MDA-MB-468, HT29, HCT116 cells, and C2C12 myoblasts; particularly, comparison of the pharmacological properties of compounds 1-3 allowed us to perform an assessment of simple structure-activity relationships which could be related to the different cytotoxicity.

Isolation of Lepadins A, B, and L and Structure Elucidation of Lepadin L
Several specimens of the colonial ascidian C. lepadiformis, collected in the bay of Pozzuoli (Naples) in 2018, were exhaustively extracted with methanol and, then, with chloroform. The concentrated extracts were combined, concentrated under reduced pressure, and partitioned between ethyl acetate and butanol. This procedure allowed to obtain three organic extracts with different polarity in which a primarily separation of compounds could be observed. Medium pressure flash chromatography of the ethyl acetate soluble material on a silica gel column, followed by repeated RP-18 high-performance liquid chromatography (HPLC) separations of the fractions eluted with EtOAc:MeOH 7:3, yielded the new lepadin L (3), together with the known compounds, lepadins A (1) and B (2) in the pure form. The known compounds 1 and 2 were readily identified by comparison of their physical and spectral data with those reported in the literature [9,10]. Despite the availability of only minute amounts of the new natural alkaloid 3, its relative configuration  (Table 1) showed typical resonances of a decahydroquinoline core. The 13 C NMR spectrum (Table 1) displayed signals for all 20 carbons, including an ester carbonyl carbon at δ C 171.8. The general features of the proton spectrum of lepadin L (3) clearly resembled those of other lepadins, as illustrated by (i) the large band of alkyl chain methylene protons at δ H 1.30, (ii) the diagnostic resonances of perhydroquinoline ring system (H-2, H-3, H-4a, H-8a), (iii) the typical oxymethine proton at δ H 5.18, and (iv) a secondary aliphatic methyl group (δ H 1.33, d, J = 6.0 Hz). The analysis of COSY, HSQC, and HMBC 2D NMR spectra of 3 led to the assignment of all protonic and carbon resonances through the key recorded homonuclear and heteronuclear correlations ( Table 1). The 2D NMR spectra allowed to highlight a close correlation between the known lepadin A (1) and the novel lepadin L (3). Indeed, compounds 1 and 3 showed similar features due to the presence of the same decahydroquinoline core and the 2-hydroxyacetate moiety linked at C3. The most significant differences in the 13 C NMR spectra of lepadin L and A were confined to the signals of the substituent at C-5 (C1 -C8 ). Similarly, the 1 H NMR spectra of these two compounds mainly differed for the resonances of two oxymethines of an acyclic 1,2 diol (δ H 3.45 and δ H 3.85). These data together with information provided by analysis of 2D NMR data allowed the sequential assignment of all the protons of the alkyl chain at C-5; indeed, 1 H-1 H spin system could be further extended to the side chain by following the chain of proton couplings from H-5 through to H-8 . The COSY spectrum evidenced H-5 to be coupled to a de-shielded methine proton resonating at δ H 5.46 (H-1 , dd, J = 15.8 and 9.3 Hz), which is in turn coupled to a methine at δ H 5.63 (H-2 , dd, J = 15.8 and 10.7 Hz). Furthermore, the latter proton was correlated to the oxymethine proton at δ H 3.85 (H-3 , dd, J = 16.0 and 3.0 Hz) and this with another oxymethine proton resonating at δ H 3.45 (H-4 , m), allowing us to locate the hydroxyl groups at C-3 , C-4 . Finally, the sequence was extended to the H-8 , taking advantage of HSQC and HMBC information.
The relative stereochemistry of lepadin L was established by a combination of 2D NOESY ( Figure 2) experiment and the comprehensive analysis of the J H-H coupling constant values. A diagnostic NOE cross peak between 1 H NMR signals for H-4a and H-8a indicated a cisoid ring fusion of decahydroquinoline unit for 3.
The cis-fused azadecalin ring system can exist in two chair-chair conformers [9,12]. The bicyclic ring conformation of 3 was established from NOESY experiments ( Figure 1). In detail, the NOESY spectrum (Table 1 and Figure 2) of 3 contained key cross-peaks between signals for the paired 1,3-syn diaxial protons, H-2 ax -H-4 ax and H-4a-H-6 ax . These data are in agreement with the most stable conformation of cis-decahydroquinoline core in which the C-8a-N bond is situated axially with respect to the cyclohexane chair and with the side chain attached at C-5 equatorially oriented. It should be noted that this is also the favored conformation of cis-azadecalin ring systems in other lepadins.
The NOESY cross peaks between H-2 ax and H-8a showed these hydrogens adopted a diaxial disposition. The small coupling constant ( 3 J = 2.4 Hz) between H-2 and H-3 revealed that they adopted a syn configuration and H-3 had to have an equatorial position, and the hydroxyacetyl group axially situated. By considering the information above, the protons H-2, H-3, H-4a, and H-8a were determined to be all syn-configured. The relative stereochemistry of lepadin L was established by a combination of 2D NOESY ( Figure 2) experiment and the comprehensive analysis of the JH-H coupling constant values. A diagnostic NOE cross peak between 1 H NMR signals for H-4a and H-8a indicated a cisoid ring fusion of decahydroquinoline unit for 3.
The cis-fused azadecalin ring system can exist in two chair-chair conformers [9,12]. The bicyclic ring conformation of 3 was established from NOESY experiments ( Figure 1). In detail, the NOESY spectrum (Table 1 and Figure 2) of 3 contained key cross-peaks between signals for the paired 1,3-syn diaxial protons, H-2ax-H-4ax and H-4a-H-6ax. These data are in agreement with the most stable conformation of cis-decahydroquinoline core in which the C-8a-N bond is situated axially with respect to the cyclohexane chair and with the side chain attached at C-5 equatorially oriented. It should be noted that this is also the favored conformation of cis-azadecalin ring systems in other lepadins.
The NOESY cross peaks between H-2ax and H-8a showed these hydrogens adopted a diaxial disposition. The small coupling constant ( 3 J = 2.4 Hz) between H-2 and H-3 revealed that they adopted a syn configuration and H-3 had to have an equatorial position, and the hydroxyacetyl group axially situated. By considering the information above, the protons H-2, H-3, H-4a, and H-8a were determined to be all syn-configured.
Moreover, scalar coupling of 15.8 Hz observed between H-1′ and H-2′ showed that the double bond had the E geometry.
Moreover, scalar coupling of 15.8 Hz observed between H-1 and H-2 showed that the double bond had the E geometry.
To establish the relative configuration at C-3 /C-4 , we prepared the acetonide derivative 4 by treating lepadin L (3) with 2,2-dimethoxypropane and a catalytic amount of Dowex Mar. Drugs 2022, 20, 65 5 of 13 50W-X8 (Scheme 1). As is well known, the difference in the chemical shifts of the methyl groups in the dioxolane ring is always greater for the cis-acetonide (∆δ 0.12-0.14), coming from the 1,2-anti isomer, when compared to the trans-acetonide (∆δ 0.01-0.04), coming from the 1,2-syn isomer [20]. The observed ∆δ value of 0.11 ppm between the two methyl groups in 4 indicated the cis configuration for the dioxolane ring and, subsequently, the anti configuration of the 1,2-diol in lepadin L (3). To establish the relative configuration at C-3′/C-4′, we prepared the acetonid ative 4 by treating lepadin L (3) with 2,2-dimethoxypropane and a catalytic am Dowex 50W-X8 (Scheme 1). As is well known, the difference in the chemical shif methyl groups in the dioxolane ring is always greater for the cis-acetonide (Δδ 0.1 coming from the 1,2-anti isomer, when compared to the trans-acetonide (Δδ 0.0 coming from the 1,2-syn isomer [20]. The observed Δδ value of 0.11 ppm between methyl groups in 4 indicated the cis configuration for the dioxolane ring and quently, the anti configuration of the 1,2-diol in lepadin L (3).

In Vitro Evaluation of Cytotoxic Effects of Lepadins 1-3 on Different Cancer Cell Li
The   Figure 3 where the cell survival percentages are graphically compared. As it can be observed, lepadin A was shown to be the most cytotoxic agent in the series; it exerted a strong cytotoxic effect toward A375, HCT116 human cells, and C2C12 mouse myoblasts ( Figure 3, panels A-C), whereas it appeared moderately active towards HT29 cells, and completely inactive towards MDA-MB-468 cells (Figure 3, panels D and E). In contrast, lepadins B and L showed weak or no activity against the tested cancer cell lines ( Figure 3, panels A-E).

Further Insights into the Cytotoxic Effects of Lepadin A on A375 Cells
In recent years, the use of target therapy and immunotherapy drugs has made it possible to improve both the quality and duration of life of numerous cancer patients suffering from aggressive and advanced forms of cancer such as metastatic melanoma [21]. Despite the undoubted progress made, most of patients treated with these innovative drugs show a poor prognosis due to emergence of resistant clones of cancer cells that proliferate regardless of treatments. This finding suggests that the identification of new anticancer drugs to complement current anti-melanoma therapies represent one the greatest challenges in the coming years. For this reason, we decided to analyze the impact of lepadin A on A375 cells and the first experiment was that to determine the lethal dose 50 (IC 50 ). To this aim, A375 cells were incubated in the presence of increasing concentrations of compound 1; again, 5-FU has been used in this assay as positive control. The results showed by this assay confirmed the strong cytotoxicity exerted by lepadin A; accordingly, we have observed that compound 1 decreased cell survival in a dose-dependent manner (Figure 4), showing an IC 50 value of 45.5 ± 4.0 µM, comparable to that obtained for the largely used anticancer chemotherapy drug 5-FU (IC 50 = 22.9 ± 4.0 µM). Then, to evaluate the underlying mechanism of the highly significant effect of lepadin A on A375 cells viability, we have performed further in vitro experiments, as described below.

Further Insights into the Cytotoxic Effects of Lepadin A on A375 Cells
In recent years, the use of target therapy and immunotherapy drugs has made it sible to improve both the quality and duration of life of numerous cancer patients su ing from aggressive and advanced forms of cancer such as metastatic melanoma [21] spite the undoubted progress made, most of patients treated with these innovative d show a poor prognosis due to emergence of resistant clones of cancer cells that prolife regardless of treatments. This finding suggests that the identification of new antica drugs to complement current anti-melanoma therapies represent one the greatest c lenges in the coming years. For this reason, we decided to analyze the impact of lep A on A375 cells and the first experiment was that to determine the lethal dose 50 (IC50 this aim, A375 cells were incubated in the presence of increasing concentrations of c pound 1; again, 5-FU has been used in this assay as positive control. The results sho by this assay confirmed the strong cytotoxicity exerted by lepadin A; accordingly, we h

Impact on Cell Migration
In the light of the interesting effects exerted by lepadin A on A375 cell line, we have evaluated the impact of this alkaloid on migration of A375 cells through a wound-healing assay. The wound-healing assay is a simple and inexpensive method to study directional cell migration in vitro, mimicking the cell migration during wound healing in vivo [22]. In our study, the plates containing confluent A375 cells have been scratched with a tip to generate a wound and then incubated for 16 h in the presence of complete medium supplemented or not with 25 µM of lepadin A. The images have been obtained using a contrast phase microscopy; the number of migrated cells has been determined by counting the cells moved into the wound. As shown in Figure 5, lepadin A decreases the cover rate of the scratch width and strongly inhibits cell migration.

Impact on Cell Migration
In the light of the interesting effects exerted by lepadin A on A375 cell line, we have evaluated the impact of this alkaloid on migration of A375 cells through a wound-healing assay. The wound-healing assay is a simple and inexpensive method to study directional cell migration in vitro, mimicking the cell migration during wound healing in vivo [22]. In our study, the plates containing confluent A375 cells have been scratched with a tip to generate a wound and then incubated for 16 h in the presence of complete medium supplemented or not with 25 µM of lepadin A. The images have been obtained using a contrast phase microscopy; the number of migrated cells has been determined by counting the cells moved into the wound. As shown in Figure 5, lepadin A decreases the cover rate of the scratch width and strongly inhibits cell migration.

Impact on Cell Migration
In the light of the interesting effects exerted by lepadin A on A375 cell line, we have evaluated the impact of this alkaloid on migration of A375 cells through a wound-healing assay. The wound-healing assay is a simple and inexpensive method to study directional cell migration in vitro, mimicking the cell migration during wound healing in vivo [22]. In our study, the plates containing confluent A375 cells have been scratched with a tip to generate a wound and then incubated for 16 h in the presence of complete medium supplemented or not with 25 µM of lepadin A. The images have been obtained using a contrast phase microscopy; the number of migrated cells has been determined by counting the cells moved into the wound. As shown in Figure 5, lepadin A decreases the cover rate of the scratch width and strongly inhibits cell migration.

Cell Cycle Analysis
Then, we have performed the cell cycle analysis by flow cytometer based on propidium iodide (PI) staining ( Figure 6). A375 cells were incubated in the presence of 10 µM 5-* Figure 5. Wound-healing assay. The images were obtained using a contrast phase microscopy. Scale bar, 200 µm. The experiment was carried out in triplicate. For each well, various images derived from different regions were acquired. The data are presented as the mean ± SD (n = 3), * p < 0.005.

Cell Cycle Analysis
Then, we have performed the cell cycle analysis by flow cytometer based on propidium iodide (PI) staining ( Figure 6). A375 cells were incubated in the presence of 10 µM 5-FU, or 40 µM of lepadin A and, after 24 h, cells were washed and incubated in the presence of propidium iodide. The percentage of cells in the cell cycle phases was determined using a flow cytometer. The obtained results (Figure 7) showed that lepadin A induced G2/M phase cell cycle arrest in A375 cells at 40 µM. Figure 6A,B showed that the cell number in both S and G2/M phase increased in A375 lepadin A-treated cells suggesting that compound 1 interferes with S phase and leads to G2/M phase cell cycle arrest. FU, or 40 µM of lepadin A and, after 24 h, cells were washed and incubated in the presence of propidium iodide. The percentage of cells in the cell cycle phases was determined using a flow cytometer. The obtained results (Figure 7) showed that lepadin A induced G2/M phase cell cycle arrest in A375 cells at 40 µM. Figure 6A,B showed that the cell number in both S and G2/M phase increased in A375 lepadin A-treated cells suggesting that compound 1 interferes with S phase and leads to G2/M phase cell cycle arrest.

Clonogenic Assay
It is known that the self-renewal process of normal stem cells is deregulated in cancer stem cells resulting in a continuous expansion of self-renewing cancer cells and tumor formation. Therefore, agents targeting the defective self-renewal pathways in cancer cells might lead to improved outcomes in the treatment of cancer [23]. Thus, we have performed a clonogenic assay with the aim to investigate the ability of lepadin A to impair self-renewing capacity of melanoma A375 cells. To this purpose, growing A375 cells were incubated with increasing concentration (0, 10, 20, and 40 µM) of the natural alkaloid 1 for 24 h (Figure 8), and then detached, counted, and seeded in new wells (500 cells for each well). After 15 days, plates were stained with crystal violet to determine the number of colonies. Plating efficiency and surviving factor have been calculated as previously described [24]. These results demonstrate that lepadin A causes a dose-dependent decrease of propidium iodide. The percentage of cells in the cell cycle phases was determined using a flow cytometer. The obtained results (Figure 7) showed that lepadin A induced G2/M phase cell cycle arrest in A375 cells at 40 µM. Figure 6A,B showed that the cell number in both S and G2/M phase increased in A375 lepadin A-treated cells suggesting that compound 1 interferes with S phase and leads to G2/M phase cell cycle arrest.

Clonogenic Assay
It is known that the self-renewal process of normal stem cells is deregulated in cancer stem cells resulting in a continuous expansion of self-renewing cancer cells and tumor formation. Therefore, agents targeting the defective self-renewal pathways in cancer cells might lead to improved outcomes in the treatment of cancer [23]. Thus, we have performed a clonogenic assay with the aim to investigate the ability of lepadin A to impair self-renewing capacity of melanoma A375 cells. To this purpose, growing A375 cells were incubated with increasing concentration (0, 10, 20, and 40 µM) of the natural alkaloid 1 for 24 h (Figure 8), and then detached, counted, and seeded in new wells (500 cells for each well). After 15 days, plates were stained with crystal violet to determine the number of colonies. Plating efficiency and surviving factor have been calculated as previously described [24]. These results demonstrate that lepadin A causes a dose-dependent decrease

Clonogenic Assay
It is known that the self-renewal process of normal stem cells is deregulated in cancer stem cells resulting in a continuous expansion of self-renewing cancer cells and tumor formation. Therefore, agents targeting the defective self-renewal pathways in cancer cells might lead to improved outcomes in the treatment of cancer [23]. Thus, we have performed a clonogenic assay with the aim to investigate the ability of lepadin A to impair selfrenewing capacity of melanoma A375 cells. To this purpose, growing A375 cells were incubated with increasing concentration (0, 10, 20, and 40 µM) of the natural alkaloid 1 for 24 h (Figure 8), and then detached, counted, and seeded in new wells (500 cells for each well). After 15 days, plates were stained with crystal violet to determine the number of colonies. Plating efficiency and surviving factor have been calculated as previously described [24]. These results demonstrate that lepadin A causes a dose-dependent decrease of cell clonogenity, suggesting that it is able to impair self-renewing capacity of A375 cells (Figure 8).
Herein, the whole reported data showed that lepadin A is able to impair viability and inhibit cell migration as well as self-renewal ability of A375 cells. Moreover, we found that A375 cells treated with lepadin A were significantly segregated into G2/M cell cycle phase, suggesting that it could interfere with tubulin assembling during mitotic spindle formation. Although this study is in its infancy in determining the mechanism of action of lepadin A, our data suggest that such kind of molecules could be used as lead compounds to generate a new class of microtubule-binding agents able to block proliferation of cancer cells. Further studies are ongoing to clarify the lepadin A mechanism of action as well as the activity of other lepadins on different kind of cancer cell lines.
Mar. Drugs 2022, 20, x 9 of 13 of cell clonogenity, suggesting that it is able to impair self-renewing capacity of A375 cells (Figure 8). Herein, the whole reported data showed that lepadin A is able to impair viability and inhibit cell migration as well as self-renewal ability of A375 cells. Moreover, we found that A375 cells treated with lepadin A were significantly segregated into G2/M cell cycle phase, suggesting that it could interfere with tubulin assembling during mitotic spindle formation. Although this study is in its infancy in determining the mechanism of action of lepadin A, our data suggest that such kind of molecules could be used as lead compounds to generate a new class of microtubule-binding agents able to block proliferation of cancer cells. Further studies are ongoing to clarify the lepadin A mechanism of action as well as the activity of other lepadins on different kind of cancer cell lines.

General Experimental Procedures
High-resolution MS (positive mode) was performed on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo-Fisher, San Josè, CA, USA). The spectra were recorded by infusion into the ESI source using MeOH as solvent. 1 H NMR and 2D NMR experiments were carried out at 700 MHz on a Bruker Avance Neo spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA); chemical shifts were referenced to the residual solvent signal (CD3OD: δH = 3.31, δC = 49.0). Two and three bond 1 H-13 C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3 J of 8 Hz. Through-space 1 H connectivities were evidenced using a NOESY experiment with a mixing time of 300 ms. A Jasco P-2000 polarimeter (Jasco Europe s.r.l., Cremella, Italy) at the sodium D line was used to measure optical rotations. High performance liquid chromatography (HPLC) separation was achieved on a Knauer K-501 apparatus equipped with a Knauer K-2301 RI detector (LabService Analytica s.r.l., Anzola dell'Emilia, Italy).

Collection, Extraction, and Isolation
Specimens of C. lepadiformis were collected in the bay of Pozzuoli (Napoli, Italy 40°49′39.61″ N 14°09′11.56″ E) in the spring of 2018 at a depth of approximatively 10 m, by

General Experimental Procedures
High-resolution MS (positive mode) was performed on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo-Fisher, San Josè, CA, USA). The spectra were recorded by infusion into the ESI source using MeOH as solvent. 1 H NMR and 2D NMR experiments were carried out at 700 MHz on a Bruker Avance Neo spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA); chemical shifts were referenced to the residual solvent signal (CD 3 OD: δ H = 3.31, δ C = 49.0). Two and three bond 1 H-13 C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3 J of 8 Hz. Throughspace 1 H connectivities were evidenced using a NOESY experiment with a mixing time of 300 ms. A Jasco P-2000 polarimeter (Jasco Europe s.r.l., Cremella, Italy) at the sodium D line was used to measure optical rotations. High performance liquid chromatography (HPLC) separation was achieved on a Knauer K-501 apparatus equipped with a Knauer K-2301 RI detector (LabService Analytica s.r.l., Anzola dell'Emilia, Italy).

Collection, Extraction, and Isolation
Specimens of C. lepadiformis were collected in the bay of Pozzuoli (Napoli, Italy 40 • 49 39.61 N 14 • 09 11.56 E) in the spring of 2018 at a depth of approximatively 10 m, by hand, using scuba. They were frozen immediately after collection and stored at −20 • C until extraction. The taxonomical identification of the organisms was carried out by Mr. Arturo Facente. A voucher specimen is deposited at the Department of Pharmacy, University of Naples "Federico II", Naples, Italy.

Synthesis of Lepadin L Acetonide (4)
A solution of lepadin L (3, 0.5 mg) in 2,2-dimethoxypropane (500 µL) was treated with Dowex 50W-X8 (H + form, 20 mg) in catalytic amount, and the mixture was stirred for 2 h at room temperature. After this time, the resin was removed by filtration and the solvent from the filtrate was dried in vacuo affording compound 4 (0.5 mg). Moreover, traces of the related derivative endowed with a hydroxyl group at C-3 instead of the 2-hydroxyacetate ester functionality was observed.

Cell Culture
The human cell lines (A375, MDA-MB-468, HT29, HCT116, and C2C12) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in 1640 medium supplemented with 10% FBS and a 1% P/S solution. LS174T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) foetal bovine serum (FBS; Gibco, Waltham, MA, USA) and a 1% (v/v) penicillin-streptomycin (P/S) solution (Gibco). HCT116 and HT29 cells were cultured in McCoy's 5 • medium supplemented with 10% FBS and a 1% P/S solution. SW480 and SW620 cells were cultured in Leibovitz's L-15 medium supplemented with 10% FBS and a 1% P/S solution. All the cells were cultured at 37 • C in a humidified atmosphere with 5% CO 2 .

Cell Viability Assay
For the initial screening, cancer cells (1.5 × 10 4 /well) were seeded into 24-well plates and treated with lepadins A, B, and L (1-3) or 100 mM of 5FU for 24 h. Subsequently, medium was removed from well, cells were washed with PBS and then incubated at 37 • C in the presence of complete cell growth medium containing 0.5 mg/mL of MTT reagent. After 1 h, the medium was removed, the wells were washed with PBS before adding DMSO to lyse the cells and dissolve the formazan crystals. Cell viability was assessed by determination of the optical density (OD) at 595 nm using a microplate reader (550 Microplate Reader, Bio Rad CA, USA).

Clonogenic Assay
Melanoma cells were incubated with increasing concentration of lepadin A for 24 h and then tripsinized. Cells were counted and seeded on P35 dishes (500 cells for each plate). After 15 days, plates were incubated for 5 min at 37 • C in the presence of a crystal violet solution (0.2% w/v dissolved in a solution containing 20% methanol) to stain the colonies grown in each plate. The influence of the compounds on the clonogenicity was expressed as percent of control according to the formula: number of colonies generated after treatment with the compounds/number of colonies in control with the vehicle.

Cell Cycle Analysis
A375 cells (60.000 cells) were plated on P35 wells and incubated in the presence of 40 µM lepadins A, B, and L, or 10 µM of 5-FU for 24 h. Then, cell plates were washed with PBS and incubated in the presence of 50 µg/mL of propidium iodide (PI) dissolved in 0.1% Na-citrate, containing 0.1% NP40. Cell plates were stored in the dark for 15 min to allow cell lysis and the staining of nuclei. After this time, cell nuclei were collected and analyzed using a flow cytometer (FACS CANTO, BD Bioscience Franklin Lakes, NJ, USA). The distribution of cells into cycle phases was calculated using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Statistical Analysis
The results were expressed as mean ± SD. The differences between the experimental and control groups were compared using one-way ANOVA followed by Dunnett's multiple comparisons test. All statistical analyses were performed using OriginPro 8.0 2021 software. The p value < 0.05 was considered statistically significant. * p < 0.05; ** p < 0.01.

Conclusions
An exhaustive examination of the chemical constituents of the Mediterranean ascidian Clavelina lepadiformis allowed us to expand the knowledge on the alkaloid composition of this marine organism with the addition of a new decahydroquinoline-based compound named lepadin L (3). The three isolated alkaloids, the known lepadins A and B (1 and 2), as well as the new lepadin L (3), were evaluated for their antiproliferative activity against a panel of cancer cell lines (A375, MDA-MB-468, HT29, HCT116 cells, and C2C12 myoblasts). Our pharmacological studies demonstrated that lepadin A behaved as strong cytotoxic agent toward A375, HCT116 cells, and C2C12 myoblasts, whereas lepadins B and L showed weak or no activity against all the tested cancer cell lines. Lepadin A strongly inhibits migration of A375 cells and interferes with S phase inducing G2/M phase cell cycle arrest in this cell line. Furthermore, the result of clonogenity assay highlighted compound 1 inhibits self-renewal capability of A375 cells. Together, these results suggested that lepadin A behaves as an antitumoral agent able to block cells cycle, cell migration, cell renewal ability, and to induce cell death. These findings agree with previous results showing that lepadin A, and to lesser extent lepadin B, was cytotoxic against several tumoral cell lines [10]. However, data we reported give important news about the antitumoral activity of these natural compounds and pave the way to use such molecules as lead compound for development of new potent antitumoral drugs. Further studies will be performed in the coming years in order to identify physiological targets of lepadin A, to deeply investigate its mechanism of action and in vivo effectiveness. The isolation of the new compound lepadin L, a metabolite closely related to the well-known lepadin A, as well as the results of our in-depth studies have allowed us to highlight important structural requirements for the cytotoxic activity of these natural compounds. In particular, it has been highlighted that the presence of two hydroxyl groups instead of a double bond on the substituent at C-5 in compound 3 causes a dramatic decrease of cytotoxicity and that, therefore, the polarity of these compounds is crucial for the fulfillment of the cytotoxic activity. These results further confirm that marine ascidians constitute an immense source of chemodiversity that can be beneficially exploited to discover new chemical entities for drug development. Funding: This research was funded by a grant from Regione Campania-POR Campania FESR 2014/2020 "Combattere la resistenza tumorale: piattaforma integrata multidisciplinare per un approccio tecnologico innovativo alle oncoterapie-Campania Oncoterapie" (Project Nr B61G18000470007) and a grant from Department of Pharmacy, University of Naples Federico II, Bando Contributo alla Ricerca Anno 2021 "-Targeting PTP1B and/or AR enzymes with marine-derived small molecules and foodstuffs constituents as natural inhibitor in search for novel therapeutics against type 2 diabetes mellitus (T2DM)" (project acronym PTP1B-AR EnNatIn). This work was also funded in part by University of Florence ("Fondi di Ateneo 2021").