key: cord-0016961-100ydw29
authors: Alzarea, Sami I.; Elmaidomy, Abeer H.; Saber, Hani; Musa, Arafa; Al-Sanea, Mohammad M.; Mostafa, Ehab M.; Hendawy, Omnia Magdy; Youssif, Khayrya A.; Alanazi, Abdullah S.; Alharbi, Metab; Sayed, Ahmed M.; Abdelmohsen, Usama Ramadan
title: Potential Anticancer Lipoxygenase Inhibitors from the Red Sea-Derived Brown Algae Sargassum cinereum: An In-Silico-Supported In-Vitro Study
date: 2021-04-10
journal: Antibiotics (Basel)
DOI: 10.3390/antibiotics10040416
sha: aee1d26035470711ba22c88425846ccd5f7fd96a
doc_id: 16961
cord_uid: 100ydw29

LC-MS-assisted metabolomic profiling of the Red Sea-derived brown algae Sargassum cinereum “Sargassaceae” dereplicated eleven compounds 1–11. Further phytochemical investigation afforded two new aryl cresol 12–13, along with eight known compounds 14–21. Both new metabolites, along with 19, showed moderate in vitro antiproliferative activity against HepG2, MCF-7, and Caco-2. Pharmacophore-based virtual screening suggested both 5-LOX and 15-LOX as the most probable target linked to their observed antiproliferative activity. The in vitro enzyme assays revealed 12 and 13 were able to inhibit 5-LOX more preferentially than 15-LOX, while 19 showed a convergent inhibitory activity toward both enzymes. Further in-depth in silico investigation revealed the molecular interactions inside both enzymes’ active sites and explained the varying inhibitory activity for 12 and 13 toward 5-LOX and 15-LOX.

Worldwide, the macroalgal genus Sargassum C. Agardh (1820) includes over 537 species, as well as 426 infra-specific names [1] . At present, 361 of the species names have been Analysis of S. cinereum crude extract led to a putative identification of several hits ( Figure 1 ). The molecular ion mass peaks at m/z 215.1283 and 277.2162 [M − H] + , for the predicted molecular formulas C11H20O4 and C18H30O2 gave hits of (5R,7S,8S)-communiol A 1, and hedaol A 2, respectively, that were previously isolated from Sargassum spp [10, 11] . The mass ion peaks at m/z 307.2624 and 343.2276 correspond to the suggested molecular formulas C20H34O2, and C22H30O3 [M+H]+ fit a fatty acid, and hydroquinone anti-inflammatory derivative compound arachidonic acid 3, and sargachromanol A 4, that was previously isolated from Sargassum pallidum, and Sargassum siliquastrum, respectively [12, 13] . The ion mass peaks at m/z 395.2950, 425.3420, 427.3576, and 487.3060 [M + H] + for the predicted molecular formulas C27H38O2, C29H44O2, C29H46O2, and C29H42O6 gave hits of the antiviral plastoquinones 2-geranylgeranyl-6-methylbenzoquinone 5, which was isolated from Sargassum micracanthum [14] , the anticancer steroidal nucleus of 24-ethylcholesta-4,24(28)-dien-3,6-dione 6, saringosterone 7, which were isolated from Sargassum carpophyllum, and Sargassum asperfolium, respectively [15, 16] , and the antioxidant meroditerpenoids of nahocol A 8, which were isolated from Sargassum siliquastrum [17] . Two major ion peaks with the m/z values of 445.3682 and 459.2749 [M + H] + with molecular formulas C29H48O3 and C27H38O6 were detected and dereplicated as 24xi-hydroperoxy-24-vinylcholesterol 9 and sargathunbergol A 10, respectively, which were isolated earlier from Sargassum carpophyllum, and Sargassum thunbergii, respectively [15, 18] . Analysis of S. cinereum crude extract led to a putative identification of several hits ( Figure 1 ). The molecular ion mass peaks at m/z 215.1283 and 277.2162 [M − H] + , for the predicted molecular formulas C 11 H 20 O 4 and C 18 H 30 O 2 gave hits of (5R,7S,8S)-communiol A 1, and hedaol A 2, respectively, that were previously isolated from Sargassum spp [10, 11] . The mass ion peaks at m/z 307.2624 and 343.2276 correspond to the suggested molecular formulas C 20 H 34 O 2, and C 22 H 30 O 3 [M + H]+ fit a fatty acid, and hydroquinone anti-inflammatory derivative compound arachidonic acid 3, and sargachromanol A 4, that was previously isolated from Sargassum pallidum, and Sargassum siliquastrum, respectively [12, 13] . The ion mass peaks at m/z 395. 2950 O 6 gave hits of the antiviral plastoquinones 2-geranylgeranyl-6-methylbenzoquinone 5, which was isolated from Sargassum micracanthum [14] , the anticancer steroidal nucleus of 24-ethylcholesta-4,24(28)-dien-3,6-dione 6, saringosterone 7, which were isolated from Sargassum carpophyllum, and Sargassum asperfolium, respectively [15, 16] , and the antioxidant meroditerpenoids of nahocol A 8, which were isolated from Sargassum siliquastrum [17] . Two major ion peaks with the m/z values of 445.3682 and 459.2749 [M + H] + with molecular formulas C 29 H 48 O 3 and C 27 H 38 O 6 were detected and dereplicated as 24xi-hydroperoxy-24vinylcholesterol 9 and sargathunbergol A 10, respectively, which were isolated earlier from Sargassum carpophyllum, and Sargassum thunbergii, respectively [15, 18] .

In addition, the mass ion peaks at m/z 553.2681 [M − H] + , for the predicted molecular formula C 25 H 46 O 11 S was dereplicated sulfoglycolipid derivative 1-O-(11-Hexadecenoyl)-3-O-(6 -sulfo-α-D-quinovopyranosyl) glycerol 11, which was previously detected in Sargassum hemiphyllum ( Figure 1 ) [19] . 

Based on the physicochemical and chromatographic properties, the spectral analyses from UV, 1 H, and DEPT-Q NMR, as well as comparisons with the literature and some authentic samples, the crude alcoholic extract of S. cinereum afforded the new aryl cresol 12-13, along with the known O-cresol 14 [20] , m-cresol 15 [21] . Additionally, arachidonic acid 16 [22] , eicosenoic acid 17 [22] , 1-O-arachidonyl-glycerol 18 [23] , 1-O-arachidonyl-3-O-(α-D-glucopyranosyl) glycerol 19 [23] , 7-β-methyl androstenol 20 [24] , and 1-deoxy-β-Dpsicosofuranose 21 [25] , were identified ( Figure 2 ). All characterized compounds 14 and 15 were isolated herein for the first time from the genus Sargassum (Figure 2, Figures S3-S28 ). Analysis of the HRESIMS, 1D and 2D NMR data of compounds 12-13 suggested a possible plastoquinones core scaffold [11] . The HRESIMS data for compound 12 showed an adduct pseudo molecular ion peak at m/z 314. 2607 NMR data also showed eight aliphatic methylene groups at δ H 1.20-2.8 δ C 20.5-33.7 (Table 1) , three olefinic methine groups at δ H 5.31-5.35 (6H, m) δ C 127.9-129.4. These signals are suggestive characteristics for 4,7,11-pentadecenyl moiety, where the heteronuclear multiple-bond correlation (HMBC) experiment of 12 ( Figure 3 ) confirmed the position of the three olefinic methine groups at 4,7,11 of the alkene side-chain. Moreover, the HMBC experiment showed the 3 J-HMBC correlation of the proton H-1 δ H 2.26 (δ C 33.4) with the quaternary carbonyl carbon C-4 (δ C 134.5). Accordingly, compound 12 was identified as 4-(1-(4,7,11-pentadecenyl)-o-cresol. The molecular formula of compound 13 was identical to that of 12 b HRESIMS (C22H34O). The 1 H and 13 C NMR data was also very close to those of com The molecular formula of compound 13 was identical to that of 12 based on HRESIMS (C22H34O). The 1 H and 13 C NMR data was also very close to those of compound 12 for the 4,7,11-pentadecenyl moiety but differed in the resonated chemical shifts of the ) correlations of compound 12. The molecular formula of compound 13 was identical to that of 12 based on HRESIMS (C 22 H 34 O). The 1 H and 13 C NMR data was also very close to those of compound 12 for the 4,7,11-pentadecenyl moiety but differed in the resonated chemical shifts of the aromatic attached methyl group of the core tri-substituted benzene unit (Table 1 ). Comparing the DEPT-Q 13 C NMR data of compound 13 with those of 12 showed a downfield shifting of carbons C-7 (∆δ C + 2.1), compared with those of compound 12 (Table 1 ). This suggested a positional difference of the location of the aromatic attached methyl group in the trisubstituted benzene unit versus 12 (Table 1 and Supplementary File 1( Figure S2 and S8-S12)). The assignment of the location of the aromatic attached methyl group in 13 was aided by the HMBC experiment. A 3 J-HMBC correlation ( Figure 4 ) of compound 13 proton H-7 δ H 1.23 (δ C 31.9) with the quaternary carbonyl carbon C-4 (δ C 134.5) and a 4 J-HMBC correlation of the proton H-7 δ H 1.23 (δ C 31.9) with the methylene carbon C-1 (δ C 33.7) confirmed the meta-location of an aromatic attached methyl group at the cresol moiety. Accordingly, compound 13 was identified as 4-(1-(4,7,11-pentadecenyl)-m-cresol. The molecular formula of compound 13 was identical to that of HRESIMS (C22H34O). The 1 H and 13 C NMR data was also very close to those 12 for the 4,7,11-pentadecenyl moiety but differed in the resonated chemic aromatic attached methyl group of the core tri-substituted benzene unit (T paring the DEPT-Q 13 C NMR data of compound 13 with those of 12 showe shifting of carbons C-7 (ΔδC + 2.1), compared with those of compound 12 suggested a positional difference of the location of the aromatic attached m the tri-substituted benzene unit versus 12 (Table 1 and Supplementary Fi and S8-S12)). The assignment of the location of the aromatic attached meth was aided by the HMBC experiment. 

The isolated compounds 12-21 were in vitro screened for their antip tivity against hepatic, breast, and colorectal carcinoma cell lines (HepG2 Caco-2, respectively) using the sulforhodamine B (SRB) assay. Results compounds 12, 13, and 19 were able to inhibit the growth of all tested cel ately with IC50 values ranged from 11.2 ± 0.6 to 21.6 ± 1.3 µM (Table 2) . 

HepG2 MCF-7 12 14.5 ± 0.8 * 17.6 ± 0.9 * 1 13

13 The molecular formula of compound 13 was identical to that of 12 based on RESIMS (C22H34O). The 1 H and 13 C NMR data was also very close to those of compound 2 for the 4,7,11-pentadecenyl moiety but differed in the resonated chemical shifts of the romatic attached methyl group of the core tri-substituted benzene unit (Table 1) . Comaring the DEPT-Q 13 C NMR data of compound 13 with those of 12 showed a downfield hifting of carbons C-7 (ΔδC + 2.1), compared with those of compound 12 (Table 1) . This uggested a positional difference of the location of the aromatic attached methyl group in he tri-substituted benzene unit versus 12 (Table 1 and Supplementary File 1( Figure S2 nd S8-S12)). The assignment of the location of the aromatic attached methyl group in 13 as aided by the HMBC experiment. A 3 J-HMBC correlation ( Figure 4 ) of compound 13 roton H-7 δH 1.23 (δC 31.9) with the quaternary carbonyl carbon C-4 (δC 134.5) and a 4 -HMBC correlation of the proton H-7 δH 1.23 (δC 31.9) with the methylene carbon C-1′ (δC 3.7) confirmed the meta-location of an aromatic attached methyl group at the cresol oiety. Accordingly, compound 13 was identified as 4-(1-(4,7,11-pentadecenyl)-m-cresol. 

The isolated compounds 12-21 were in vitro screened for their antiproliferative acivity against hepatic, breast, and colorectal carcinoma cell lines (HepG2, MCF-7, and aco-2, respectively) using the sulforhodamine B (SRB) assay. Results showed that ) correlations of compound 13.

The isolated compounds 12-21 were in vitro screened for their antiproliferative activity against hepatic, breast, and colorectal carcinoma cell lines (HepG2, MCF-7, and Caco-2, respectively) using the sulforhodamine B (SRB) assay. Results showed that compounds 12, 13, and 19 were able to inhibit the growth of all tested cell lines moderately with IC 50 values ranged from 11.2 ± 0.6 to 21.6 ± 1.3 µM (Table 2) . Table 2 . In vitro antiproliferative activity of the isolated compounds, 12-21 expressed as IC 50 ± (SSEM) µM.

HepG2 MCF-7 Caco-2 12 14.5 ± 0.8 * 17.6 ± 0.9 * 18.2 ± 0.7 * 13

13

The IC 50 value of compounds against each cancer cell line, which was defined as the concentration (µM) that caused a 50% inhibition of cell growth in vitro, data were expressed as mean ± SEM (n = 3). One-way analysis of variance (ANOVA) followed by Dunnett's test using PASW Statistics ® version 18 (Quarry Bay, Hong Kong) was applied. GraphPad Prism software version 6 (La Jolla, CA, USA) was used for statistical calculations. * Statistically significant at p < 0.05. Doxorubicin is a positive control. 

Characterization of the biological target for a certain molecule is a true challenge. However, the continuous development of in silico tools, including molecular modeling and virtual screening, has significantly improved the success rate of finding suitable molecular targets. Many online target identification platforms are currently available, and their search protocols are either structural-based or ligand-based. PharmMapper is one of these online platforms that can screen and suggest the most probable protein targets of a query molecule based on its pharmacophore model [26] . The basic principle of pharmacophorebased screening is that the binding of certain molecules with their protein targets is mainly determined by key pharmacophore maps (i.e., spatial arrangement of structural features). Thus, molecules that shapes are able to fit with these pharmacophore maps have the highest probability to bind the same protein target. Consequently, PharmMapper was used to propose a proper protein target for compounds 12, 13 and 19. 5-LOX and 15-LOX were found to be the top-scoring hits for these metabolites. As discussed in the introduction, these enzymes have been shown a direct link to the development of many cancers, e.g., breast, colorectal, liver, skin cancers [6] [7] [8] [27] [28] [29] [30] . Herein, compounds 12, 13 and 19 showed considerable inhibitory activity towards the human breast, colorectal, and liver cancer cell lines, and hence, they were selected for further in vitro and in silico validations against 5-LOX and 15-LOX.

To validate the preliminary virtual screening prediction, compounds 12, 13 and 19 were assayed for their 5-LOX and 15-LOX inhibitory activities. Interestingly the three compounds achieved potent enzyme inhibition toward 5-LOX (IC 50 1.3 ± 0.1 to 2.1 ± 0.4 µM, Table 3 ). However, their activity against 15-LOX was weaker, particularly compounds 12 and 13 (IC 50 25.3 ± 0.4 and 23.6 ± 0.3 µM, respectively) that were more selective for 5-LOX (Table 3) . Moreover, they showed inhibitory constants (K i ) ranged from 0.7 ± 0.2 to 17.4 ± 0.2 µM (Table 3) , and these values were most agree with the competitive inhibition of both enzymes [31] .

The results of enzyme inhibition assay were also correlated with those of the antiproliferative ones for HepG2 and MCF-7, and Caco-2. Overexpression of 5-LOX has been reported in breast, liver and colorectal cancers [27] [28] [29] . Furthermore, 15-LOX has been reported to be overexpressed in a number of tumors like prostate and breast cancers. Hence, these enzymes can be considered promising targets for cancer therapy.

5-LOX has a hydrophobic active site [9] that harbors a catalytic iron (Fe +2 ), and such hydrophobicity is essential to allow efficient binding with the hydrophobic substrate Molecular docking experiments revealed that these compounds could bind with the 5-LOX's active site efficiently, with binding scores ranged from −8.9 to −9.3 kcal/mol ( Figure 5 ), and their bindings were even better than the co-crystalized ligands (Table 3) . Additionally, the phenolic moiety of both compounds was involved in H-bonding with HIS-600, similarly to the co-crystallized redox-type inhibitor, nordihydroguaiaretic acid (NDGA) ( Figure 5) .

Compounds 12 and 13s hydrophobic side chains were able to adapt themselves inside the hydrophobic U-shaped active site, where they took convergent orientations but slightly different from that of AA ( Figure 5 

The marine algae S. cinereum was collected during January 2020 along the shore of the Red Sea in Hurghada, Egypt. The samples were collected in sterilized polyethylene bags and kept in an icebox for transportation to the laboratory. Samples were washed thoroughly with sterile distilled water to remove any associated debris. A voucher specimen (2020-BuPD 55) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Egypt.

The solvents used in this work included n-hexane (n-hex., boiling point b.p. 60-80 °C), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (n-but.), and methanol (MeOH) were purchased from El-Nasr Company for Pharmaceuticals and Chemicals (Egypt). High-performance liquid chromatography (HPLC) and deuterated solvents used for chromatographic and spectroscopic analyses were purchased from Sigma-Aldrich (Saint Louis, MO, USA), including HPLC-methanol, HPLC-water, HPLCacetonitrile, deuterium oxide (D2O), methanol (CD3OD), and dimethyl sulfoxide (DMSO-d6). Column chromatography (CC) was performed using silica gel 60 (63-200 µm, E. Merck, Sigma-Aldrich), and Sephadex LH-20 (0.25-0.1 mm, GE Healthcare, Sigma-Aldrich, Steinheim, Germany), while silica gel GF254 for thin-layer chromatography (TLC) (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt) was employed for vacuum liquid chromatography (VLC). Thin-layer chromatography (TLC) was carried out using precoated silica gel 60 GF254 plates (E. Merck, Darmstadt, Germany; 20 × 20 cm, 0.25 mm in thickness). Spots were visualized by spraying with para-anisaldehyde (PAA) reagent (85:5:10:0.5 absolute EtOH:sulfuric acid:G.A.A.:para-anisaldehyde), followed by heating at 110 °C [32] . For the biological study, doxorubicin (Sigma-Aldrich, Germany) was used as a positive control, while the HepG2, MCF-7, and Caco-2 cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA; HPACC, Salisbury, UK) and were routinely subcultured twice per week. 

The marine algae S. cinereum was collected during January 2020 along the shore of the Red Sea in Hurghada, Egypt. The samples were collected in sterilized polyethylene bags and kept in an icebox for transportation to the laboratory. Samples were washed thoroughly with sterile distilled water to remove any associated debris. A voucher specimen (2020-BuPD 55) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Egypt.

The solvents used in this work included n-hexane (n-hex., boiling point b.p. 60-80 • C), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (n-but.), and methanol (MeOH) were purchased from El-Nasr Company for Pharmaceuticals and Chemicals (Egypt). High-performance liquid chromatography (HPLC) and deuterated solvents used for chromatographic and spectroscopic analyses were purchased from Sigma-Aldrich (Saint Louis, MO, USA), including HPLC-methanol, HPLC-water, HPLC-acetonitrile, deuterium oxide (D 2 O), methanol (CD 3 OD), and dimethyl sulfoxide (DMSO-d 6 ) . Column chromatography (CC) was performed using silica gel 60 (63-200 µm, E. Merck, Sigma-Aldrich), and Sephadex LH-20 (0.25-0.1 mm, GE Healthcare, Sigma-Aldrich, Steinheim, Germany), while silica gel GF254 for thin-layer chromatography (TLC) (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt) was employed for vacuum liquid chromatography (VLC). Thin-layer chromatography (TLC) was carried out using precoated silica gel 60 GF254 plates (E. Merck, Darmstadt, Germany; 20 × 20 cm, 0.25 mm in thickness). Spots were visualized by spraying with para-anisaldehyde (PAA) reagent (85:5:10:0.5 absolute EtOH:sulfuric acid:G.A.A.:para-anisaldehyde), followed by heating at 110 • C [32] . For the biological study, doxorubicin (Sigma-Aldrich, Germany) was used as a positive control, while the HepG2, MCF-7, and Caco-2 cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA; HPACC, Salisbury, UK) and were routinely subcultured twice per week.

Proton 1 H and distortionless enhancement by polarization transfer-Q (DEPT-Q) 13 C NMR spectra were recorded at 400 and 100 MHz, respectively. Tetramethylsilane (TMS) was used as an internal standard in deuterium oxide (D 2 O), methanol (CD 3 OD), and dimethyl sulfoxide (DMSO-d 6 ), using the residual solvent peak (δ H = 4.78), (δ H = 3.34, 4.78 and δ C = 49.9) and (δ H = 2.50 and δ C = 39.5) as references, respectively. Measurements were performed on a Bruker Advance III 400 MHz with BBFO Smart Probe and a Bruker 400 MHz EON nitrogen-free magnet (Bruker AG, Billerica, MA, USA). Carbon multiplicities were determined using a DEPT-Q experiment. The ultraviolet radiation (UV) spectrum in methanol was obtained using a Shimadzu UV 2401PC spectrophotometer (Shimadzu Corporation-UV-2401PC/UV-2501PC, Kyoto, Japan). Infrared (IR) spectra were measured using a Jasco FTIR 300E infrared spectrophotometer. HRESIMS data were obtained using an Acquity ultra-performance liquid chromatography system coupled to a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer (Waters, Milford, MA, USA). HPLC chromatographic separations were conducted using an Agilent 1260 Infinity preparative pump (G1361A), Agilent 1260 diode array detector VL (G1315 D), Agilent 1260 Infinity Thermostand column compartment (G1361 A), Agilent 1260 Infinity preparative autosampler (G2260A) and a YMC-Pack ODS-A A-324 column (i.d. 10 × 300 mm, YMC, Kyoto, Japan).

Sargassum cinereum (0.5 kg) was collected and air-dried in the shade for one month. After drying, the brown algae were finely powdered using an OC-60B/60B grinding machine (60-120 mesh, Henan, China). The finely powdered algae extracted by maceration using 70% methanol (3 L, 3×, seven days each) at room temperature, and concentrated under vacuum at 45 • C using a rotary evaporator (Buchi Rotavapor R-300, Cole-Parmer, Vernon Hills, IL, USA) to afford 75 g crude extract. The dry extract was suspended in 100 mL distilled water (H 2 O) and successively portioned with solvents of different polarities (n-Hex., DCM, EtOAc, and n-but.). The organic phase in each step separately evaporated under reduced pressure to afford the corresponding fractions I (8.0 g), II (1.5 g), III (1.5 g) and IV (3.0 g), respectively, while the remaining mother liquor was then concentrated down to give the aqueous fraction (V). All resulting fractions were kept at 4 • C for biological and phytochemical investigations.

The crude methanolic extract from S. cinereum was prepared at 1 mg/mL for mass spectrometry analysis. The recovered methanolic extract was subjected to metabolic analysis using LC-HRESIMS according to Abdelmohsen et al. 2014 [33] . An Acquity ultraperformance liquid chromatography system connected to a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer (Waters, Milford, M.A. USA) was used. Positive and negative ESI ionization modes were utilized to carry out the high-resolution mass spectrometry coupled with a spray voltage at 4.5 kV, the capillary temperature at 320 • C, and mass range from m/z 150-1500. The MS dataset was processed, and data were extracted using MZmine 2.20 based on the established parameters [22] . Mass ion peaks were detected and accompanied by chromatogram builder and chromatogram deconvolution. The local minimum search algorithm was addressed, and isotopes were also distinguished via the isotopic peaks of grouper. Missing peaks were displayed using the gap-filling peak finder. An adduct search along with a complex search was carried out. The processed data set was next subjected to molecular formula prediction and peak identification. The positive and negative ionization mode data sets from the respective extract were dereplicated against the Dictionary of Natural Products (DNP) databases.

Fraction I (8 g) was subjected to normal VLC fractionation using silica gel GF 254 (column 6 × 30 cm, 50 g). Elution was performed using n-hex.:EtOAc gradient mixtures in order of increasing polarities (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 60, 80 and 100%, 500 mL each). The effluents from the column were collected in fractions (100 mL each), and each collected fraction was concentrated and monitored by TLC using the system n-hex.:EtOAc 8:2 and PAA reagent. Similar fractions were grouped and concentrated under reduced pressure to provide three subfractions (I 1 -I 3 ). Subfraction II 2 (3.0 g) was further fractionated on silica gel 60 (100 × 1 cm, 50 g). Elution was performed using n-hex.:EtOAc gradient mixtures in the order of increasing polarities (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10%, 1 L each, FR 3 mL min −1 ), to afford four sub-subfractions (II 2-1 -II 2-4 ). Sub-subfraction II 2-1 (50 mg) was further fractionated on silica gel 60 (100 × 1 cm, 20 g). Elution was performed using n-hex.:EtOAc isocratic mixture (1%, 500 mL, FR 3 mL min −1 ) to afford compound 17 (20 mg). Sub-subfractions II 2-2 , and II 2-4 (70, 30 mg each) was further fractionated on C-18 RP-HPLC using H 2 O-CH 3 CN (10-60%, 30 min, 5 mL/min) to afford compound 12 (20 mg), 13 (10 mg), 14 (10 mg), 15 (7 mg). Sub-subfraction II 2-3 (100 mg) was further fractionated on silica gel 60 (100 × 1 cm, 20 g). Elution was performed using n-hex.:EtOAc isocratic mixture (5%, 500 mL, FR 3 mL min −1 ) to afford compound 16 (50 mg). Finally, subfraction II 3 was further fractionated on silica gel 60 (100 × 1 cm, 20 g). Elution was performed using n-hex.:EtOAc isocratic mixture (1%, 500 mL, FR 3 mL min −1 ) to afford compound 20 (30 mg) . Fraction II (1.5 g) was subjected to normal VLC fractionation on a silica gel (column 6 × 30 cm, 50 g). Elution was performed using DCM:MeOH gradient mixtures in the order of increasing polarities (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 60, 80 and 100%, 1 L each). The effluents were collected in fractions (100 mL each); each fraction was concentrated and monitored by TLC using the system DCM:MeOH 9.5:0.5 and PAA reagent. Similar fractions were grouped and concentrated under reduced pressure to provide two subfractions (II 1 -II 2 ), which were further purified on a Sephadex LH 20 column (0.25-0.1 mm, 100 × 0.5 cm, 100 g), which eluted with MeOH to afford compound 18 (16 mg) , and 19 (6 mg), separately.

Crystallization of fractions IV was performed separately using CH 2 CL 2 and afforded compounds 21 (2 g 

The antiproliferative activity of the isolated compounds 12-21 was measured by the sulforhodamine B (SRB) assay as described by Skehan et al. 1990 [34] , and Vichai and Kirtikara 2006 [35] , on the breast (MCF-7), liver (HepG2) and colorectal (Caco-2) cancer cell lines. Cells were seeded in 96-well microtiter plates at an initial concentration of 3 × 10 3 cell/well in 150 µL, fresh medium and left for 24 h to attach to the plates. Different concentrations 0, 5, 12.5, 25, 50 µg/mL of the respective compound were added. The plates were incubated for 48 h. The cells were fixed with 50µL cold trichloroacetic acid (10% final concentration) for 1 h at 4 • C. The plates were washed with distilled water (automatic washer Tecan, Neustadt, Germany) and stained with 50 µL 0.4% SRB dissolved in 1% acetic acid for 30 min., at room temperature. Then they were washed with 1% acetic acid and airdried. The dye was solubilized with 100 µL/well of 10 M Tris base (pH 10.5). The optical density of each well was measured spectrophotometrically at 570 nm using an ELISA microplate reader (Sunrise Tecan reader, Neustadt, Germany). Doxorubicin was used as a positive control. The mean background absorbances were automatically subtracted, and the mean values of each drug concentration were calculated. The experiment was repeated three times, and then the IC 50 values were calculated.

The ability of the isolated compounds 12, 13, and 19 to inhibit 5-LOX and 15-LOX enzymes (IC 50 and K i values, µM) was determined using human recombinant enzyme assay kits (catalog no 60,402 and 10011263, Cayman Chemical, Ann Arbor, MI, USA) following manufacturer's specifications [36] . Stock solutions were freshly prepared before use, and buffer solution (0.1 M Tris-HCl, PH, 7.4) was used. 10 µL of each compound were prepared, dissolved in the least amount of DMSO and diluted with the stock solution to be in concentrations of (0.001, 0.1, 1, 5, 10 µM) in a final volume of 210 mL. The kinetic parameters for both 5-LOX and 15-LOX were determined by measuring the increase in absorbance at 238 nm in an Agilent 8453 diode array spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Substrate concentration was ranged from 5 to 50 µM. Substrate concentrations (5, 10, 20, 30, 40 , 50 µM) were monitored in triplicate for each sample [37] . Doxorubicin was used as a positive control.

The crystal structures of both 5-LOX and 15-LOX (PDB: 6N2W and 4NRE) were used for the docking analysis using an AutoDock Vina docking machine [38] . The co-crystallized ligands nordihydroguaiaretic acid (NDGA) and AA were used to determine the binding sites. The ligand to binding site shape matching root means square (RMSD) threshold was set to 2.0 Å. The interaction energies were determined using the Charmm force field (v.1.02) with 10.0 Å as a non-bonded cutoff distance and distance-dependent dielectric. Then, 5.0 Å was set as an energy grid extending from the binding site [39] . The tested compounds were energy minimized inside the selected binding pocket. The editing and visualization of the generated binding poses were performed using Pymol software [40] .

Molecular dynamic simulations (MDS) for ligand enzyme complexes were performed according to the previous protocol [41] , using the Nanoscale Molecular Dynamics (NAMD) 2.6 software [42] , applying the CHARMM27 force field [43] . Hydrogen atoms were added to the protein structures using the psfgen plugin included in the Visual Molecular Dynamic (VMD) 1.9 software [44] . Afterward, the whole system was solvated using TIP3P water particles and 0.15 M NaCl. The energy of the generated systems was first minimized and gradually heated to 300 K and equilibrated for 200/s. Subsequently, the MDS was continued for 20 ns, and the trajectory was stored every 0.1 ns and further analyzed with the VMD 1.9 software. The MDS output was sampled every 0.1 ns to evaluate the conformational changes of the entire system to analyze the root mean square deviation (RMSD) and root mean square fluctuation (RMSF). The topologies and parameters of the tested compounds were prepared using the VMD force field toolkit (ffTK) and the online software ligand reader and modeler (http://www.charmm-gui.org/?doc=input/ligandrm, accessed on 15 January 2021) [45] . MDS-derived binding free energies (∆G) were calculated using the free energy perturbation (FEP) method through the web-based software Absolute Ligand Binder along with MDS using NAMD software [45, 46] . Moreover, ∆G was calculated using another web-based software utilizing neural networking in its calculations, namely KDEEP (https://www.playmolecule.org/Kdeep/, accessed on 16 January 2021) [47] .

All in vitro experiments were performed in triplicate. Pooled data were presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. The differences among various treatment groups were determined by ANOVA, followed by Dunnett's test using PASW Statistics ® version 18 (Quarry Bay, Hong Kong). A difference of p < 0.05 was considered statistically significant and shown by a *symbol. The IC 50 values were determined using a nonlinear regression curve fitting analysis using GraphPad Prism software version 6 (La Jolla, CA, USA).

Phytochemical investigation of the brown algae S. cinereum with the guidance of LC-HRESIMS dereplication afforded two new phenolic derivatives 12 and 13, along with the known 19, which exhibited moderate in vitro antiproliferative activity against HepG2, MCF-7, and Caco-2 cancer cell lines and considerable selective inhibition toward 5-LOX over 15-LOX. A series of in silico experiments (docking, MDS, and binding free energy calculations) were carried out to explore the mode of interaction of these compounds inside the active site of each enzyme. The present study shows the potential of marine natural products in providing unique metabolites with potent biological activities and highlighted the power of in silico investigations to facilitate drug discovery and development processes.

Supplementary Materials: The following are available online. Table S1 : Dereplicated metabolites from LC-HRESIMS analysis of Sarragassum cinnerum; Figure S1 : LC-HRESIMS Chromatogram of the dereplicated metabolites of Sarragassum cinnerum (positive); Figure S2 : LC-HRESIMS Chromatogram of the dereplicated metabolites of Sarragassum cinnerum (negative); Figure S3 : 1 H NMR spectrum of compound 12 measured in DMSO-d6 at 400 MHz; Figure S4 : DEPT-Q NMR spectrum of compound 12 measured in DMSO-d6 at 100 MHz; Figure S5 : HSQC spectrum of compound 12 measured in DMSO-d6; Figure S6 : HMBC spectrum of compound 12 measured in DMSO-d6; Figure S7 : HRESIMS spectrum of compound 12; Figure S8 : 1 H NMR spectrum of compound 13 measured in DMSO-d6 at 400 MHz; Figure S9 : DEPT-Q NMR spectrum of compound 13 measured in DMSO-d6 at 100 MHz; Figure S10 : HSQC spectrum of compound 13 measured in DMSO-d6; Figure S11 : HMBC spectrum of compound 13 measured in DMSO-d6; Figure S12 : HRESIMS spectrum of compound 13; Figure S13 

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The authors would like to extend their sincere appreciation to the central laboratory at Jouf University for support this study.

The authors declare no conflict of interest.