New sesquiterpenes from the soft coral Litophyton arboreum

The methanol extract of soft corals L. arboreum collected from the Egyptian Red Sea coast was fractionated by silica gel column chromatography to obtain five fractions eluted with combinations of the organic solvents with increasing polarity. These sub-fractions were further separated and purified using reversed-phase open column chromatography and HPLC (ODS) to yield eleven compounds (1–11) as shown in Fig. 1, including seven sesquiterpenes; 11-hydroxy-8-oxo-β-cyperon (1) [14], 8α,11-dihydroxy-β-cyperon (2), 5-epi-7α-hydroxy-( +)-oplopanone (3), alismoxide (4) [10], 5β,8β-epidioxy-11-hydroxy-6-eudesmene (5) [15], chabrolidione B (6) [16], 7-oxo-tri-nor-eudesm-5-en-4β-ol (7) [17], two sterols; 7β-acetoxy-24-methyl-cholesta-5,24(28)-diene-3β,19-diol (8) [10], nebrosteroid M (9) [18], two glycerol derivatives; chimyl alcohol (10) [10]; batyl alcohol (11) [19]. Their structures were identified by intensive spectroscopic analyses, in addition to comparing their physical and chemical properties with those reported.

Compound 2, [α]_D²³ + 96.15 (c 2.6, MeOH), exhibited a molecular formula of C₁₅H₂₂O₃ determined by positive ion-mode HR-ESI–MS that showed a sodiated molecular ion peak at m/z: 273.1461 [M + Na]⁺ (Calcd for C₁₅H₂₂O₃Na⁺: 273.1461). The IR spectrum of 2 displayed an absorption band at 3393 cm⁻¹ for hydroxyl groups. The UV spectrum showed an absorption band at 296 nm for a conjugated system. The NMR data of compound 2 were closely related to compound 1 [14], except for the chemical shift of C-8 that was highly upfield-shifted from 198.6 in compound 1 to 66.8 in compound 2, suggesting the presence of a hydroxyl group at C-8. The ¹H-NMR spectrum of compound 2 (Table 1) showed one doublet olefinic proton signal at δ_H 6.95 (d, J = 1.7 Hz) due to long-range coupling with H-8 assignable for H-6. The four singlet methyl signals at δ_H 1.11, 1.85, 1.88 and 2.02 were assignable for H₃-14, 13, 12, and 15, respectively. The ¹³C-NMR and DEPT spectra (Table 1) showed the characteristic carbon signals at δ_C 198.5 (C-3), 158.1 (C-7), 155.4 (C-5), 128.8 (C-4) and 120.3(C-6), confirming that compound 2 was an eudesmane-type sesquiterpenoid with 4,6-dien-3-one moiety as 1 [14]. In addition, four methyls at δ_C 32.0, 30.7, 22.4, and 11.2 were assignable for C-12, C-13, C-14, and C-15, respectively. Three methylenes at δ_C 36.6 (C-1), 34.2 (C-2) and 48.8 (C-9), a quaternary carbon at δ_C 35.9 (C-10), a hydroxylated methine carbon at δ_C 66.8 (C-8), and a hydroxylated quaternary carbon at δ_C 74.4 (C-11) were also assigned by comparing with the chemical shift values of 1. The HSQC and ¹H-¹H COSY analyses were conducted to determine the ¹J_CH, ²J_HH and ³J_HH connectivities. The HMBC correlations from H-8 with C-6 and 7, from H-6 with C-4, 5, 7, 8, 10, and 11, from H-12/13 with C-7 and 11, from H-14 with C-1, 5, 9 and 10, and from H-15 with C-3, 4 and 5 confirmed the planar structure of 2 (Fig. 2). The axial-axial coupling of H-9α (δ_H 1.91, dd, J = 12.4, 10.6 Hz) indicated the β-axial configuration of H-8. The NOE correlation between H₃-14 and H-8 confirmed that they have the same orientations. The absolute configuration of C-8 and C-10 is determined to be 8S and 10R by ECD calculation, as shown in Fig. 3. Consequently, from the above results, compound 2 was revealed to be a previously undescribed compound, namely 8α,11-dihydroxy-β-cyperon.

Compound 3 [α]_D²³ –3.64 (c = 3.3, MeOH) exhibited a molecular formula of C₁₅H₂₆O₃ determined by positive ion-mode HR-ESI–MS that showed a sodiated molecular ion peak at m/z: 277.1773 [M + Na]⁺ (Calcd for C₁₅H₂₆O₃Na⁺: 277.1774). The IR spectrum of compound 3 displayed the existence of hydroxy (3419 cm⁻¹) and ketone (1705 cm⁻¹) functionalities. The index of hydrogen deficiency suggested a two-ring system. The ¹H-NMR spectrum of 3 (Table 1) showed the presence of a triplet of doublets signal at δ_H 2.08 (td, J = 12.5, 5.4 Hz, H-1) and a doublet of doublets proton signal at δ_H 2.15 (J = 12.5, 8.8 Hz, H-6). These large coupling constants indicated a trans-diaxial linkage of the rings. In addition, two doublet methyls at δ_H 0.76 (d, J = 6.8 Hz, H-12) and δ_H 0.92 (d, J = 6.8 Hz, H-13), and a septet signal at δ_H 1.43 (sept, J = 6.8 Hz, H-11) were assignable for an isopropyl function. Two singlet methyl signals at δ_H 1.15 and δ_H 2.21 were assigned for H-14 and H-15, respectively, based on the difference in chemical shift values. The ¹³C-NMR and DEPT spectra (Table 1) showed the presence of 15 carbon signals, indicating the sesquiterpenoid nature of compound 3. Four methyl carbon signals at δc 18.4, 17.2, 19.3, and 30.2 were assignable for C-12, 13, 14, and 15, respectively. Besides, four methylene carbon signals at δc 26.6, 29.0, 30.4 and 38.1 were assignable for C-2, 8, 3, and 9, respectively. In addition, the ¹³C-NMR revealed the presence of four additional methine carbon signals resonated at δ_C 51.8 (C-1), 50.7 (C-5), 50.4 (C-6) and 37.7 (C-11), two hydroxylated quaternary carbon signals at δ_C 75.7 (C-7), 73.7 (C-10) and one ketonic carbonyl carbon at δ_C 215.1 corresponding to C-4. Full inspection of the compound was carried out through 2D analyses, including ¹H-¹H COSY, HSQC and HMBC analyses as shown in Fig. 4. These data were closely related to oplopanane sesquiterpenes [20], except the downfield shift of C-7 to δ_C 75.6 confirmed the hydroxylation of C-7. The NOESY correlations (Fig. 4) of H-6 with H-5, H-12, and H₃-14 showed the same orientation of them. The absolute configuration was determined to be 1S, 5S, 6R, 7S and 10S by ECD calculation, as shown in Fig. 5. Consequently, from the above-mentioned data, the structure of compound 3 was concluded as a previously undescribed compound, namely 5-epi-7α-hydroxy-( +)-oplopanone.

The growth-inhibitory effect of compounds 1–11 of L. arboreum on the lung adenocarcinoma (A549), breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) was examined. Compound 9 exhibited potent cytotoxic activity against A549 and MCF-7 cell lines (IC₅₀ = 16.5 ± 1.3 and 24.7 ± 2.2 μg/ml, respectively) as compared with the standard antitumor agent etoposide (IC₅₀ 28.4 ± 4.5 and 22.2 ± 4.2 μg/mL, respectively).

Compounds 4 and 8 showed potent cytotoxicity against the A549 cell line (IC₅₀ = 17.0 ± 2.5, and 13.5 ± 2.1 μg/ml, respectively) and moderate cytotoxic activities toward the MCF-7 cell line (IC₅₀ = 35.6 ± 1.9 and 33.1 ± 1.0 μg/mL, respectively), and the HepG2 cell line (IC₅₀ = 34.7 ± 1.5, and 36.7 ± 1.6 μg/mL, respectively). The remaining compounds showed very weak or no activity toward the tested cell lines (Table 2).

The growth-inhibitory effects of the isolated compounds on the L. major promastigotes were assessed. Compounds 5, 8, and 9 showed weak anti-leishmanial activity with IC₅₀ values of 70.2 ± 1.5, 67.4 ± 2.4, and 77.5 ± 1.3 μg/mL, respectively, compared to the standard anti-leishmanial agent miltefosine (IC₅₀ 7.7 ± 2.1 μg/mL), although the other compounds showed no anti-leishmanial activity (Table 2).

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.

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).

Colorless oil; [α]_D²³ + 96.15 (c 2.6, MeOH); UV λ_max nm (log ɛ) (MeOH): 296 (4.04); IR (film) v_max 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).

Colorless viscous oil; [α]_D²³ -3.64 (c 3.3, MeOH); IR (film) v_max 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).

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

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

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.