Polyoxypregnane Ester Derivatives and Lignans from Euphorbia gossypina var. coccinea Pax.

From the aerial parts of Euphorbia gossypina var. coccinea Pax., eight new pregnane glycosides (euphogossypins A–H, 1–8) of the cynanforidine and deacetylmetaplexigenin aglycons, two new lignans (gossypilignans A and B, 9 and 10), and four known compounds, namely, the pregnane 12-O-benzoyldeaxcylmetaplexigenin (11), the lignan 9α-hydroxypinoresinol (12), and the flavonoids naringenin (13) and quercitrin (14) were isolated. The structure elucidation of the new compounds was carried out by a spectroscopic analysis, including HRMS, 1D (1H, 13C JMOD), and 2D NMR (HSQC, 1H–1H COSY, HMBC, and NOESY) experiments. The obtained pregnane glycosides were substituted with acetyl and benzoyl ester moieties, and sugar chains containing thevetose, cymarose, digitoxose, and glucose monosaccharides. All of the compounds are described for the first time from E. gossypina var. coccinea. The isolated pregnanes and lignans were tested for their antiproliferative activity on HeLa cells using the MTT assay; the compounds exerted no significant effect against the tumor cells.


Introduction
Plants belonging to the genus Euphorbia are known to possess considerable chemical, medicinal, and economic importance [1]. Terpenes, including diterpenes and triterpenes, steroids, cerebrosides, glycerols, and phenolic compounds are characteristic constituents of Euphorbia species [2]. In the course of our work focusing on the isolation of special metabolites from various Euphorbia species, the chemical composition of Euphorbia gossypina var. coccinea Pax. (Euphorbiaceae) was thoroughly investigated [3]. The aim of our work was the identification of the special metabolites of the plant. This plant is a perennial, much-branched, succulent, herbaceous, evergreen shrub native to Kenya and Tanzania. Preparations of E. gossypina have long been used in traditional medicine for the treatment of swollen legs and general body pain. Moreover, it is applied as eye drops in the treatment of conjunctivitis and warts [4]. The diluted latex of small twigs is taken to treat laryngitis [5]. In Somalia, the latex is applied to treat mange in livestock [6]. There is no literature data on the phytochemistry and pharmacology of E. gossypina var. coccinea.
Pregnane glycosides are C21 steroidal natural compounds, in which the pregnane part is attached to different sugars. These compounds demonstrate a fair degree of diversity in their aglycone part with different numbers and types of sugar units being attached to the aglycone at position C-3 [7]. The sugar part of these compounds can be a mono-or disaccharide, or an oligosaccharide chain arranged mainly linearly to the pregnane skeleton through an acetal linkage. The characteristic monosaccharides of pregnane glycosides are D-glucose, L-rhamnose, D-cymarose, D-oleandrose, D-allose, and D-digitoxose. The occurrence of pregnane glyosides is characteristic of the Asclepiadaceae, Apocynaceae, Malpighiaceae, Ranunculaceae, and Zygophyllaceae families [8]. Pregnane glycosides are reported to possess noteworthy pharmacological properties, such as immunosuppressant, cytotoxic, antidepressant, anti-inflammatory, antioxidant, antibacterial, antifungal, and antiproliferative activities [8]. Therefore, the isolated compounds were tested for their antiproliferative activity against the HeLa cell line using the MTT assay.

Isolation of Compounds
The dried and ground aerial parts of the plant were extracted with methanol. After evaporation of the methanol, the extract was dissolved in 50% aqueous methanol, and a solvent-solvent partition was performed with n-hexane, CHCl3, and EtOAc. The CHCl3 extract was separated by normal (NP) and reversed-phase (RP) vacuum liquid chromatography (VLC), and then it was purified by preparative thin layer chromatography (prep. TLC) and high-performance liquid chromatography (HPLC) to yield eight new pregnane glycosides (1)(2)(3)(4)(5)(6)(7)(8), two new lignans (9, 10), one known pregnane and one lignan, and two known flavonoids ( Figure 1).

Structure Elucidation of the Compounds
The structure determination of the isolated compounds was carried out by an extensive spectroscopic analysis, including one-and two-dimensional NMR and HRMS measurements.
HRESIMS and NMR data of compounds 4-6 suggested that they were tetrasaccharide derivatives due to the presence of four anomeric carbon and proton signals, one more than observed for compounds 1-3. The additional sugar unit was identified as D-glucopyranose ( Table 2). The polyoxypregnane ester aglycone was the same as in   The NMR data of compound 7 were very similar to that of 2, with the only difference between them being the replacement of the benzoyl moiety into an acetyl substituent at C-12. Therefore, the aglycone of 7 was identified as metaplexigenin (Table 3) [11]. The connecting sugar chain was determined as β-D-thevetopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)β-D-digitoxopyranoside based on the 1D and 2D NMR spectral data. Compound 7 was, thus, characterized as metaplexigenin β-D-thevetopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-digitoxopyranoside and named euphogossypin G. Compound 8 wore the same C-3 trisaccharide sugar chain, β-D-thevetopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-cymaropyranoside, as 3 (Table 3). By the NMR data, it was determined to be a deacetylmetaplexigenin derivative and named euphogossypin H. Gossypilignan A (9) was obtained as a pale yellow, amorphous solid. The molecular formula of 9 was determined as C 22 Na,429.1884). The 1 H and 13 C JMOD NMR data indicated the presence of four methoxy (δ H 2 × 3.82, s, and 2 × 3.83, s; δ C 4 × 56.8), two methyl (δ H 0.68, d, J = 6.9 Hz, and 0.76, d, J = 7.0 Hz; δ C 10.0 and 12.1), one methylene (δ H 3.35 and 3.45; δ C 67.3), and three methine groups (δ H 1.77, m, 2.62, m, 3.52, d; δ C 37.0, 37.2, and 57.9) (Table 4). Additionally, the 1 H NMR spectrum of 9 showed aromatic protons at δ H 6.64, s and 6.66, s (2H each), implying that the aromatic rings were tetrasubstituted. The 13 C NMR spectrum of 9 also supported the presence of six oxygenated aromatic carbons at δ C 149.1, 149.2, and 134.7 (each 2C). According to the HMBC correlations from H-2, H-6, H-2 , and H-6 to C-7, both aromatic rings were attached to C-7. The 1 H-1 H COSY correlations of H-7/H-8/H-8 /H-7 , H-8/H-9, and H-8 /H-9 , as well as the HMBC correlations from H-7 to C-8, C-8 , and C-9, indicated the presence of a 2,3-dimethylbutane moiety (Figure 3). By comparison, the skeleton of 9 was found to be the same as that of kadangustin J [12]. According to the chemical shift of δ C 67.3 (C-7 ), a hydroxy group should be placed at C-7 . Four methoxy groups were located at C-3, C-5, C-3 , and C-5, respectively, while C-4 and C-4 were substituted with hydroxy groups. Thus, the structure of compound 9 was determined to be 4,4-di-(4-hydroxy-3,5-dimethoxyphenyl)-2,3-dimethylbutanol and named gossypilignan A. As compound 9 was a 7,7-diaryl-8,8 -dimethylbutan-1-ol lignan, it had two chiral centers. Based on the investigation of Davidson et al., synand anti-isomers of such compounds can be distinguished based on the significant differences between their 1 H NMR data [13]; therefore, as in the case of 9, both methyl groups were β-oriented, proving that this compound was a syn-isomer. The molecular formula of 10 was determined as C 22 H 28 O 7 by the HRESIMS pseudoion at m/z 427.1734 [M + Na] + . The 1 H NMR spectrum of 10 showed four aromatic hydrogens as two similar systems, one at δ H 6.69 (2H, br s), and another at 6.63 (2H, br s), indicating the presence of two 1,3,4,5-tetrasubstituted benzene rings ( Table 4). The chemical shifts observed for aromatic hydrogens along with the presence of four singlets corresponding to methoxy hydrogens at δ H 2 × 3.84 and 2 × 3.86 (s, 3H each) indicated the presence of 3,5-dimethoxy-4-hydroxyphenyl groups in this compound. The 13 C NMR data corroborated the structural determination of these aromatic rings. Moreover, 1 H NMR spectral data suggested a nonsymmetric tetrahydrofuran lignan, through signals corresponding to two methyl groups at δ H 1.00 (d, J = 6.4 Hz) and 0.63 (d, J = 7.0 Hz) in addition to two oxybenzyl methines at δ H 4.64 (d, J = 9.3) and 5.47 (d, J = 4.4 Hz). The attachment of aromatic rings to the tetrahydrofuran ring was determined by an HMBC experiment. In the HMBC spectrum, interactions were observed between H-2 and C-7 and C-4 , between H-6 /C-4 and C-2 , and between H-2 and C-7. These data indicated the presence of the two 3,5-dimethoxy-4-hydroxyphenyl structural parts at C-7 and C-7 , respectively. Based on previous literature data [14,15], the coupling constant of 9.3 Hz for the doublet at δ   The known compounds were identified as 12-O-benzoyldeacylmetaplexigenin, 9αhydroxypinoresinol [16], naringenin [17], and quercitrin [18]. All compounds were isolated for the first time from the plant. Such polyoxypregnane ester derivatives were isolated previously, mainly from Asclepiadaceae species (e.g., Cynanchum wilfordi and Leptadenia hastata) [9]. Though Euphorbia species are frequently characterized by the abundant presence of various terpenoids, this is only the second report of pregnanes from a plant belonging to the Euphorbiaceae family. Previously, two pregnane glycosides were identified from the aerial parts of Croton ruizianus. Their aglycon was 3β,14β,15β,16α-tetrahydroxypregnan-20-one, while the connecting sugar parts were  [19]. Pregnane glycosides isolated from E. gossypina var. coccinea were substituted with thevetose, cymarose, digitoxose, and glucose-containing linear sugar chains.
The 7,7-diarylbutanol seco-lignans are an interesting class of lignans with both chemical and pharmacological importance. Cytotoxic, anti-HIV-1, and antioxidant activities have been previously reported for this class of natural products [12,20,21].

General Experimental Procedures
NMR spectra were recorded in CDCl 3 , CD 3 OD, and C 5 D 5 N on a Bruker Avance DRX 500 spectrometer (Billerica, MA, USA) at 500 MHz ( 1 H) and 125 MHz ( 13 C). The signals of the deuterated solvents were taken as reference. The chemical shift values (δ) were given in ppm and coupling constants (J) were in Hz. Two-dimensional (2D) experiments were performed with standard Bruker software. In the COSY, HSQC, and HMBC experiments, gradient-enhanced versions were used. High-resolution MS spectra were acquired on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (Waltham, MA, USA) equipped with ESI ion source in positive ionization mode. The data were acquired and processed with MassLynx software.
Column chromatography (CC) was performed on polyamide (MP Biomedicals, Irvine, CA, USA). Normal-phase vacuum liquid chromatography (VLC) was carried out on silica gel (Kieselgel 60 GF 254 , 15 µm, Merck, Darmstadt, Germany). Thin-layer chromatography was performed on Kieselgel 60 RP-18 F 254 and Kieselgel 60 F 254 (Merck, Darmstadt, Germany). Spots were detected under UV light (245 nm and 336 nm) and made visible with vanillin sulfuric acid reagent and heating at 105 • C for 1 min. The high-performance liquid chromatography (HPLC) separation was carried out on a Waters HPLC (Waters 600 controller, Waters 600 pump, and Waters 2998 photodiode array detector), using RP (LiChrospher RP-18, 5 µm, 250 × 4 mm, Merck, Darmstadt, Germany) column. In the case of the RP-HPLC separation, the mobile phase consisted of MeOH (solvent A) and H 2 O (solvent B). The flow rate was 1 mL/min. The data were acquired and processed with the Empower software.
All solvents used for CC were of at least analytical grade (VWR Ltd., Szeged, Hungary). Ultra-pure water was prepared with a Milli-Q water purification system (Millipore, France).

Plant Material
Aerial parts of Euphorbia gossypina var. coccinea Pax. were collected in Kenya (GPS coordinates 1 • 24 24.31777 S, 36 • 42 53.86125 E), Africa, in July 2018, and identified by Patrick Chalo Mutiso, a taxonomist (Department of Biological Sciences, Faculty of Science and Technology, University of Nairobi). A voucher specimen (no. UON 2018/249) was deposited at the Herbarium of the School of Biological Sciences, University of Nairobi, Kenya.