General experimental procedures

1H and 13C-NMR spectra were measured on a JEOL spectrometer at 600 and 150 MHz, respectively. HR-ESI–MS data were noted on a Thermo Fisher Scientific LTQ Orbitrap XL spectrometry. Silica gel for column chromatography (70–230) was used for fine separation (E. Merck, Darmstadt, Germany). Silica gel 60 pre-coated plates F254 (E. Merck, Germany) were used for TLC detection. Reversed-phase (RP-C18) silica gel purchased from Nacalai Tesque, Kyoto, Japan was utilized for reversed-phase column chromatography separation. Inertsil ODS-3 column (GL Science, Tokyo, Japan) was used for HPLC analyses using a refractive index detector (RID-6A, Shimadzu, Kyoto, Japan). The stable 3D molecular models were obtained using the MMFF94s force field.

The cancer-cell lines (lung adenocarcinoma (A549), breast cancer (MCF-7), and hepatocellular carcinoma (HepG2)) were obtained from the National Institute of Biomedical Innovation's Japanese Collection of Research Bioresources (JCRB) Cell Bank. The Institute of Tropical Medicine at Nagasaki University in Japan provides Leishmania major. Dimethyl sulfoxide, Dulbecco’s modified Eagleʹs medium, fetal bovine serum, medium 199, miltefosine, etoposide, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and kanamycin were obtained from Nacalai Tesque, Kyoto, Japan. Becton Dickinson provided the 96-well plates (Franklin Lakes, NJ, USA).

Soft coral material

The Litophyton arboreum soft coral was collected at a depth of 10–15 m using a diving technique (SCUBA) in March 2018 in front of the National Institute of Oceanography and Fisheries at Hurghada province on the Egyptian Red Sea Coast. The sample was collected and identified by Abdallah Alian Department of Zoology, Faculty of Science, Al-Azhar University, Assiut-Branch, Assiut, Egypt. The sample was stored in a freezer until the time of extraction. A voucher specimen was deposited with the symbol of LA.5 at the Department of Zoology, Faculty of Science, Al-Azhar University, Assiut-Branch, Assiut, Egypt.

Extraction and isolation

L. arboreum ( ̴ 1.8 kg wet wt.) was cut into small pieces and macerated in methanol until it was exhausted. The methanolic extract has been concentrated using reduced pressure to get a dried residue (38 g). The total methanolic extract was fractionated using vacuum liquid-column chromatography (CC) filled with silica gel. The elution was carried out with the solvents [n-hexane (3L), n-hexane-chloroform (1:1) (3L), chloroform (3L), EtOAc (3L), and MeOH (3L)], successively, produced n-hexane (1.0 g), n-hexane-chloroform (1:1) (9.0 g), chloroform (2.5 g), EtOAc (5.5 g), and MeOH (18.0 g) fractions.

The n-hexane-chloroform (1:1) fraction was subjected to silica gel CC and eluted with EtOAc in n-hexane (0–100% of EtOAc gradients) and further with MeOH in EtOAc (0–100% of MeOH gradients) and yielded 22 sub-fractions (F1-F22).

The sub-fraction F3 (3.5 g), eluted with n-hexane–EtOAc (80:20), was chromatographed over reversed-phase CC and eluted with MeOH-H2O (0–100% of MeOH gradients) to afford ten sub-fractions (F3-1⁓F3-10). The subfraction F3-5 (415 mg) eluted with 50% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 50:50, and afforded compounds 1 (7.0 mg) and 2 (4.0 mg). The subfraction F3-6 (400 mg) eluted with 50% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 60:40, and afforded compound 4 (40.0 mg).

The sub-fraction F5 (2.2 g), eluted with n-hexane–EtOAc (60:40), was chromatographed over reversed-phase CC and eluted with MeOH-H2O (0–100% of MeOH gradients) to afford ten sub-fractions (F5-1⁓F5-10). The subfraction F5-4 (200 mg) eluted with 40% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 40:60, and afforded compound 5 (10.5 mg). The subfraction F5-5 (240 mg) eluted with 50% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 50:50, and afforded compounds 6 (7.7 mg) and 7 (24.0 mg). The subfraction F5-9 (879 mg) eluted with 90% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 90:10, and afforded compounds 8 (17.6 mg), 9 (5.6 mg), 10 (11.5 mg) and 11 (20.0 mg).

The sub-fraction F9 (710.8 g), eluted with n-hexane–EtOAc (20:80), was chromatographed over reversed-phase CC and eluted with MeOH-H2O (0–100% of MeOH gradients) to afford ten sub-fractions (F9-1⁓F9-10). The subfraction F9-5 (150 mg) eluted with 50% MeOH, was finally purified on a reversed-phase HPLC with MeOH–H2O, 50:50, and afforded compound 3 (3.5 mg).

8β,11-Dihydroxy-β-cyperon (2)

Colorless oil; [α]D23 + 96.15 (c 2.6, MeOH); UV λmax nm (log ɛ) (MeOH): 296 (4.04); IR (film) vmax 3393, 2965, 1658, 1541, 1374, 1132, 1095, 1071, 1032 cm−1; 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz): see Table 1; HR-ESI–MS (positive ion-mode) m/z: 273.1461 [M + Na]+ (Calcd 273.1461 for C15H22O3Na).

5-epi-7α-Hydroxy-( +)-oplopanone (3)

Colorless viscous oil; [α]D23 -3.64 (c 3.3, MeOH); IR (film) vmax 3419, 2936, 1705, 1374, 1132, 1033, 501 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz): see Table 1; HR-ESI–MS (positive ion-mode) m/z: 277.1773 [M + Na]+ (Calcd 277.1774 for C15H26O3Na).

Evaluation of cytotoxicity

Cytotoxic activity was determined against different cell lines, A549, MCF-7 and HepG2, using the colorimetric cell viability MTT method described previously [21, 22].

Evaluation of antileishmanial assay

The antileishmanial action was assessed using MTT colorimetric cell viability assay method described previously [23, 24].

Density functional theory calculations

Using Omega2 software (OMEGA, 2.5.1.4; OpenEye Scientific Software: Santa Fe, NM, USA, 2013), conformational analysis was executed to unveil the possible conformers within an energy window of 10 kcal/mol for compounds 2 and 3. All generated conformers were optimized employing the B3LYP/6-31G* level of theory with the aid of Gaussian09 software [25]. Relying on the optimized structures, frequency computations were performed to reveal the nature of the local minimum of the inspected structures and estimate the corresponding Gibbs free energies. Employing a polarizable continuum model (PCM), time-dependent density functional theory (TDDFT) calculations were established at the B3LYP/6-31G* level of theory utilizing methanol as a solvent to compute the first fifty excitation states. Utilizing Gaussian band shapes with sigma = 0.20–30 eV, electronic circular dichroism (ECD) spectra were finally generated with the help of SpecDis 1.71 software (SpecDis 2017) [26, 27]. The extracted ECD spectra were subjected to Boltzmann average.