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Ergostane-type steroids from the Cameroonian ‘white tiama’ Entandrophragma angolense
Journal Pre-proofs Ergostane-type steroids from the Cameroonian ‘white tiama’ Entandrophragma angolense Gervais Mouthé Happi, Steven Collins N. Wouamba, Mohamed Ismail, Simeon Fogue Kouam, Marcel Frese, Bruno Ndjakou Lenta, Norbert Sewald PII: DOI: Reference:
S0039-128X(20)30009-X https://doi.org/10.1016/j.steroids.2020.108584 STE 108584
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
8 October 2019 5 January 2020 20 January 2020
Please cite this article as: Happi, G.M., Wouamba, S.C.N., Ismail, M., Kouam, S.F., Frese, M., Lenta, B.N., Sewald, N., Ergostane-type steroids from the Cameroonian ‘white tiama’ Entandrophragma angolense, Steroids (2020), doi: https://doi.org/10.1016/j.steroids.2020.108584
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Ergostane-type
steroids
from
the
Cameroonian
‘white
tiama’
Entandrophragma angolense
Gervais Mouthé Happi1,2,*, Steven Collins N. Wouamba1, Mohamed Ismail2, Simeon Fogue Kouam1, Marcel Frese2, Bruno Ndjakou Lenta1, Norbert Sewald2
1
Department of Chemistry, Higher Teacher Training College, University of Yaounde I, P. O.
Box 47, Yaounde, Cameroon 2
Organic and Bioorganic Chemistry, Faculty of Chemistry, Bielefeld University, D-33501
Bielefeld, Germany
------Correspondence: [email protected], [email protected] (G. M. Happi)
1
Abstract Two new ergostane-type steroids named tiamenones A and B (1–2) were isolated from the bark of Entandrophragma angolense (Meliaceae) along with ten known compounds identified as 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3), 3β,7α,20β-trihydroxyergosta-5,24(28)-diene (4), 3β,5α-dihydroxyergosta-7,22-diene (5), stigmasterol, β-sitosterol, β-amyrin, oleanolic acid, asperphenamate, sucrose and daucosterol. The structures of the isolated compounds have been established using NMR spectroscopic and mass spectrometric analyses. The assignment of relative and absolute configurations of compound 1 was achieved by a NOESY experiment and comparison of its NMR data with those of known compound reported in literature. Compounds 1–3, β-amyrin and asperphenamate were evaluated for their antibacterial potencies against five bacterial model strains viz. Escherichia coli DSMZ 1058, Pseudomonas agarici DSMZ 11810, Bacillus subtilis DSMZ 704, Micrococcus luteus DMSZ 1605 and Staphylococcus warneri DSMZ 20036 and their cytotoxicity on two cell lines KB3-1 and HT-29. No potencies were exhibited by the tested compounds even at the highest concentration of 0.5 mg/mL. Compounds 1–3 were found to be potential HIV-1 inhibitors based on their molecular docking analyses.
Keywords: Entandrophragma angolense, Meliaceae, tiamenones A and B, antibacterial, cytotoxicity, HIV-1 inhibitors
2
1. Introduction The genus Entandrophragma (Meliaceae), also called “tiama” by local population, is native to Africa and well reputed for its medicinal potential in the treatment of several illnesses including malaria or rheumatism [1–3]. It comprises ten species, including E. angolense “white tiama” found in Cameroon [4]. Our recent publication covers significant information on phytochemical and biological investigations of this genus over the last half-century [5]. Phytochemical studies into the species E. angolense known as ‘white tiama’ in Cameroon, revealed the presence of limonoids [6–9], tirucallane triterpenoids [10] and other minor classes of metabolites such phenolic compounds and fatty acids [4,11]. Briefly, limonoids and protolimonoids have been the most reported metabolite classes and were defined as chemotaxonomic markers of the genus [5]. Additionally, numerous triterpenoids, sesquiterpenoids and steroids have been also reported from the genus so far. Five of the ten distinct Entandrophragma species, including E. angolense, are found in Cameroon [4]. In recent years, our research work on the species E. cylindricum (‘sapele’) and E. congoënse (‘black tiama’) led to the report of novel triterpenoids with significant anti-inflammatory and antiplasmodial activities, as well as known limonoids and steroids [1–3,12]. In our continuous search for bioactive metabolites from the genus Entandrophragma collected in Cameroon, a phytochemical investigation on hydro-ethanolic extract of E. angolense was performed. We herein report the isolation and structure elucidation of two new steroids namely tiamenones A (1) and B (2), as well as their molecular docking studies to the enzyme pyruvate kinase PKM2. This enzyme plays a role in the inhibition of HIV-1 as a rate limiting enzyme in glycolysis, where host cell kinases were found important for the development of viral infections [13,14].
2. Experimental 2.1. General experimental procedures Optical rotation indices (in degrees) were determined on a JASCO DIP-3600 digital polarimeter (JASCO, Tokyo, Japan) using a 10 cm cell. IR spectra were determined on a JASCO Fourier transform IR-420 spectrometer (JASCO). Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH and infrared spectra on a JASCO 302-A spectrophotometer (Thermo Scientific, Waltham, MA, USA). EI mass spectra were recorded using an Autospec X (Vacuum Generators, Manchester, UK). Samples were dissolved in dichloromethane and aluminium crucibles were filled with this solution and the solvent was evaporated. The aluminium crucibles were introduced by push rod. Ions were accelerated by 8 kV in EI mode. 3
The mass axis was externally calibrated with PFK (perfluorokerosine) as calibration standard. ESI accurate mass measurements were acquired using an Agilent 6220 (Agilent Technologies, Santa Clara, CA, USA) in extended dynamic range mode equipped with a Dual-ESI source, operating with a spray voltage of 2.5 kV. Nitrogen served both as nebulizer gas and dry gas. Nitrogen was generated by a nitrogen generator NGM 11. Samples were dissolved in acetonitrile and introduced with a 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a C18 Hypersil Gold column (length: 50 mm, diameter: 2.1 mm, particle size: 1,9 μm) with a short gradient (in 4 min from 0% B to 98% B, back to 0% B in 0.2 min, total run time 7.5 min) at a flow rate of 250 μL/min and column oven temperature of 40°C. HPLC solvent A consists of 94.9% water, 5% acetonitrile and 0.1% formic acid, solvent B of 5% water, 94.9% acetonitrile and 0.1% formic acid. The mass axis was externally calibrated with ESI-L Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) as calibration standard. The 1H- and 13CNMR spectra were recorded on Bruker DRX 600
spectrometer (Bruker, Rheinstetten,
Germany) in CDCl3. Chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as internal standard, while coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel 230–400 mesh, Merck, (Merck, Darmstadt, Germany), silica gel 70–230 mesh (Merck) and sephadex LH-20 (Sigma-Aldrich). Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 aluminum foil (Merck).
2.2. Plant material The bark of E. angolense was collected in November 2016 at Bertoua, East Region of Cameroon. The plant was identified by the botanist Mr. Victor Nana at the National Herbarium of Cameroon, where a voucher specimen (N°29031/SRF/Cam) was deposited.
2.3. Extraction and isolation Dried and powdered bark of E. angolense (~3.0 kg) were macerated twice with a hydroethanolic mixture of EtOH/H2O (1:1, v/v) for 48 h each, at room temperature. Evaporation of solvent mixture produced a crude extract (81.0 g), which was dissolved again in water and partitioned with hexane (Hex), ethyl acetate (EtOAc) and butanol (n-BuOH) to afford three fractions A (26 g), B (35 g) and C (20 g), respectively. The oily fraction A was mainly contained fatty acids and was not investigated further. The semi-polar fraction B was subjected to silica gel column chromatography using of gradient of ethyl acetate in hexane to yield 186 fractions (ca. 150 mL, each) which were combined into 18 sub-fractions B1-B18 based on their TLC 4
profiles. The mixture of known steroids β-sitosterol and stigmasterol (16 mg), their glucoside derivative daucosterol, the triterpene β-amyrin (31 mg), the sugar sucrose (8 mg), as well as the peptide asperphenamate (12 mg) were all precipitated as white powders from the sub-fractions B2 (2.6 g, Hex/EtOAc 19:1), B3 (1.8 g, Hex/EtOAc 8:1), B14 (2.9 g, EtOAc), B12 (2.2 g, Hex/EtOAc 1:3), B10 (3 g, Hex/EtOAc 1:1), respectively. The sub-fractions B6 (4,3 g, Hex/EtOAc 3:1) and B8 (2,8 g, Hex/EtOAc 7:3) were further purified using silica gel column chromatography eluting with the same solvent polarity used previously for their elution to afford compound 4 (8 mg) and oleanolic acid (7 mg) from B6, as well as compound 5 (6 mg) from B8. Compound (3) (11 mg) was filtered off as white precipitate from fraction B4 (3.8 g, Hex/EtOAc 17:3) and the yellowish filtrate was subjected to sephadex LH-20 column chromatography eluting with the mixture of dichloromethane-methanol (3:2) to afford compounds 1 (4 mg) and 2 (3 mg). 2.3.1. Tiamenone A (1) Oil; [α]20D + 3.6 (c 0.28, CHCl3); UV (MeOH) λmax (log ε) 288 (2.63), 235 (0.41), 220 (0.11); IR (KBr) νmax 2957, 2361, 1661, 1030, 798 cm-1; 1H and 13C-NMR data, see Table 1; HR-ESIMS: m/z 449.3027 [M + Na]+ (calcd for C28H42O3Na, 449.3026). 2.3.2. Tiamenone B (2) Oil; [α]20D + 40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 327 (0.37), 313 (0.51), 298 (0.67), 286 (0.79), 225 (2.98); IR (KBr) νmax 3366, 2357, 1641, 1092 cm-1; 1H and 13C-NMR data, see Table 1; HR-ESI-MS: m/z 435.3231 [M + Na]+ (calcd for C28H44O2Na, 435.3234).
2.4. Docking experiments The X-ray crystal structure data of pyruvate kinase M2 (PKM2) was downloaded from the protein data bank (PDB entry: 3gr4). The docking study was performed using YASARA structure. The crystal structure was optimized by removing the water molecules and ligands, the structure was further energy minimized using the AMBER14 force field with a 10Å cut-off and PME was used for long range electrostatic. Structures of the different tested compounds were built using YASARA structure and energy minimized using YASARA2 force field before using in docking experiments. Docking was performed using Autodock [15]. The simulation cell was defined around the binding domain at the interface between monomer subunits of the PKM2 for docking of compounds 1–3. Docking results were analyzed based on the B-factor
5
(binding energy) calculated by YASARA structure and interaction with the amino acid residues of the target binding pocket.
3. Results and discussion Compound 1 was isolated as an oil with a positive specific rotation αD20 + 3.6° and the composition C28H42O3 deduced from its accurate mass determined by HR-ESI-MS {m/z 449.3027 [M + Na]+ (calcd for C28H42O3Na, 449.3026)}. The UV and IR spectra showed absorptions at 288 nm and 1661 cm-1, respectively, revealing the presence in the molecule a ketone conjugated with double bonds. The 1H NMR spectrum (Table 1) contains singlets for three methyl groups at δ 0.91, 1.13 and 1.34 attached to quaternary carbons; two doublets of methyl groups at δ 0.90 and 1.01 attached to a tertiary carbon; two doublets at δ 3.35 and 3.65, resp., corresponding to an oxymethylene moiety; and signals of three olefinic protons. These protons resonate as a singlet at δ 5.67 and as two double doublets at δ 6.10 (dd, J = 2.6 and 9.5 Hz) and 6.16 (dd, J = 1.5 and 9.9 Hz) indicating a cis-double bond. The 13C NMR spectrum (Table 1) contains signals of 28 carbon atoms, which were sorted using the HMQC spectrum into one ketone carbonyl (δ 199.7), four sp2 carbons (δ 164.0, 141.4, 127.8 and 123.5), five methyls (δ 27.4, 18.3, 17.3, 16.3 and 13.5), one oxymethylene (δ 63.3), eight methylenes, five methines and four quaternary sp3 carbon atoms. All these data suggested 1 to be a steroid derivative similar to the known 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) that was also isolated in the course of this investigation. Compounds 1 and 3 share the same tetracyclic moiety (rings A–D) containing a 4,6-dien-3-one system indicated by the presence of protons at δ 5.67 (H-4), 6.10 (H-6), 6.16 (H-7) and carbons appearing at δ 199.7 (C-3), 123.5 (C-4), 164.0 (C-5), 127.8 (C-6) and 141.4 (C-7). This system was further supported by the HMBC cross-peaks from the proton H-4 at δ 5.67 to the carbon signals at δ 199.7 (C-3), 164.0 (C-5) and 127.8 (C-6), as well as the cross-peaks between the proton H-7 at δ 6.16 and the carbon signals at δ 164.0 (C-5) and 127.8 (C-6). Additionally, the HMBC correlations (Fig. 2) between the methyl group at δ 0.91 (H3-18) and the carbon signals at δ 59.7 (C-17), 40.0 (C12) and 43.6 (C-13); those between the methyl group at δ 1.13 (H3-19) and the carbon signals at δ 50.6 (C-9), 36.0 (C-10) and 33.9 (C-1); together with the COSY correlations (Fig. 2) between H-1/H-2, H-8/H-9/H-11/H-12 and H-14/H2-15/H2-16/H-17, allowed to establish unambiguously the tetracyclic nucleus of compound 1 as shown in Figure 1. However, the uncommon side chain connected at C-17 has not yet been reported in any steroid derivatives hitherto. The literature survey and comparison of 13C NMR data indicated that compound 1 and the dammarane-type saponin operculinoside A reported previously share the same side chain 6
[16]. The carbon signals at δ 86.1 (C-20), 88.3 (C-24) and 63.3 (C-28) revealed the presence of a 24-hydroxymethyltetrahydrofuran moiety in the side chain. This partial conclusion was confirmed by the cross-peaks observed in the HMBC spectrum (Fig. 2) between the methyl groups at δ 0.90 and 1.01 (H3-26 and H3-27, resp.) to the carbon signal at δ 88.3 (C-24), from the methyl group at δ 1.34 (H3-21) to the carbon signal at δ 86.1 (C-20), as well as from the tertiary oxymethylene protons at δ 3.35 and 3.65 (H2-28) to the carbon appearing at δ 29.7 (C23), 88.3 (C-24) and 34.7 (C-25). The relative configuration of 1 was established based on its NOESY spectrum (Fig. 3) in which the correlations observed between the proton signals of H3-19/H-8 and H-8/H-18, confirmed their β-orientation. Additionally, the spatial correlations from H-9/H-14/H-17 put forward their α-orientation. However, we observed that the side is stabilized via hydrogen bond, the missing NOE cross-peaks between H-17 and H3-21 suggested that they are on the opposite faces while the cross-peaks between the proton signals of H3-21, H-22β, H-23β and H-25 show that they are placed on the same face (Fig. 3). This partial conclusion on the orientation of the isopropyl group connected in C-24 was supported by the comparison of NMR data of compound 1 with those of operculinoside A that had been structurally confirmed by X-ray diffraction [16]. We observed that both compounds share very close chemical shift values of 1H and 13C from C-20 to C-28 (for 1) or C-31 (for operculinoside A); and especially, identical proton coupling constants (J) for H-25, H3-26, H3-27 and H2-28. Based on these accepted β-orientations of H321 and the isopropyl group in C-24, both C-20 and C-24 were assigned with S configurations. The new ergostane-type steroid (20S,24S)-20,24-epoxy-24-hydroxymethylergostane-4,6-dien3-one was given the trivial name tiamenone A (1) . Compound 2 was obtained as an oil with a positive specific rotation αD20 + 40°. Its formula C28H44O2 was deduced from the HR-ESI-MS spectrum {m/z 435.3231 [M + Na]+ (calcd for C28H44O2Na, 435.3234)}. The IR spectrum exhibited vibration frequencies of a hydroxyl group (3366 cm-1) and of a conjugated ketone (1641 cm-1). Careful comparison of 1H and 13C NMR data (Table 1) of compounds 2 and 3 showed that they share the same side chain with a striking difference in the tetracyclic moiety. In this regard, analysis of the 13C NMR spectra of 2 and 3 (Table 1) revealed that the signals of the double bond Δ6,7 at δ 127.7 (C-6) and 141.2 (C-7) in 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) are replaced by two methylene carbon signals at δ 32.9 (C-6) and 31.9 (C-7) in 2. This was confirmed by the HMQC and HMBC NMR data, as well as the two mass units difference between both compounds 2 and 3. The heteronuclear correlations between the olefinic proton signal at δ 5.73 (s, H-4) and the carbon signals at δ 7
199.7 (C-3), 171.5 (C-5), 38.6 (C-10) and 32.9 (C-6) confirmed an enone moiety in ring A as presented in 2 (see Fig. 1), as well as an aliphatic chain from C-6 to C-9, strongly supported by the COSY correlations (Fig. 2) in this section of the ring B. The absolute configuration of C-20 was assigned to be S with respect of the biosynthesis evidence of ergostane-type steroids and comparison with the literature. The compound 20S-hydroxy-ergostane-4,24(28)-dien-3-one 2 was given the trivial name tiamenone B. A literature survey indicated that the secondary metabolites with a 2-(hydroxymethyl)-2isopropyl-5-methyltetrahydrofuran moiety in the side chain are very rare. To the best of our knowledge, a small number of dammarane-type triterpenoids has been reported so far from Euphorbia supina [17] and Operculina turpethum [16]. However, tiamenone A (1) is reported here as the first steroid with a 2-(hydroxymethyl)-2-isopropyl-5-methyltetrahydrofuran moiety. A proposed biosynthetic pathway for tiamenone A (1) is depicted in Figure 4. Since compound 1 is an ergostane-type steroid, the formation of its side chain could start from the core structure of 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) in which an enzymatic oxidation of the terminal exomethylene Δ24,28 took place to afford an epoxide. A cyclization may occur through an attack of this epoxide at C-24 by the hydroxyl group at C-20 to obtain compound 1. In addition to 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) [18,19], the other known compounds were identified as 3β,7α,20β-trihydroxyergosta-5,24(28)-diene (4) [20], 3β,5αdihydroxyergosta-7,22-diene (5) [21,22], stigmasterol and β-sitosterol [23], daucosterol [24], asperphenamate [25], sucrose [26], β-amyrin [27] and its derivative oleanolic acid [28] based on the comparison of their spectral data with those reported in the literature. New compounds tiamenones A (1) and B (2), as well as the known products namely 20Shydroxy-4,6,24(28)-ergostatrien-3-one (3), β-amyrin and asperphenamate were tested for antibacterial activity against five bacterial strains including Escherichia coli DSMZ 1058, Pseudomonas agarici DSMZ 11810, Bacillus subtilis DSMZ 704, Micrococcus luteus DMSZ 1605 and Staphylococcus warneri DSMZ 20036, using gentamicin as positive control and their cytotoxicity were evaluated against KB3-1 and HT-29 cell lines using griseofulvin as positive control. All tested compounds did not show any antibacterial or cytotoxic even at a concentration as high as 0.5 mg/mL. Based on previous studies on a number of new ergostane-type steroids discovered with potent anti-HIV activity; proteomic approach postulated pyruvate kinase M2 (PKM2) as the target enzyme in the glucose metabolism pathway to compromise the replication of HIV-1 [29,30], 8
docking of compounds 1–3 against pyruvate kinase M2 compared to the activator reference compound 1-[(2,6-difluorophenyl)sulfonyl]-4-(2,3-dihydro-1,4-benzodioxin-6-ylsulfonyl)piperazine (DYY) from the crystal structure of pyruvate kinase M2 (PDB: 3gr4), have revealed high affinity for compound 1 (8.31 kcal mol-1) compared to the reference (Table 2). An H-bond was also detected to the Arg400 residue located at the interface between two monomers of pyruvate kinase M2, in addition to the hydrophobic interactions with amino acid residues within the activator binding pocket at the interface between the protein subunits (Fig. 5). Likewise, compound 3 had a binding energy comparable to the reference compound. In a similar manner to compound 1; two H-bonds were detected with Arg400 and Glu397 residues (Fig. 6). Those results suggest a possible role of compounds 1 and 2 in targeting pyruvate kinase M2. Conversely, compound 2 showed lower binding energy and no interaction could be established with the target binding site (Fig. 7). Docking was also performed with the reference compound DYY to confirm the correct binding position. The compound bound to pyruvate kinase M2 at the interface between the two monomers in a similar fashion to compounds 1–3. A binding energy of 8.21 was calculated for DYY and two H-bonds with Arg400 and Tyr390 residues were detected (Fig. 8). The docking results suggest that there might be a possible role of compounds 1 and 2 in targeting the activator binding site in pyruvate kinase M2 when compared to the reference compound DYY.
4. Conclusion In summary, the phytochemical investigation of the bark of Entandrophragma angolense led to the isolation and characterization of two new ergostane-type steroids namely tiamenones A and B (1 and 2) along with ten known compounds. To the best of our knowledge, compound 1 is reported as the first steroid with a 2-(hydroxymethyl)-2-isopropyl-5-methyltetrahydrofuran moiety and its pausible biosynthesis is proposed herein. Although no antibacterial and cytotoxic effects were depicted for the isolated compounds, their report provides further information to enrich the chemistry of the genus Entandrophragma and varies the chemotaxonomic potential of steroids. Moreover, the molecular docking studies suggested that the new compounds 1 and 2 might be potential anti-HIV inhibitors. This study further supports the identification of E. angolense as source of steroids and the chemical investigation as well as in vitro assays of the extracts and the compounds from other plant parts (leaves, root and fruits) could give further evidence on the active principles of the plant and confirm its use in traditional medicine.
9
Acknowledgements This work was supported by the German Academic Exchange Service (DAAD) with funds from the Federal Ministry for Economic Cooperation and Development (BMZ) through the postdoctoral scholarship to G.M.H., the postgraduate scholarship to S.C.N.W. and the equipment subsidies to S.K.F., all in the frame of the Yaounde-Bielefeld Graduate School of Natural
Products
with
Antiparasite
and
Antibacterial
Activities
(YaBiNaPA,
www.yabinapa.de), project N° 57316173. The authors are grateful to Carmela Michalek, Bielefeld University, for the biological activity testing and to the NMR and MS departments at Bielefeld University for the spectral measurements.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at https:// References [1] G.M. Happi, S.F. Kouam, F.M. Talontsi, M. Lamshöft, S. Zühlke, J.O. Bauer, C. Strohmann, M. Spiteller, J. Nat. Prod. 78 (2015) 604–614. [2] G.M. Happi, F.M. Talontsi, H. Laatsch, S. Zühlke, B.T. Ngadjui, M. Spiteller, S.F. Kouam, Fitoterapia 124 (2018) 17–22. [3] S.F. Kouam, S. Kusari, M. Lamshöft, O.K. Tatuedom, M. Spiteller, Phytochemistry 83 (2012) 79–86. [4] J. Bickii, G.R.F. Tchouya, J.C. Tchouankeu, E. Tsamo, Afr. J. Trad. Compl. Alt. 4 (2007) 135–139. [5] G.M. Happi, B.T. Ngadjui, I.R. Green, S.F. Kouam, J. Pharm. Pharmacol. 70 (2018) 14311460. [6] V.C. Njar, J.K. Adesanwo, Y. Raji, Planta Med. 61 (1995) 91–92. [7] N.T. Kipassa, H. Okamura, M. Doe, Y. Morimoto, T. Iwagawa, M. Nakatani, Heterocycles 75 (2008) 157–164. [8] T.K. Nsiama, H. Okamura, T. Hamada, Y. Morimoto, M. Doe, T. Iwagawa, M. Nakatani, Phytochemistry 72 (2011) 1854–1858. [9] W.Y. Zhang, F.L. An, M.M. Zhou, M.H. Chen, K.L. Jian, O. Quasie, M.H. Yang, J. Luo, L.Y. Kong, RSC Adv. 6 (2016) 97160–97171. [10] A.T. Orisadipe, A.A. Adesomoju, M. D’Ambrosio, A. Guerriero, J.I. Okogun, Phytochemistry 66 (2005) 2324–2328. [11] A.T. Orisadipe, N.N. Ibekwe, A.A. Adesomoju, J.I. Okogun, Afr. J. Pure Appl. Chem. 6 (2012) 184–187.
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for compounds 1–3 in CDCl3
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22α 22β 23α 23β 24 25 26 27 28
1 δC 33.9 33.9 199.7 123.5 164.0 127.8 141.4 37.3 50.6 36.0 20.6 40.0 43.6 53.5 23.3 23.1 59.7 13.5 16.3 86.1 27.4 37.8 29.7 88.3 34.7 18.3 17.3 63.3
δH (J in Hz) 1.72b, 2.00b 2.45b, 2.62b 5.67, s 6.10, dd (2.6, 9.6) 6.16, dd (1.5, 9.6) 2.22, m 1.22, m 1.43b, 1.56b 1.27b, 2.14b 1.25, m 1.80b 1.88b 1.65b 0.91, s 1.13, s 1.34, s 1.64b 1.98b 1.74, m 1.91, m 1.87b 0.90, d (6.9) a 1.01, d (6.9) a 3.35, d (11.4) 3.65, d (11.4)
2 δC 35.7 33.9 199.7 123.8 171.5 32.9 31.9 34.9 53.7 38.6 20.8 40.0 42.3 56.0 23.7
δH (J in Hz) 1.70b, 2.04b 2.32b, 2.57b 5.73, s 2.29, 2.41 1.04, 1.86 1.58, m 0.92, m 1.42, 1.55, m 1.26, 2.16, m 1.04, m 1.17b, 1.68b
3 δC 33.9 33.9 199.6 123.6 163.4 127.7 141.2 37.0 50.6 36.0 20.7 39.8 43.6 53.5 23.2
δH (J in Hz) 1.71b, 2.01b 2.45b, 2.58b 5.68, s 6.12 dd (2.3, 9.8) 6.15 dd (1.8, 9.8) 2.22, m 1.21, m 1.48b, 1.60b 1.27b, 2.16b 1.25, m 1.33b, 1.85b
22.3 57.8 13.6 17.4 75.0 26.3 42.7
1.67b, 1.84b 1.51, m 0.91, s 1.19, s 1.31, s 1.55
22.3 57.7 13.5 16.2 74.9 26.2 42.4
1.27b, 2.14b 1.53, m 0.96, s 1.12, s 1.32, s 1.56
28.9
2.02
28.9
2.02
156.2 33.9 21.9 21.9 106.3
2.28, m 1.04, d (7.0) a 1.03, d (7.0) a 4.68, brs 4.74, brs
156.1 33.8 21.8 21.8 106.3
2.24, m 1.03, d (7.0) a 1.03, d (7.0) a 4.69, brs 4.75, brs
a
Values are exchangeable in the same column. bOverlapped signals are reported without multiplicities.
12
Table 2: Binding energy calculated for the docked compounds 1–3 in comparison to the reference compound DYY.
Compound
Binding energy (Kcal mol-1)
1
8.31
2
7.02
3
7.97
DYY
8.21
13
Figure Legends: Fig. 1: Ergostane-type steroids isolated from bark of E. angolense. Fig. 2: 1H-1H COSY and main HMBC correlations for 1 and 2. Fig. 3: Key NOESY correlations for 1. Fig. 4: Proposed biosynthesis pathway for compounds 1 and 2. Fig. 5: (A) Docking of compound 1 (blue) to pyruvate kinase M2 (grey), showing the Hbonding (yellow dots) with Arg400 residue (white). (B) Interaction between compound 1 and PKM2 using Ligplot+. Fig. 6: (A) Docking of compound 3 (blue) to pyruvate kinase M2 (grey), showing the Hbonding (yellow dots) with Arg400 and Glu397 residue (white). (B) Interaction between compound 3 and PKM2 using Ligplot+. Fig. 7: (A) Docking of compound 2 (blue) to pyruvate kinase M2 (grey), no H-bonding to the protein residues was detected. (B) Interaction between compound 2 and PKM2 using Ligplot+. Fig. 8: Docking of the reference compound DYY (blue) to pyruvate kinase M2 (grey), where H-bonding (yellow dots) with Arg400 and Tyr390 residue (white) were detected.
14
Fig. 1: Ergostane-type steroids isolated from bark of E. angolense.
15
Fig. 2: 1H-1H COSY and main HMBC correlations for 1 and 2.
16
Fig. 3: Key NOESY correlations for 1.
17
Fig. 4: Proposed biosynthesis pathway for compound 1.
18
A
B
Fig. 5: (A) Docking of compound 1 (blue) to pyruvate kinase M2 (grey), showing the H-bonding (yellow dots) with Arg400 residue (white). (B) Interaction between compound 1 and PKM2 using Ligplot+.
19
A
B
Fig. 6: (A) Docking of compound 3 (blue) to pyruvate kinase M2 (grey), showing the H-bonding (yellow dots) with Arg400 and Glu397 residue (white). (B) Interaction between compound 3 and PKM2 using Ligplot+.
20
A
B
Fig. 7: (A) Docking of compound 2 (blue) to pyruvate kinase M2 (grey), no H-bonding to the protein residues was detected. (B) Interaction between compound 2 and PKM2 using Ligplot+.
21
Fig. 8: Docking of the reference compound DYY (blue) to pyruvate kinase M2 (grey), where Hbonding (yellow dots) with Arg400 and Tyr390 residue (white) were detected.
Graphical Abstract
Highlights
Two new steroids tiamenones A and B were obtained from Entandrophragma angolense
For the first time a steroid with a 20,24-epoxy-24-hydroxymethyl moiety was isolated
We reported the molecular docking studies of tiamasterols A, B and amasterol to enzyme pyruvate kinase PKM2
22
S0039-128X(20)30009-X https://doi.org/10.1016/j.steroids.2020.108584 STE 108584
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
8 October 2019 5 January 2020 20 January 2020
Please cite this article as: Happi, G.M., Wouamba, S.C.N., Ismail, M., Kouam, S.F., Frese, M., Lenta, B.N., Sewald, N., Ergostane-type steroids from the Cameroonian ‘white tiama’ Entandrophragma angolense, Steroids (2020), doi: https://doi.org/10.1016/j.steroids.2020.108584
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© 2020 Published by Elsevier Inc.
Ergostane-type
steroids
from
the
Cameroonian
‘white
tiama’
Entandrophragma angolense
Gervais Mouthé Happi1,2,*, Steven Collins N. Wouamba1, Mohamed Ismail2, Simeon Fogue Kouam1, Marcel Frese2, Bruno Ndjakou Lenta1, Norbert Sewald2
1
Department of Chemistry, Higher Teacher Training College, University of Yaounde I, P. O.
Box 47, Yaounde, Cameroon 2
Organic and Bioorganic Chemistry, Faculty of Chemistry, Bielefeld University, D-33501
Bielefeld, Germany
------Correspondence: [email protected], [email protected] (G. M. Happi)
1
Abstract Two new ergostane-type steroids named tiamenones A and B (1–2) were isolated from the bark of Entandrophragma angolense (Meliaceae) along with ten known compounds identified as 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3), 3β,7α,20β-trihydroxyergosta-5,24(28)-diene (4), 3β,5α-dihydroxyergosta-7,22-diene (5), stigmasterol, β-sitosterol, β-amyrin, oleanolic acid, asperphenamate, sucrose and daucosterol. The structures of the isolated compounds have been established using NMR spectroscopic and mass spectrometric analyses. The assignment of relative and absolute configurations of compound 1 was achieved by a NOESY experiment and comparison of its NMR data with those of known compound reported in literature. Compounds 1–3, β-amyrin and asperphenamate were evaluated for their antibacterial potencies against five bacterial model strains viz. Escherichia coli DSMZ 1058, Pseudomonas agarici DSMZ 11810, Bacillus subtilis DSMZ 704, Micrococcus luteus DMSZ 1605 and Staphylococcus warneri DSMZ 20036 and their cytotoxicity on two cell lines KB3-1 and HT-29. No potencies were exhibited by the tested compounds even at the highest concentration of 0.5 mg/mL. Compounds 1–3 were found to be potential HIV-1 inhibitors based on their molecular docking analyses.
Keywords: Entandrophragma angolense, Meliaceae, tiamenones A and B, antibacterial, cytotoxicity, HIV-1 inhibitors
2
1. Introduction The genus Entandrophragma (Meliaceae), also called “tiama” by local population, is native to Africa and well reputed for its medicinal potential in the treatment of several illnesses including malaria or rheumatism [1–3]. It comprises ten species, including E. angolense “white tiama” found in Cameroon [4]. Our recent publication covers significant information on phytochemical and biological investigations of this genus over the last half-century [5]. Phytochemical studies into the species E. angolense known as ‘white tiama’ in Cameroon, revealed the presence of limonoids [6–9], tirucallane triterpenoids [10] and other minor classes of metabolites such phenolic compounds and fatty acids [4,11]. Briefly, limonoids and protolimonoids have been the most reported metabolite classes and were defined as chemotaxonomic markers of the genus [5]. Additionally, numerous triterpenoids, sesquiterpenoids and steroids have been also reported from the genus so far. Five of the ten distinct Entandrophragma species, including E. angolense, are found in Cameroon [4]. In recent years, our research work on the species E. cylindricum (‘sapele’) and E. congoënse (‘black tiama’) led to the report of novel triterpenoids with significant anti-inflammatory and antiplasmodial activities, as well as known limonoids and steroids [1–3,12]. In our continuous search for bioactive metabolites from the genus Entandrophragma collected in Cameroon, a phytochemical investigation on hydro-ethanolic extract of E. angolense was performed. We herein report the isolation and structure elucidation of two new steroids namely tiamenones A (1) and B (2), as well as their molecular docking studies to the enzyme pyruvate kinase PKM2. This enzyme plays a role in the inhibition of HIV-1 as a rate limiting enzyme in glycolysis, where host cell kinases were found important for the development of viral infections [13,14].
2. Experimental 2.1. General experimental procedures Optical rotation indices (in degrees) were determined on a JASCO DIP-3600 digital polarimeter (JASCO, Tokyo, Japan) using a 10 cm cell. IR spectra were determined on a JASCO Fourier transform IR-420 spectrometer (JASCO). Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH and infrared spectra on a JASCO 302-A spectrophotometer (Thermo Scientific, Waltham, MA, USA). EI mass spectra were recorded using an Autospec X (Vacuum Generators, Manchester, UK). Samples were dissolved in dichloromethane and aluminium crucibles were filled with this solution and the solvent was evaporated. The aluminium crucibles were introduced by push rod. Ions were accelerated by 8 kV in EI mode. 3
The mass axis was externally calibrated with PFK (perfluorokerosine) as calibration standard. ESI accurate mass measurements were acquired using an Agilent 6220 (Agilent Technologies, Santa Clara, CA, USA) in extended dynamic range mode equipped with a Dual-ESI source, operating with a spray voltage of 2.5 kV. Nitrogen served both as nebulizer gas and dry gas. Nitrogen was generated by a nitrogen generator NGM 11. Samples were dissolved in acetonitrile and introduced with a 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a C18 Hypersil Gold column (length: 50 mm, diameter: 2.1 mm, particle size: 1,9 μm) with a short gradient (in 4 min from 0% B to 98% B, back to 0% B in 0.2 min, total run time 7.5 min) at a flow rate of 250 μL/min and column oven temperature of 40°C. HPLC solvent A consists of 94.9% water, 5% acetonitrile and 0.1% formic acid, solvent B of 5% water, 94.9% acetonitrile and 0.1% formic acid. The mass axis was externally calibrated with ESI-L Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) as calibration standard. The 1H- and 13CNMR spectra were recorded on Bruker DRX 600
spectrometer (Bruker, Rheinstetten,
Germany) in CDCl3. Chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as internal standard, while coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel 230–400 mesh, Merck, (Merck, Darmstadt, Germany), silica gel 70–230 mesh (Merck) and sephadex LH-20 (Sigma-Aldrich). Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 aluminum foil (Merck).
2.2. Plant material The bark of E. angolense was collected in November 2016 at Bertoua, East Region of Cameroon. The plant was identified by the botanist Mr. Victor Nana at the National Herbarium of Cameroon, where a voucher specimen (N°29031/SRF/Cam) was deposited.
2.3. Extraction and isolation Dried and powdered bark of E. angolense (~3.0 kg) were macerated twice with a hydroethanolic mixture of EtOH/H2O (1:1, v/v) for 48 h each, at room temperature. Evaporation of solvent mixture produced a crude extract (81.0 g), which was dissolved again in water and partitioned with hexane (Hex), ethyl acetate (EtOAc) and butanol (n-BuOH) to afford three fractions A (26 g), B (35 g) and C (20 g), respectively. The oily fraction A was mainly contained fatty acids and was not investigated further. The semi-polar fraction B was subjected to silica gel column chromatography using of gradient of ethyl acetate in hexane to yield 186 fractions (ca. 150 mL, each) which were combined into 18 sub-fractions B1-B18 based on their TLC 4
profiles. The mixture of known steroids β-sitosterol and stigmasterol (16 mg), their glucoside derivative daucosterol, the triterpene β-amyrin (31 mg), the sugar sucrose (8 mg), as well as the peptide asperphenamate (12 mg) were all precipitated as white powders from the sub-fractions B2 (2.6 g, Hex/EtOAc 19:1), B3 (1.8 g, Hex/EtOAc 8:1), B14 (2.9 g, EtOAc), B12 (2.2 g, Hex/EtOAc 1:3), B10 (3 g, Hex/EtOAc 1:1), respectively. The sub-fractions B6 (4,3 g, Hex/EtOAc 3:1) and B8 (2,8 g, Hex/EtOAc 7:3) were further purified using silica gel column chromatography eluting with the same solvent polarity used previously for their elution to afford compound 4 (8 mg) and oleanolic acid (7 mg) from B6, as well as compound 5 (6 mg) from B8. Compound (3) (11 mg) was filtered off as white precipitate from fraction B4 (3.8 g, Hex/EtOAc 17:3) and the yellowish filtrate was subjected to sephadex LH-20 column chromatography eluting with the mixture of dichloromethane-methanol (3:2) to afford compounds 1 (4 mg) and 2 (3 mg). 2.3.1. Tiamenone A (1) Oil; [α]20D + 3.6 (c 0.28, CHCl3); UV (MeOH) λmax (log ε) 288 (2.63), 235 (0.41), 220 (0.11); IR (KBr) νmax 2957, 2361, 1661, 1030, 798 cm-1; 1H and 13C-NMR data, see Table 1; HR-ESIMS: m/z 449.3027 [M + Na]+ (calcd for C28H42O3Na, 449.3026). 2.3.2. Tiamenone B (2) Oil; [α]20D + 40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 327 (0.37), 313 (0.51), 298 (0.67), 286 (0.79), 225 (2.98); IR (KBr) νmax 3366, 2357, 1641, 1092 cm-1; 1H and 13C-NMR data, see Table 1; HR-ESI-MS: m/z 435.3231 [M + Na]+ (calcd for C28H44O2Na, 435.3234).
2.4. Docking experiments The X-ray crystal structure data of pyruvate kinase M2 (PKM2) was downloaded from the protein data bank (PDB entry: 3gr4). The docking study was performed using YASARA structure. The crystal structure was optimized by removing the water molecules and ligands, the structure was further energy minimized using the AMBER14 force field with a 10Å cut-off and PME was used for long range electrostatic. Structures of the different tested compounds were built using YASARA structure and energy minimized using YASARA2 force field before using in docking experiments. Docking was performed using Autodock [15]. The simulation cell was defined around the binding domain at the interface between monomer subunits of the PKM2 for docking of compounds 1–3. Docking results were analyzed based on the B-factor
5
(binding energy) calculated by YASARA structure and interaction with the amino acid residues of the target binding pocket.
3. Results and discussion Compound 1 was isolated as an oil with a positive specific rotation αD20 + 3.6° and the composition C28H42O3 deduced from its accurate mass determined by HR-ESI-MS {m/z 449.3027 [M + Na]+ (calcd for C28H42O3Na, 449.3026)}. The UV and IR spectra showed absorptions at 288 nm and 1661 cm-1, respectively, revealing the presence in the molecule a ketone conjugated with double bonds. The 1H NMR spectrum (Table 1) contains singlets for three methyl groups at δ 0.91, 1.13 and 1.34 attached to quaternary carbons; two doublets of methyl groups at δ 0.90 and 1.01 attached to a tertiary carbon; two doublets at δ 3.35 and 3.65, resp., corresponding to an oxymethylene moiety; and signals of three olefinic protons. These protons resonate as a singlet at δ 5.67 and as two double doublets at δ 6.10 (dd, J = 2.6 and 9.5 Hz) and 6.16 (dd, J = 1.5 and 9.9 Hz) indicating a cis-double bond. The 13C NMR spectrum (Table 1) contains signals of 28 carbon atoms, which were sorted using the HMQC spectrum into one ketone carbonyl (δ 199.7), four sp2 carbons (δ 164.0, 141.4, 127.8 and 123.5), five methyls (δ 27.4, 18.3, 17.3, 16.3 and 13.5), one oxymethylene (δ 63.3), eight methylenes, five methines and four quaternary sp3 carbon atoms. All these data suggested 1 to be a steroid derivative similar to the known 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) that was also isolated in the course of this investigation. Compounds 1 and 3 share the same tetracyclic moiety (rings A–D) containing a 4,6-dien-3-one system indicated by the presence of protons at δ 5.67 (H-4), 6.10 (H-6), 6.16 (H-7) and carbons appearing at δ 199.7 (C-3), 123.5 (C-4), 164.0 (C-5), 127.8 (C-6) and 141.4 (C-7). This system was further supported by the HMBC cross-peaks from the proton H-4 at δ 5.67 to the carbon signals at δ 199.7 (C-3), 164.0 (C-5) and 127.8 (C-6), as well as the cross-peaks between the proton H-7 at δ 6.16 and the carbon signals at δ 164.0 (C-5) and 127.8 (C-6). Additionally, the HMBC correlations (Fig. 2) between the methyl group at δ 0.91 (H3-18) and the carbon signals at δ 59.7 (C-17), 40.0 (C12) and 43.6 (C-13); those between the methyl group at δ 1.13 (H3-19) and the carbon signals at δ 50.6 (C-9), 36.0 (C-10) and 33.9 (C-1); together with the COSY correlations (Fig. 2) between H-1/H-2, H-8/H-9/H-11/H-12 and H-14/H2-15/H2-16/H-17, allowed to establish unambiguously the tetracyclic nucleus of compound 1 as shown in Figure 1. However, the uncommon side chain connected at C-17 has not yet been reported in any steroid derivatives hitherto. The literature survey and comparison of 13C NMR data indicated that compound 1 and the dammarane-type saponin operculinoside A reported previously share the same side chain 6
[16]. The carbon signals at δ 86.1 (C-20), 88.3 (C-24) and 63.3 (C-28) revealed the presence of a 24-hydroxymethyltetrahydrofuran moiety in the side chain. This partial conclusion was confirmed by the cross-peaks observed in the HMBC spectrum (Fig. 2) between the methyl groups at δ 0.90 and 1.01 (H3-26 and H3-27, resp.) to the carbon signal at δ 88.3 (C-24), from the methyl group at δ 1.34 (H3-21) to the carbon signal at δ 86.1 (C-20), as well as from the tertiary oxymethylene protons at δ 3.35 and 3.65 (H2-28) to the carbon appearing at δ 29.7 (C23), 88.3 (C-24) and 34.7 (C-25). The relative configuration of 1 was established based on its NOESY spectrum (Fig. 3) in which the correlations observed between the proton signals of H3-19/H-8 and H-8/H-18, confirmed their β-orientation. Additionally, the spatial correlations from H-9/H-14/H-17 put forward their α-orientation. However, we observed that the side is stabilized via hydrogen bond, the missing NOE cross-peaks between H-17 and H3-21 suggested that they are on the opposite faces while the cross-peaks between the proton signals of H3-21, H-22β, H-23β and H-25 show that they are placed on the same face (Fig. 3). This partial conclusion on the orientation of the isopropyl group connected in C-24 was supported by the comparison of NMR data of compound 1 with those of operculinoside A that had been structurally confirmed by X-ray diffraction [16]. We observed that both compounds share very close chemical shift values of 1H and 13C from C-20 to C-28 (for 1) or C-31 (for operculinoside A); and especially, identical proton coupling constants (J) for H-25, H3-26, H3-27 and H2-28. Based on these accepted β-orientations of H321 and the isopropyl group in C-24, both C-20 and C-24 were assigned with S configurations. The new ergostane-type steroid (20S,24S)-20,24-epoxy-24-hydroxymethylergostane-4,6-dien3-one was given the trivial name tiamenone A (1) . Compound 2 was obtained as an oil with a positive specific rotation αD20 + 40°. Its formula C28H44O2 was deduced from the HR-ESI-MS spectrum {m/z 435.3231 [M + Na]+ (calcd for C28H44O2Na, 435.3234)}. The IR spectrum exhibited vibration frequencies of a hydroxyl group (3366 cm-1) and of a conjugated ketone (1641 cm-1). Careful comparison of 1H and 13C NMR data (Table 1) of compounds 2 and 3 showed that they share the same side chain with a striking difference in the tetracyclic moiety. In this regard, analysis of the 13C NMR spectra of 2 and 3 (Table 1) revealed that the signals of the double bond Δ6,7 at δ 127.7 (C-6) and 141.2 (C-7) in 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) are replaced by two methylene carbon signals at δ 32.9 (C-6) and 31.9 (C-7) in 2. This was confirmed by the HMQC and HMBC NMR data, as well as the two mass units difference between both compounds 2 and 3. The heteronuclear correlations between the olefinic proton signal at δ 5.73 (s, H-4) and the carbon signals at δ 7
199.7 (C-3), 171.5 (C-5), 38.6 (C-10) and 32.9 (C-6) confirmed an enone moiety in ring A as presented in 2 (see Fig. 1), as well as an aliphatic chain from C-6 to C-9, strongly supported by the COSY correlations (Fig. 2) in this section of the ring B. The absolute configuration of C-20 was assigned to be S with respect of the biosynthesis evidence of ergostane-type steroids and comparison with the literature. The compound 20S-hydroxy-ergostane-4,24(28)-dien-3-one 2 was given the trivial name tiamenone B. A literature survey indicated that the secondary metabolites with a 2-(hydroxymethyl)-2isopropyl-5-methyltetrahydrofuran moiety in the side chain are very rare. To the best of our knowledge, a small number of dammarane-type triterpenoids has been reported so far from Euphorbia supina [17] and Operculina turpethum [16]. However, tiamenone A (1) is reported here as the first steroid with a 2-(hydroxymethyl)-2-isopropyl-5-methyltetrahydrofuran moiety. A proposed biosynthetic pathway for tiamenone A (1) is depicted in Figure 4. Since compound 1 is an ergostane-type steroid, the formation of its side chain could start from the core structure of 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) in which an enzymatic oxidation of the terminal exomethylene Δ24,28 took place to afford an epoxide. A cyclization may occur through an attack of this epoxide at C-24 by the hydroxyl group at C-20 to obtain compound 1. In addition to 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3) [18,19], the other known compounds were identified as 3β,7α,20β-trihydroxyergosta-5,24(28)-diene (4) [20], 3β,5αdihydroxyergosta-7,22-diene (5) [21,22], stigmasterol and β-sitosterol [23], daucosterol [24], asperphenamate [25], sucrose [26], β-amyrin [27] and its derivative oleanolic acid [28] based on the comparison of their spectral data with those reported in the literature. New compounds tiamenones A (1) and B (2), as well as the known products namely 20Shydroxy-4,6,24(28)-ergostatrien-3-one (3), β-amyrin and asperphenamate were tested for antibacterial activity against five bacterial strains including Escherichia coli DSMZ 1058, Pseudomonas agarici DSMZ 11810, Bacillus subtilis DSMZ 704, Micrococcus luteus DMSZ 1605 and Staphylococcus warneri DSMZ 20036, using gentamicin as positive control and their cytotoxicity were evaluated against KB3-1 and HT-29 cell lines using griseofulvin as positive control. All tested compounds did not show any antibacterial or cytotoxic even at a concentration as high as 0.5 mg/mL. Based on previous studies on a number of new ergostane-type steroids discovered with potent anti-HIV activity; proteomic approach postulated pyruvate kinase M2 (PKM2) as the target enzyme in the glucose metabolism pathway to compromise the replication of HIV-1 [29,30], 8
docking of compounds 1–3 against pyruvate kinase M2 compared to the activator reference compound 1-[(2,6-difluorophenyl)sulfonyl]-4-(2,3-dihydro-1,4-benzodioxin-6-ylsulfonyl)piperazine (DYY) from the crystal structure of pyruvate kinase M2 (PDB: 3gr4), have revealed high affinity for compound 1 (8.31 kcal mol-1) compared to the reference (Table 2). An H-bond was also detected to the Arg400 residue located at the interface between two monomers of pyruvate kinase M2, in addition to the hydrophobic interactions with amino acid residues within the activator binding pocket at the interface between the protein subunits (Fig. 5). Likewise, compound 3 had a binding energy comparable to the reference compound. In a similar manner to compound 1; two H-bonds were detected with Arg400 and Glu397 residues (Fig. 6). Those results suggest a possible role of compounds 1 and 2 in targeting pyruvate kinase M2. Conversely, compound 2 showed lower binding energy and no interaction could be established with the target binding site (Fig. 7). Docking was also performed with the reference compound DYY to confirm the correct binding position. The compound bound to pyruvate kinase M2 at the interface between the two monomers in a similar fashion to compounds 1–3. A binding energy of 8.21 was calculated for DYY and two H-bonds with Arg400 and Tyr390 residues were detected (Fig. 8). The docking results suggest that there might be a possible role of compounds 1 and 2 in targeting the activator binding site in pyruvate kinase M2 when compared to the reference compound DYY.
4. Conclusion In summary, the phytochemical investigation of the bark of Entandrophragma angolense led to the isolation and characterization of two new ergostane-type steroids namely tiamenones A and B (1 and 2) along with ten known compounds. To the best of our knowledge, compound 1 is reported as the first steroid with a 2-(hydroxymethyl)-2-isopropyl-5-methyltetrahydrofuran moiety and its pausible biosynthesis is proposed herein. Although no antibacterial and cytotoxic effects were depicted for the isolated compounds, their report provides further information to enrich the chemistry of the genus Entandrophragma and varies the chemotaxonomic potential of steroids. Moreover, the molecular docking studies suggested that the new compounds 1 and 2 might be potential anti-HIV inhibitors. This study further supports the identification of E. angolense as source of steroids and the chemical investigation as well as in vitro assays of the extracts and the compounds from other plant parts (leaves, root and fruits) could give further evidence on the active principles of the plant and confirm its use in traditional medicine.
9
Acknowledgements This work was supported by the German Academic Exchange Service (DAAD) with funds from the Federal Ministry for Economic Cooperation and Development (BMZ) through the postdoctoral scholarship to G.M.H., the postgraduate scholarship to S.C.N.W. and the equipment subsidies to S.K.F., all in the frame of the Yaounde-Bielefeld Graduate School of Natural
Products
with
Antiparasite
and
Antibacterial
Activities
(YaBiNaPA,
www.yabinapa.de), project N° 57316173. The authors are grateful to Carmela Michalek, Bielefeld University, for the biological activity testing and to the NMR and MS departments at Bielefeld University for the spectral measurements.
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for compounds 1–3 in CDCl3
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22α 22β 23α 23β 24 25 26 27 28
1 δC 33.9 33.9 199.7 123.5 164.0 127.8 141.4 37.3 50.6 36.0 20.6 40.0 43.6 53.5 23.3 23.1 59.7 13.5 16.3 86.1 27.4 37.8 29.7 88.3 34.7 18.3 17.3 63.3
δH (J in Hz) 1.72b, 2.00b 2.45b, 2.62b 5.67, s 6.10, dd (2.6, 9.6) 6.16, dd (1.5, 9.6) 2.22, m 1.22, m 1.43b, 1.56b 1.27b, 2.14b 1.25, m 1.80b 1.88b 1.65b 0.91, s 1.13, s 1.34, s 1.64b 1.98b 1.74, m 1.91, m 1.87b 0.90, d (6.9) a 1.01, d (6.9) a 3.35, d (11.4) 3.65, d (11.4)
2 δC 35.7 33.9 199.7 123.8 171.5 32.9 31.9 34.9 53.7 38.6 20.8 40.0 42.3 56.0 23.7
δH (J in Hz) 1.70b, 2.04b 2.32b, 2.57b 5.73, s 2.29, 2.41 1.04, 1.86 1.58, m 0.92, m 1.42, 1.55, m 1.26, 2.16, m 1.04, m 1.17b, 1.68b
3 δC 33.9 33.9 199.6 123.6 163.4 127.7 141.2 37.0 50.6 36.0 20.7 39.8 43.6 53.5 23.2
δH (J in Hz) 1.71b, 2.01b 2.45b, 2.58b 5.68, s 6.12 dd (2.3, 9.8) 6.15 dd (1.8, 9.8) 2.22, m 1.21, m 1.48b, 1.60b 1.27b, 2.16b 1.25, m 1.33b, 1.85b
22.3 57.8 13.6 17.4 75.0 26.3 42.7
1.67b, 1.84b 1.51, m 0.91, s 1.19, s 1.31, s 1.55
22.3 57.7 13.5 16.2 74.9 26.2 42.4
1.27b, 2.14b 1.53, m 0.96, s 1.12, s 1.32, s 1.56
28.9
2.02
28.9
2.02
156.2 33.9 21.9 21.9 106.3
2.28, m 1.04, d (7.0) a 1.03, d (7.0) a 4.68, brs 4.74, brs
156.1 33.8 21.8 21.8 106.3
2.24, m 1.03, d (7.0) a 1.03, d (7.0) a 4.69, brs 4.75, brs
a
Values are exchangeable in the same column. bOverlapped signals are reported without multiplicities.
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Table 2: Binding energy calculated for the docked compounds 1–3 in comparison to the reference compound DYY.
Compound
Binding energy (Kcal mol-1)
1
8.31
2
7.02
3
7.97
DYY
8.21
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Figure Legends: Fig. 1: Ergostane-type steroids isolated from bark of E. angolense. Fig. 2: 1H-1H COSY and main HMBC correlations for 1 and 2. Fig. 3: Key NOESY correlations for 1. Fig. 4: Proposed biosynthesis pathway for compounds 1 and 2. Fig. 5: (A) Docking of compound 1 (blue) to pyruvate kinase M2 (grey), showing the Hbonding (yellow dots) with Arg400 residue (white). (B) Interaction between compound 1 and PKM2 using Ligplot+. Fig. 6: (A) Docking of compound 3 (blue) to pyruvate kinase M2 (grey), showing the Hbonding (yellow dots) with Arg400 and Glu397 residue (white). (B) Interaction between compound 3 and PKM2 using Ligplot+. Fig. 7: (A) Docking of compound 2 (blue) to pyruvate kinase M2 (grey), no H-bonding to the protein residues was detected. (B) Interaction between compound 2 and PKM2 using Ligplot+. Fig. 8: Docking of the reference compound DYY (blue) to pyruvate kinase M2 (grey), where H-bonding (yellow dots) with Arg400 and Tyr390 residue (white) were detected.
14
Fig. 1: Ergostane-type steroids isolated from bark of E. angolense.
15
Fig. 2: 1H-1H COSY and main HMBC correlations for 1 and 2.
16
Fig. 3: Key NOESY correlations for 1.
17
Fig. 4: Proposed biosynthesis pathway for compound 1.
18
A
B
Fig. 5: (A) Docking of compound 1 (blue) to pyruvate kinase M2 (grey), showing the H-bonding (yellow dots) with Arg400 residue (white). (B) Interaction between compound 1 and PKM2 using Ligplot+.
19
A
B
Fig. 6: (A) Docking of compound 3 (blue) to pyruvate kinase M2 (grey), showing the H-bonding (yellow dots) with Arg400 and Glu397 residue (white). (B) Interaction between compound 3 and PKM2 using Ligplot+.
20
A
B
Fig. 7: (A) Docking of compound 2 (blue) to pyruvate kinase M2 (grey), no H-bonding to the protein residues was detected. (B) Interaction between compound 2 and PKM2 using Ligplot+.
21
Fig. 8: Docking of the reference compound DYY (blue) to pyruvate kinase M2 (grey), where Hbonding (yellow dots) with Arg400 and Tyr390 residue (white) were detected.
Graphical Abstract
Highlights
Two new steroids tiamenones A and B were obtained from Entandrophragma angolense
For the first time a steroid with a 20,24-epoxy-24-hydroxymethyl moiety was isolated
We reported the molecular docking studies of tiamasterols A, B and amasterol to enzyme pyruvate kinase PKM2
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