Cytotoxic Alkylynols of the Sponge Cribrochalina vasculum: Structure, Synthetic Analogs and SAR Studies

A series of twenty-three linear and branched chain mono acetylene lipids were isolated from the Caribbean Sea sponge Cribrochalina vasculum. Seventeen of the compounds, 1–17, are new, while six, 18–23, were previously characterized from the same sponge. Some of the new acetylene-3-hydroxy alkanes 1, 6, 7, 8, 10 were tested for selective cytotoxicity in non-small cell lung carcinoma (NSCLC) cells over WI-38 normal diploid lung fibroblasts. Compound 7, presented clear tumor selective activity while, 1 and 8, showed selectivity at lower doses and 6 and 10, were not active towards NSCLC cells at all. The earlier reported selective cytotoxicity of some acetylene-3-hydroxy alkanes (scal-18 and 23), in NSCLC cells and/or other tumor cell types were also confirmed for 19, 20 and 22. To further study the structure activity relationships (SAR) of this group of compounds, we synthesized several derivatives of acetylene-3-hydroxy alkanes, rac-18, scal-S-18, R-18, rac-27, rac-32, R-32, S-32, rac-33, rac-41, rac-42, rac-43, rac-45, rac-48 and rac-49, along with other 3-substituted derivatives, rac-35, rac-36, rac-37, rac-38, rac-39 and rac-40, and assessed their cytotoxic activity against NSCLC cells and diploid fibroblasts. SAR studies revealed that the alcohol moiety at position 3 and its absolute R configuration both were essential for the tumor cell line selective activity while for its cytotoxic magnitude the alkyl chain length and branching were of less significance.


Results and Discussion
The crude extract of freeze-dried Cribrochalina vasculum material was separated on a reversed phase flash column, followed by Sephadex LH-20 gel filtration and repeated reversed phase HPLC separations guided by cytotoxicity bioassay to afford twenty-three pure compounds (1-23, Figure 1). We earlier reported on cytotoxicity of 18 (i.e., scal-18) and 23, in NSCLC cells which involved action on the insulin like growth factor receptor (IGF-1R) and inhibition of downstream proliferative signaling circuits [17,18]. The compounds could be grouped into five clusters based on the structure of the acetylene end of the molecules (substructures a-e in Figure 2) while the rest of their chains varied in length (19 to 24 carbons), saturation (15Z), point of methylation (at carbons 13, 14, 18, 19, and 21), and in a single case also in presenting a cyclopropane ring (8). The alkyl-4E-ene-1-yn-3-ols, compounds, 1, 2, 4, 5, 8, 9, 18, 19, 20, 21, 22 and 23 ( Figure 1) were isolated as scalemic mixtures based on a comparison of their optical rotations with those of 18 and 23, for which the enantiomeric excess at C-3 was determined by the modified Mosher method [19]. The alkyl-4E-ene-1-yn-3-ol, 12 and the alkyl-3Z-ne-1-yn-5-ols, 13-16 (zero optical rotation), were isolated as racemic mixtures and the alkyl-1-yn-3-ols 7 and 11 were isolated as pure 3R-enantiomers, based on the results of the modified Mosher method [19].
(3R)-18-Methylnonadec-(4E)-en-1-yn-3-ol (1) presented an HRAPGCMS pseudomolecular ion, [M + H] + , at m/z 293.2848 consistent with the molecular formula C 20 H 36 O and three degrees of unsaturation. The NMR data (Table S1 in Supporting Material) indicated the presence of a terminal acetylene, a secondary alcohol, a double bond, an aliphatic chain and terminal isopropyl moieties. Its fragments, a (C-1 to C-7) and j (C-16 to C-20) were deduced based on COSY, HSQC and HMBC correlations ( Figure 2). The two fragments are connected through an aliphatic chain whose length is deduced from the molecular formula of 1. The E-configuration of the 4,5-double bond was inferred from the 15.2 Hz coupling constant of H-4 and H-5. The 3R-absolute configuration of the chiral center of 1 was based on its negative optical rotation, which was similar to those of compounds 18 and 23 [17], for which the 3R-configurations were obtained by the modified Mosher method [19] (Figure 3, 55 and 36 enantiomeric excesses, respectively). Based on these arguments the structure of compound 1 was determined to be a scalemic mixture in which (3R)-18-methylnonadec-(4E)-en-1-yn-3-ol was the major enantiomer.  (Table S2) revealed similarity to that of 1 but indicated that the methyl branching was in the middle of the chain in contrast to 1 where it localized to the end of the chain. Fragments a, f (C-12 to C-16) and i (C-18 to C- 19), were assigned based on COSY, HSQC and HMBC correlations ( Figure 2). The three fragments were connected through the remaining methylene groups. The methyl branching site (partial structure f ) could not be ascertained by NMR analysis, due to the overlapping signals in the aliphatic region, but rather deduced from the fragmentation patterns of the EIMS data ( Figure 4) [20]. The EIMS spectrum of compound 2 presented two key fragments at m/z 193 and 221 corresponding to the fragmentation before and after the methyl group ( Figure S14), which along with the absence of the ion at m/z 207, securely suggests that the methyl group is connected to C-14. Based on similar considerations as for 1, the structure of 2 was assigned as a mixture of enantiomers at C-3, where the (3R)-14-methylnonadec-(4E)-en-1-yn-3-ol enantiomer was the major one. The absolute configuration of C-14 was not determined.    (Table S3) with those of 2 (Table S2) revealed that 3, did not present secondary alcohol protons and carbon, but rather a doubly conjugated ketone (δ 177.9, C-3) with a terminal acetylene (δ 3.20 s, H-1; 78.8 CH, C-1; 79.8 C, C-2) and disubstituted E-olefin (δ 6.17 d, J = 16.0 Hz, H-4; 7.23 dt, J = 16.0, 7.0 Hz, H-5; 2.30 q, J = 7.0 H, H 2 -6; 131.4 CH, C-4; 156.0 CH, C-5; 32.7 CH 2 , C-6). The rest of the proton and carbon chemical shifts of 3 were similar to those of 2. COSY, HSQC and HMBC correlations (Table S3) allowed the assignment of fragments b, f and i. The later fragments, of 3, could be assembled by comparison of the NMR and EIMS [20] (Figure S18) of 3 with those of 2. Thus, the structure of 3 was assigned as 14-methylnonadec-(4E)-en-1-yn-3-one. The absolute configuration of C-14 was not determined.
(3R,E)-12-cis-(2-Hexylcyclopropyl)dodec-4-en-1-yn-3-ol (8) [21], in addition to a terminal methyl group, chain of methylenes and a (4E)-ene-3-ol-1-yne spin system similar to that of 18. COSY, HSQC and HMBC correlations (Table S8) established fragments, a (C-1 to C-7), h (C-11 to C-16) and i (C-18 to C-20) but failed to establish their connection. The above presented fragments and carbon chemical shift calculated by ChemDraw ( Figure S50) suggested that the cyclopropane ring is situated between C-13 and C-15 but did not allow conclusive determination of its position. The fragmentations in the EIMS presented single ion fragments down to m/z 201 and multiple (m, m-2, m-4) ions from m/z 191 down to m/z 51 ( Figure S51). The ions at m/z 219 and 201 suggest a loss of a C 6 H 13 radical from the molecular ion or [M − H 2 O] + ion, respectively. The next three multiple ions (each composed of m, m-2, m-4 ions, m/z 181, 189, 187; 177, 175, 173 and 163, 161, 159) were weaker in intensity than the ions on both sides ( Figure S51). This might be explained by three possible bond cleavages of a cyclopropane fused to C-13 and C-14 of 8 ( Figure S53a,b) and derived from its molecular ion or [M − H 2 O] + ion. Based on these arguments the cyclopropane ring was assigned to positions 13 and 14 [22]. The negative sign of the optical rotation of 8 suggests a 3R absolute configuration of its chiral center similar to the above-described compounds. Thus, compound 8 was assigned as (3R,E)-12-cis-(2-hexylcyclopropyl)dodec-4-en-1-yn-3-ol.
(3R)-13-Methylhenicos-(4E)-en-1-yn-3-ol (9) was isolated as a colorless oil that presented an HRAPGCMS pseudomolecular ion, [M + H] + , at m/z 321.3122 corresponding to the molecular formula C 22 H 40 O and three degrees of unsaturation. Its NMR data (Table S9) were similar to that of 2 indicating that the methyl branching was in the middle of the chain. Fragments a, f (C-12 to C-14) and i (C-19 to C-21) were assigned based on COSY, HSQC and HMBC correlations ( Figure 2). The three fragments should thus, be connected through the remaining methylene groups. The methyl branching site (partial structure f ) could not be ascertained by NMR analysis, due to the overlapping signals in the aliphatic region, but could rather be deduced from the fragmentation patterns of the EIMS data [20]. Compound 9 EIMS spectrum presented two sets of consecutive chain fragmentations derived from the molecular ion and water elimination product ion which produced the key fragments at m/z 207 and 179, and 189 and 161 corresponding to the fragmentation before and after the methyl group ( Figure S56). The negative sign of the optical rotation of 9 suggested a 3R absolute configuration of its chiral center similar to the above-described compounds. Thus, compound 9 was assigned as (3R)-13-methylhenicos-(4E)-en-1-yn-3-ol.
Docos-(4E,15Z)-dien-1-yn-3-one (10) was isolated as a colorless oil presenting an EIMS molecular ion at m/z 316 corresponding to the molecular formula C 22 (Table S10) established fragments b (C-1 to C-7), g (C-14 to C-17) and i (C-20 to C-22) but failed to bring about their connection. The 2D NMR correlations (Table S10) did not allow an unequivocal connection between the three fragments. However, LC-MS analysis of the periodate-permanganate oxidation products of 10 ( Figure S64), identified undecadioic acid as the heaviest product thus establishing the position of the Z-double bond between carbons 15 and 16, and the structure of compound 10 as docos-(4E,15Z)-dien-1-yn-3-one.
rac-Tetracos-(4E,15Z)-dien-1-yn-3-ol (12), a colorless oil, presented an EIMS molecular ion, M+, at m/z 346 corresponding to the molecular formula C 24 H 42 O and four degrees of unsaturation. It presented NMR data (Table S12) almost identical (fragments a, C-1 to C-7, g, C-13 to C-17, and i, C-18 to C-20, except of the 24H integration of the huge methylene signal between 1.22 and 1.32 ppm) to those of the known 21 [10,11,15], (which was also isolated in this study), suggesting extra two methylenes in 12 relative to 21. The position of the 15,16-Z-double bond was determined as for 10 by a combination of oxidation and LCMS determination of the undecadioic acid ( Figure S75) [19]. Thus, the structure of compound 12 was determined to be rac-tetracos-(4E,15Z)-dien-1-yn-3-ol.
rac-Icos-(3Z)-en-1-yn-5-ol (13) was isolated as colorless oil which presented a HRAPGC MS pseudomolecular ion, [M + H] + , at m/z 293.2831 corresponding to the molecular formula C 20 H 36 O and three degrees of unsaturation. The NMR spectra of 13 (Table S13) differed from the spectra of 1-12 (Tables S1-S12) in presenting a conjugated acetylene system (fragment d,  (Table S13) and the length of the alkyl chain based on its molecular formula calculated from the HRMS measurements, establishing the structure of 13 as rac-icos-(3Z)-en-1-yn-5-ol.
We have previously demonstrated specific cytotoxic activity for two compounds, scal-18 and 23 in multiple NSCLC cell lines, and other tumor cell lines, e.g., Ovcar and SCLC as well as in diploid lung fibroblasts WI-38 [17]. Here, the NSCLC cell line U-1810 was used alongside WI-38 to reveal the selective cytotoxicity of some of the novel compounds 1, 6-8, 10, 19, 20 and 22 (Table 1, Figure S188). Results showed that the chain length and point of alkylation or additional double-bonds in the chain did not to any large extent affect the activity, while the structure around the acetylene end of the molecule had profound effects on the specific cytotoxic activity in the tested NSCLC cell line. Table 1. Cytotoxicity analyses of the natural products and synthetic compounds. The IC 50 values were estimated from the cell viability curve and is given in µM. a The selectivity of compound in NSCLC cells (U-1810) vs. diploid fibroblasts (WI-38) was calculated at IC 50 if not otherwise indicated.  Figure 2) were equally active in NSCLC U-1810 cells, while those containing fragment b, 6 and 10, were not active and those containing fragments d and e, 13-16 and 17, were not tested, however, compounds containing fragment d have been previously shown to be similarly active to those containing fragment a [20].
Cytotoxicity analyses of the latter derivatives established (Table 1, Figure S188) that the compound with a short chain, rac-31 presents much lower toxicity to the NSCLC U-1810 cell line while, rac-32, rac-33 and especially R-32 were the most potent and tumor selective derivatives. Based on these results, we set to prepare some derivatives of rac-32 for SAR studies. Interestingly, the byproduct, rac-27, which contains an extra ethylene moiety relative to rac-18, presented cytotoxicity similar to that of rac-18 in the NSCLC U-1810 cell line, but its selective cytotoxic activity was much better as rac-27 did not affect the viability of the normal lung fibroblasts WI-38 even when very high concentrations were applied (Table 1, Figure S188).
To facilitate the preparation of the amino and thiol derivatives of rac-32, it was reacted with tosylsulfonyl chloride to afford the tosylate derivative, rac-35, in moderate yield, along with the chloride byproduct, rac-36 (Scheme 3) [24]. Treatment of rac-35 with ammonia in methanol afforded the desired amine derivative rac-37 and the methoxyl-derivative rac-38, both in moderate yields (Scheme 3). The acetylthiol derivative, rac-39, was finally produced by reaction with thioacetic acid [25] and acid hydrolysis produced the relatively unstable thiol derivative rac-40. When compounds rac-35-rac-40 were studied for cytotoxic effects in NSCLC cells (U-1810) and lung fibroblasts (WI-38), none of these synthetic analogs inhibited NSCLC cells at concentrations comparable with those of rac-32 (Table 1). In fact, some of them (rac-36 rac-38, rac-39 and rac-40) did not display any specific cytotoxicity (Table 1).  Five additional derivatives, rac-41, rac-42, rac-45, rac-48 and rac-49 were synthesized (Scheme 4) to study the influence of steric hindrance and restricted rotation next to the acetylene moiety, on the activity of the alkyl acetylene alcohols. The reaction of acetylene magnesium bromide with 2-oxo-hexadecane afforded in good yield the tertiary alcohol rac-41 [23]. Palmitaldehyde was reacted with propylene magnesium bromide to produce rac-42 in good yield. Coupling reaction of 2-(3-phenylmagnesiumbromide)-1,3-dioxolane and 2-(2-phenylmagnesium-bromide)-1,3-dioxolane with 1-bromotetradecane afforded the corresponding alkylphenyl dioxolanes 43 and 46, respectively, which upon acidic hydrolysis afforded the corresponding aldehydes, 44 and 47. Reactions of these aldehydes with ethynyl magnesium bromide resulted in the corresponding metaand ortho-alkylphenyl propargylic alcohol derivatives rac-45 and rac-48 (Scheme 4). Finally, benzaldehyde was reacted with acetylene magnesium bromide in good yield to afford rac-49. When rac-41, rac-42, rac-45, rac-48 and rac-49, were assayed for anti-tumor effect in NSCLC U-1810 cells lower cytotoxicity was evident, of one to two orders of magnitudes, relative to rac-18 and rac-32 and thus these compounds were not tested further in diploid fibroblasts ( Table 1). The latter results demonstrated that substitution of the proton of the carbinol methine-3, in rac-32, with a methyl group resulted in a product, rac-41, which presented two orders of magnitude less cytotoxic towards NSCLC U-1810 cells. Similar results were obtained for the substitution of the acetylene proton at position 1, in rac-32, with a methyl group, in rac-42 (Table 1). Restricting the rotation further along the chain, by insertion of a phenyl ring in positions 4-6, rac-45, or 4-5, rac-48, similarly resulted in an order of magnitude less potent cytotoxic activity in the tested NSCLC U-1810 cells. Exclusion of the alkyl substituent from the phenyl ring, rac-49, generated a product that did not display any anti-tumor effect in the NSCLC cells tested. A comparison of the cytotoxic activity of rac-27, rac-45 and rac-48, (Table 1) reveals that the restricted rotation imposed by the phenyl ring negatively influenced the anti-tumor potency of the resulting compounds.

General Experimental Procedures
Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded on an Agilent 8453 spectrophotometer. IR spectra were recorded on a Bruker Tensor 27 FT-IR instrument. NMR spectra were recorded on a Bruker Avance III Spectrometer at 500.13 MHz for 1 H and 125.76 MHz for 13 C and a Bruker Avance III 400 Spectrometer at 400.13 MHz for 1 H, 100.62 MHz for 13 C NMR, chemical shifts were referenced to TMS δ H and δ C = 0 ppm. DEPT, COSY-45, gTOCSY, gROESY, gHSQC, gHMQC, and gHMBC, spectra were recorded using standard Bruker pulse sequences. Low resolution mass spectra were recorded on a Waters MaldiSynapt instrument (ESI and APPI), a Waters Xevo TQD instrument (ESI), an Aviv Analytical 5975-SMB instrument (GCMS with cold EI) and Agilent Technologies GCMS5977A MDS with 7890B GC system. High resolution mass spectra were recorded on a Waters MaldiSynapt instrument (ESI and APPI) and Bruker Maxis Impact QTOF APGC instrument equipped with a Bruker SCION456 GC. HPLC separations were performed on a Merck Hitachi HPLC system (L-6200 Intelligent pump and L-4200 UV-VIS detector), a JASCO P4-2080 plus HPLC system with a Multiwavelength detector, and an Agilent 1100 Series HPLC system.

Isolation Procedure
The freeze-dried sample (206 g) was extracted with a 45:45:10 mixture of EtOAc/MeOH /H 2 O at room temperature three times. The crude extract (43 g) was evaporated to dryness and separated, in 11     General procedure for oxidation of hexadecanol and octadecanol to 24 and 30: Hexadecanol (20 g, 82.6 mmol) was dissolved in 200 mL of anhydrous dichloromethane. The mixture was added to a stirred suspension consisting of pyridinium chlorochromate (PCC) (26.8 g, 124 mmol) and Celite (26.8 g) in 250 mL of anhydrous dichloromethane. The resulting suspension was stirred for 3 h at rt and the progress of the reaction was monitored by TLC (Silica Gel 60, 9:1 petroleum ether/ethyl acetate (PE/EtOAc). Upon the disappearance of the starting material, the suspension was filtered twice through a filter paper (Whatman 2). The solvent was evaporated and the residue dissolved in 500 mL of PE. The resulting suspension was filtered through a thin layer of silica, the filter cake was rinsed with an additional 200 mL of PE and the filtrates were combined. The solvent was removed under reduced pressure to afford hexadecanal (24) (17.08 g, 71.1 mmol, 85%) as an amorphous white solid. The same procedure was used for the synthesis of octadecanal (30), from octadecanol, in an 83% yield.
General procedure for hydrolysis of MTPA esters, preparation of pure acetylenes R-18, R-32 and S-32: The appropriate MTPA ester, (S,R)-29, (R,R)-34 or (R,S)-34 were treated with NH3 in methanol in a sealed pressure resistant tube. The reaction was heated to 90 • C and the progress was monitored by TLC (Silica Gel 60, 9:1 PE/EtOAc). After two days the solvent was removed under reduced pressure and the residue was extracted with PE. The crude extract was purified by column chromatography on silica gel, with 1% step gradient from Cytotoxicity structure-activity relationship analyses of compounds in NSCLC tumor cells and fibroblasts. Stock solutions (10 mg/mL) of the compounds to be tested were made in DMSO and further diluted with cell culture media prior to structure-activity relationship (SAR) screening as earlier described [17]. To assess anti-tumor activity, the large cell lung carcinoma cell line NSCLC U-1810 (kind gift from Uppsala University) [29] and diploid lung fibroblasts WI-38 [30] were used. U-1810 cells were propagated in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MI, USA) supplemented with FBS (10% HyClone) and L-glutamine (2 mmol/L, Invitrogen) while WI-38 fibroblasts were cultured in Eagle's Minimum Essential Medium (Sigma-Aldrich) with 15% FBS and L-glutamine. For SAR assessment the cytotoxic assay 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) was applied [8]. Profiling of U-1810 cells was carried out at 80% confluency (obtained by seeding 5000 cells/well in a 96-well plate for 24 h prior to treatment) and of WI-38 at 100% confluency (obtained by seeding 18,000 cells/well in a 96-well plate for 24 h prior to compound addition), the later to mimic the behavior of fibroblasts in the human body where they are non-dividing. For SAR evaluation, different concentrations of the compounds were added to fresh media while untreated cells were treated with DMSO corresponding to the amount applied when testing the highest concentration of the different compounds. The cytotoxicity of the compounds was assessed at 72 h post addition to cells by adding MTT solution (0.5 mg/mL, Sigma-Aldrich, 4 h at 37 • C) and dissolving the resulting formazan crystals in an SDS-containing solution (10% SDS and 0.01 mol/L HCl). Absorbance was quantified at 595 nm and the compound-induced cytotoxicity was calculated relative to that observed in DMSO-treated cells. The IC 50 values were deduced from survival plots drawn in Graph Pad PRISM software vers.6 and for some of the compounds extrapolated as indicated in footnotes to Table 1. The cell survival curves for some of the compounds are shown in Figure S188.

Conclusions
The alkyl-4E-ene-3-ol-1-ynes that were isolated from the extract of the sponge were composed of different mixtures of 3R-and 3S-isomers, where the 3R-isomers dominated. The fact that the carbocation at position-3 is rather stabilized by surrounding double-and triple-bonds, and that the 3-ol-1-ynes isolated in this study were composed solely of the 3Risomer, suggests that the compounds are biosynthesized by the sponge as the 3R-isomers and were racemized along the isolation process. Furthermore, the alkyl-3Z-en-5-ol-1-ynes isolated in this study were all racemic. This group of compounds is believed to be derived from the migration of the carbocation formed at position-3, to position-5, and its quenching by water to give the racemic 5-alcohol. This suggestion is in line with the finding in this study that the 3R-isomers are more active than the 3S-isomers. The results of the cytotoxic activity of the natural and synthetic compounds revealed that the terminal acetylene and the alcohol at position 3 (with the 3R-configuration) are essential for the potency of these alkylynols and resulted in two orders of magnitude selectivity toward the NSCLC cell line over normal fibroblasts, i.e., 7, R-32, rac-32 and rac-33. The length of the alkyl chain seems to influence the activity where a short alkyl chain (rac-31), or the absence of a chain (rac-49) presented reduced cytotoxicity toward the NSCLC cell line relative to rac-32 and rac-33. The addition of an E-4,5-double bond to the alkylynol skeleton results in equally potent but less selective derivatives 1, 8, rac-18, R-18, 19, 20, 22 and 23, toward the tumor cell line. Any substitution of the 3-OH with another electronegative substituent resulted in much less potent cytotoxicity in the tested NSCLC cell line, i.e., rac-35 to rac-40. Substitution of the acetylene (at C-1) or the carbinol (at C-3) by a methyl group results in essentially non-active products (41 and 42). The presence of a 4E-double bond, in rac-18, results in equipotent products but with less selective cytotoxicity towards the tested tumor cell line, relative to the presence of 4E,6E-diene, in rac-27, or saturated chain, in rac-32. However, the introduction of a less flexible moiety, a phenyl, to 4,5-bond (rac-45 and rac-48) resulted in one order of magnitude less potent cytotoxicity in the examined NSCLC cell line.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20040265/s1, 1 D ( 1 H, 13 C) and 2D NMR (HSQC, HMBC, COSY, ROESY) spectra and HR MS data of compounds 1-17, tables of full NMR data of 1-17 and two figures with the structures of the known metabolites isolated in this study.
Author Contributions: D.K. performed the entire work of isolation, structure elucidation and synthesis, as part of his PhD thesis. A.Z., K.V., P.H. and A.S. designed and performed the cytotoxicity assays. A.Z. and K.V. summarized and analyzed obtained SAR data. K.V., R.L., M.I. and S.C. supervised the project. All authors contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript.