Cytotoxic ketosteroids from the Red Sea soft coral Dendronephthya sp.

: A marine specimen of the Red Sea soft coral Dendronephthya sp. was extracted with a mixture of n - hexane, diethyl ether, and methanol. One new cytotoxic steroid dendronestadione ( 1 ) , ﬁ ve known steroids: den - dronesterones A - C ( 2 – 4 ) , dendrotriol ( 5 ) , and cholesterol ( 6 ) along with 4 - oxo - pentanoic acid ( 7 ) and a polyhydroxy alkane hexitol ( 8 ) were isolated from the Dendronephthya sp. extract. The chemical structures of the isolated meta - bolites were elucidated by the application of several spec - troscopic techniques ( 1D, 2D NMR, IR, and UV ) and mass spectrometry.Theantiproliferativee ﬀ ectoftheisolatedcom - pounds was assessed against a panel of human cancer cell lines including HepG2, HT - 29, and PC. The obtained results indicated that compounds 1 – 4 ( dendronesterones A – C ) exhibited a higher cytotoxic e ﬀ ect than that of the other co - isolated ones. Among all examined dendronesterones, dendronesterone C showed the highest IC 50 values of 19.1 ± 1.81, 32.4 ± 2.84, and 7.8 ± 0.80 µM against the three cancer cells under investigation. Interestingly, all isolated ketos - teroids showed potent e ﬀ ects against prostate cancer cells. These ﬁ ndings highlight the role of ketosteroids as an anti - proliferative agent against the examined cells in this study.


Introduction
The increasing evidence of human diseases like pathogenic bacteria or virus infection and cancer in the current decadal demand attention to discovering new treatment methods [1].Chemotherapy, radiotherapy, and surgery are common treatment methods, while synthetic-antibiotic drugs are administered to cure microbial infection [2].
However, the administration of synthetic drugs shows side effects [3].Natural products derived from plants or marine invertebrates have scientifically proven their merit with fewer side effects [4].
Soft corals inhabit competitive and extreme environments that produce secondary metabolites for their defense mechanism [5].Until 2012, the metabolite from soft coral represented 22% of total new marine metabolites isolated from marine invertebrates [6].Marine natural products from soft coral possess diverse molecular structures with pharmaceutical properties [7].Many of them enter the preclinical and clinical studies due to their promising in vivo and in vitro evaluation [8].The family of Xeniidae, Nephtheidae, Alcyoniidae, or Clavulariidae was the primary source of natural products [9].Roughly 179 new steroids had been discovered from soft corals worldwide from 2015 to 2020, most of which belong to hydroxysteroids [10].The noticeable bioactive activity from these newly discovered metabolites was anticancer, antibacterial, and anti-inflammatory [11].

General
The instruments' specifications and solvents and materials used in this article are detailed elsewhere [17].

Animal material
A sample of a soft bodied coral Dendronephthya sp.animal was collected in April 2019 off Jeddah city coast, Saudi Arabia.A voucher specimen (DS19-01) was deposited at the Marine biology department, Faculty of Marine Sciences, KAU.

Extraction and isolation
The squeeze-dried Dendronephthya sp.sample (900 g) was macerated in acetone at room temperature (three times, 24 h each).The extract was completely dried to provide 37.3 g of solvent-free oil residue.15.0 g of the residue was well mixed with a suitable amount of normal phase column chromatographic Si gel powder.The homogenized sample-silica gel paste was poured on the top of 1 m silica gel column (3.5 cm diameter).The fractionation process started with a non-polar solvent (n-hexane) and the polarity was increased gradually by adding increased percentages of chloroform in pet.ether and the EtOAc in pet.ether.A total of 678 fractions (25 ml each) were obtained.TLC plates were visualized with the aid of UV lamp (UV 254 ) and p-anisaldehyde-sulfuric acid reagent.PTLC glass plates were used for further purification.
Chromatographic fractionation of the animal material was performed as previously described by Alassass et al. [15].In brief, elution commenced with n-hexane, and then increased amounts of Et 2 O were added to elevate the polarity and then the polarity increased by EtOAc.The initial fraction eluted with n-hexane was repurified with PTLC (100% n-hexane), to give a pink spot up on spraying with sulfuric acid reagent.This zone was collected (1.1 mg) and assigned as 7.The fraction eluted with Et 2 O/n-hexane (1:4) was applied to PTLC using the same previous composition, the spot at R f 0.51 (reddish spot with sulfuric acid reagent) was collected (2.0 mg) and assigned as 6.The fraction eluted with EtOAc/n-hexane (1.5:8.5) was applied to PTLC (22% EtOAc in n-hexane), and the spots appeared at R f 0.73, 0.69, and 0.51 were collected and assigned to compounds 2 (1.0 mg), 3 (0.7 mg), and 4 (1.1 mg), respectively.The fraction eluted with EtOAc/n-hexane (1:4) was applied to PTLC (25% EtOAc in n-hexane), and the spot appeared at R f 0.66 was collected to give compound 1 (0.9 mg).The fraction eluted with EtOAc/n-hexane (1:3) was applied to PTLC (35% EtOAc in n-hexane), and the spot appeared at R f 0.49 was collected to give compound 5 (0.7 mg).The fraction eluted with MeOH/EtOAc/n-hexane (5:20:80) was applied to PTLC (15% MeOH in dichloromethane), and the spot appeared at R f 0.66 was collected to give compound 8 (0.9 mg).   C NMR (CHCl 3 ) (Table 1).Compounds 2-8 were characterized and identified by comparing their spectral data with the previously reported data in the literature.Those compounds were identified dendronesterones A-C (2-4), dendrotriol (5), and cholesterol (6) along with, 4-oxo-pentanoic acid (7) and the polyhydroxy alkane hexitol (8) [16,18].

Results and discussion
Sequential chromatographic fractionation and purification of the organic extract of the Red Sea soft coral resulted in the identification of eight secondary metabolites; six of them belong to steroids with C-27 and C-28 carbocyclic skeletons and the remaining compounds belong open chain alkanes.
Compound 1 was isolated as an optically active gummy substance.Its thin layer chromatography profile exhibited a positive response for steroids up on spraying with p-anisaldehyde-sulfuric acid reagent (blue turned brown color).Compound 1 showed absorption bands at 238 and 226 nm in the UV spectrum, indicating the presence of two a,bunsaturated carbonyl functions.The molecular formula was found to be C 27 H 40 O 2 from HRESIMS (requiring eight unsaturation sites) supported by twenty-seven signals appeared in the 13 C NMR spectrum.No absorption due to hydroxyl function was found in the IR absorption spectrum; however, absorption bands due a,b-unsaturated carbonyls (1,686 and 1,675 cm −1 ) and gem-dimethyl (1,368 cm −1 ) were observed.The 1 H NMR spectrum indicated the presence of one signal of secondary methyl protons resonating at δ H 1.02 ppm, four tertiary methyl protons resonating at δ H 0.85, 1.01, 1.91, and 2.13 ppm, and three olefinic protons resonating at δ H 7.16, 6.19, and 5.85 ppm (Table 1).The methylation pattern, the carbon number signals, the unsaturation degrees, and previous publications from the same genus call to the mind the probability of a steroid with a cholestane skeleton.The 13 C NMR spectrum of 1 showed the presence of signals due to carbonyl functions resonating at δ C 204.6 and 200.3 ppm, four olefinic carbon signals resonating at 158.6, 156.7, 127.4,and 125.2 ppm.DEPT NMR experiments showed the presence of five unprotonated, nine methine, eight methylene, and five methyl carbons.HSQC NMR enabled the direct connection between all carbons and protons.Therefore, compound 1 contains no protonated hetero atoms.The presence of two carbonyls and two carbon-carbon double bonds accounted for three sites of unsaturation, which pointed out to the presence of a tetracyclic carbo-structure.The 13 C NMR signals at δ C (204.6, 200.3), along with absorption bands in the IR and UV spectra suggested the presence of separate a,b-unsaturated carbonyls. 1H-1 H COSY spectrum showed a distinct  A literature survey of chemical structures isolated from the genus Dendronephthya revealed that compound 1 was not previously isolated metabolite and was identified as dendronestadione (1).
Compound 2 was isolated as an optically active substance.It gave a positive response for steroids up on spraying with p-anisaldehyde-sulfuric acid reagent (blue turned brown color) on thin layer chromatography.Compound 2 showed an absorption band at 226 nm in the UV spectrum, indicating the presence of a,b-unsaturated carbonyl function.The molecular formula was found to be C 27 H 42 O, from HREIMS (required seven unsaturation sites) supported by 27 signals appeared in the 13 C NMR spectrum.No absorption due to hydroxyl function was found in the IR absorption spectrum; however, absorption bands due a,b-unsaturated carbonyl (1,688 cm −1 ), carbon-carbon double bond (1,655 cm −1 ), and gem-dimethyl (1,360 cm −1 ) were observed.The 1 H NMR spectrum indicated the presence of three secondary methyl protons resonating at δ H 1.00, 0.87, and 0.85 ppm, two tertiary methyl protons resonating at δ H 1.01 and 1.00 ppm, and four olefinic protons resonating at δ H 7.16, 5.85, 5.22, and 5.28 ppm.The 13 C NMR spectrum of 2 suggested the presence of a,b-unsaturated ketone (δ C 200.6, 159.0, and 127.4) and carbon-carbon double bond (δ H 138.0 and 126.5).DEPT NMR experiments showed the presence of three unprotonated, eleven methine, eight methylene, and five methyl carbons.HSQC NMR enabled the direct connection between carbons and protons.The presence of one carbonyl and two carbon-carbon double bonds accounted for three sites of unsaturation which pointed to the presence of a tetracyclic carbo-structure.With the aid of 1 H-1 H COSY and HMBC NMR, the gross structure of 2 was concluded to be a cholestane skeleton with a carbonyl function at C-3 and two carbon-carbon double bonds at C-1 and C-22.The geometry of the C-22-C-23 was proved to be trans-geometry due to the large coupling constant (J = 15.3Hz).A literature survey of chemical structures isolated from the genus Dendronephthya revealed that compound 2 was previously isolated from Dendronephthya gigantea and was identified as dendronesterone A [16].Compounds 3, 4, 7, and 8 were identified by the comparison of their spectral data with those reported in the literature [18].Compound 5 was identified by the comparison of its spectral data with that reported in the literature [18].
In this work, eight compounds were isolated from the Res Sea animal, Dendronephthya sp.The antiproliferative action of these metabolites was investigated against a panel of three cancer cell lines, HepG2, HT-29, and PC.Compound 1 showed the highest cytotoxic effects, where it had IC 50 values of 19.1 ± 1.81, 32.4 ± 2.84, and 7.8 ± 0.80 µM in the treatment of HepG2, HT-29, and PC-3 cells respectively.Compound 2 showed good cytotoxic activity against PC-3 cells with an IC 50 value of 43.7 ± 3.12 µM.By contrast to compounds 2 and 3, compound 4 showed reasonable activity against all examined cells (Table 2).The data in Table 2 strictly clarified the following points: all examined steroids are more active toward PC-3 cells; decoration of the side chain with α,β-unsaturated ketone might affect the bioactivity exerted by the examined steroids; ketosteroids are more cytotoxic than the co-isolated fatty acid derivatives and the 3-hydroxy steroids.

Conclusion
The Red Sea soft coral animal Dendronephthya sp. is a rich source of the ketosteroids of C-27 and C-28 carbocyclic skeletons in addition to keto-fatty acids.All ketosteroids showed antiproliferative effects with varying degrees of activity.Compound 1, which was characterized by the presence of two α,β-unsaturated carbonyls, showed the highest potency among all compounds.It is worth noting that the maximum antiproliferative effect of all ketosteroids was observed against prostate adenocarcinoma (PC-3) cells.
) and H-2 (5.85).Other proton sequences were observed and clarified in Figure2.The HMBC correlations observed from H-1 to the carbonyl carbon at 200.3 (C-3), 38.9 (C-10), and 13.0 (C-19) established the location of an a,b-unsaturated carbonyl function (C-1-C-3).The correlations observed from H-24 to the carbonyl carbon at 204.6 (C-23), and the gem-dimethyl function signals at 156.7, 27.5, and 21.0 established the presence of the second a,b-unsaturated carbonyl moiety at the forked tail (side chain).With the aid of 1 H-1 H COSY and HMBC NMR, the gross structure of 1 was concluded to be a cholestane skeleton with two carbonyl functions at C-3 and C-23 and two carbon-carbon double bonds at C-1 and C-24.

Table 2 :
Cytotoxicity of compounds 1-8 isolated from Dendronephthya sp.Four replicates were used for each treatment.Doxorubicin = positive control. *