Prenylated Flavonoids from the Roots of Tephrosia rhodesica

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Prenylated Flavonoids from the Roots of Tephrosia rhodesica © 2020 American Chemical Society and American Society of Pharmacognosy Published version Atilaw, Yoseph; Muiva-Mutisya, Lois; Bogaerts, Jonathan; Duffy, Sandra; Valkonen, Arto; Heydenreich, Matthias; Avery, Vicky M.; Rissanen, Kari; Erdélyi, Máté; Yenesew, Abiy Atilaw, Y., Muiva-Mutisya, L., Bogaerts, J., Duffy, S., Valkonen, A., Heydenreich, M., Avery, V. M., Rissanen, K., Erdélyi, M., & Yenesew, A. (2020). Prenylated Flavonoids from the Roots of Tephrosia rhodesica. Journal of Natural Products, 83(8), 2390-2398. https://doi.org/10.1021/acs.jnatprod.0c00245 2020 Prenylated Flavonoids from the Roots of Tephrosia rhodesica Yoseph Atilaw, Lois Muiva-Mutisya, Jonathan Bogaerts, Sandra Duffy, Arto Valkonen, Matthias Heydenreich, Vicky M. Avery, Kari Rissanen, MateErdelyi,*and Abiy Yenesew* Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00245 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: Five new compoundsrhodimer (1), rhodiflavan A (2), rhodiflavan B (3), rhodiflavan C (4), and rhodacarpin (5)along with 16 known secondary metabolites, were isolated from the CH2Cl2−CH3OH (1:1) extract of the roots of Tephrosia rhodesica. They were identified by NMR spectroscopic, mass spectrometric, X-ray crystallographic, and ECD spectroscopic analyses. The crude extract and the isolated compounds 2−5, 9,15, and 21 showed activity (100% at 10 μg and IC50 =5−15 μM) against the chloroquine-sensitive (3D7) strain of Plasmodium falciparum. Tephrosia (Leguminosae) is a tropical and subtropical genus consisting of more than 400 species, 30 of which are native to Kenya. 1 These have provided a variety of new flavonoids, including flavanones, flavones, chalcones, pterocarpans, and rotenoids, most of them prenylated and showing promising antiplasmodial, antifeedant, antileishmanial, estrogenic, antitumor, and antimicrobial activities. 2−6 As part of our ongoing work on the genus Tephrosia, 6,7 we now report the first phytochemical investigation of the roots of Tephrosia rhodesica, which showed antiplasmodial activity. The investigation of the root extract led to the isolation of five new compounds (1−5) and 16 known compounds (6−21). Herein, we describe their isolation, characterization, and activity against the chloroquinesensitive (3D7) strain of Plasmodium falciparum. ■RESULTS AND DISCUSSION Applying silica gel gravity column chromatography, followed by Sephadex LH-20 gel filtration and preparative reverse-phase HPLC, 21 secondary metabolites were isolated from the roots of T. rhodesica. The structures of the isolated secondary metabolites were determined by NMR spectroscopic, mass spectrometric, and single-crystal X-ray diffractometric analyses. In addition to the five new secondary metabolites (1−5), the known tephrowatsin B (6), 8 tephrinone (7), 9 glabranin (8), 10 quercetol B (9), 11 maackiain (10), 12 6a-hydroxymaackiain (11), 13 pisatin (12), 14 tephrosin (13), 15 rotenone (14), 15 6hydroxyrotenone (15), 16 12a-hydroxyrotenone (16), 17 hildecarpin (17), 18 3-hydroxy-2-methoxy-8,9-methylenedioxypterocarpene (18), 19 isoliquirtigenin (19), 20 D-pinitol (20), 21 and tephrowatsin A (21) 8 were identified by comparison of their observed and reported spectroscopic data (see pages S22−S34 in the Supporting Information). Compound 1was isolated as white crystals from CH2Cl2− CH3OH (1:1) solution. Its molecular formula was deduced as C42H44O7based on HREIMS ([M]+m/z660.3095, calcd 660.3087) and NMR data analyses (see Table 1, as well as Figures S1−S6 in the Supporting Information). The 1H NMR signals at δH5.43 (H-2), 5.06 (H-2′′′), 2.84 and 3.07 (H-3a-b), 2.18 and 2.31 (H-3′′′), and 4.66 (H-4′′′), along with the 13C NMR signals at δC78.6 (C-2), 75.4 (C-2′′′), 43.7 (C-3), 36.9 (C-3′′′), 196.3 (C-4), and 26.8 (C-4′′′) were consistent with a flavanone-flavan dimer core structure 22,23 that was corroborated by the UV absorptions of λmax = 230, 290, and 350 nm. The NMR data (Table 1) indicated the presence of two sets of unsubstituted aromatic rings, two sets of prenyl groups, two hydroxy, and two methoxy groups. This is consistent with a 5hydroxyflavanone moiety possessing an unsubstituted ring B, along with a ring A substituted with prenyl at C-8 (δC108.1) and two hydroxy groups, δH12.61 (HO-5) and 6.80 (HO-7) at C-5 (δC159.3) and C-7 (δC162.8). Accordingly, C-6 (δC 109.8) connects the flavanone to the flavan moiety. Whereas the placement of a hydroxy group at C-7 was based on biogenetic considerations, 24 and on the chemical shift of the HO-5 hydrogen (δH12.61), suggesting its involvement in an intramolecular hydrogen bond; the placement of the prenyl group at C-8 (δC108.1) rather than at C-6 (δC109.8) was Received: March 5, 2020 Articlepubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Downloaded via UNIV OF JYVASKYLA on August 14, 2020 at 05:46:29 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. based on the HMBC correlation of CH2−1′′ (δH3.18) with C7(δC162.8), C-8 (δC108.1), C-8a (δC157.6), C-3′′ (δC 131.6), C-2′′ (δC122.5), and of H-2 (δH5.43) with C-4 (δC 196.3), C-1′(δC139.1), C-2′/C-6′(δC125.9), C-8a (δC 157.6). The identity of the other half of the molecule was established as a 5,7-dimethoxy-8-prenylflavan moiety based on the NMR data (Table 1). Hence, its 1H NMR spectrum revealed the presence of an unsubstituted aromatic ring (ring Table 1. NMR Spectroscopic Data (800 MHz, CDCl3) for Rhodimer (1) position δC, type δH(J, in Hz) HMBC 2 78.6, CH 5.43, dd (2.9, 13.1) C-4, C-1′, C-2′/6′, C-8a 3 43.7, CH22.84, dd (2.9, 17.1) 3.07, dd (13.1, 17.1) C-2, C-4, C-1′, C-4, C-4a, C-1′ 4 196.3, CO 4a 100.4, C 5 159.3, C−O 6 109.8, C 7 162.8, C−O 8 108.1, C 8a 157.6, C−O 1′139.1, C 2′/6′125.9, CH 7.47, m C-2, C-2′/6′, C-3′/5′ 3′/5′128.7, CH 7.43, m C-1′, C-3′/5′ 4′128.5, CH 7.39, m C-2′/6′ 1′′ 21.8, CH23.18, br t C-7, C-8, C-8a, C-3′′, C-2′′ 2′′ 122.5, CH 5.15, br t C-1′′, C-4′′, C-5′′ 3′′ 131.6, C 4′′ 17.8, CH31.57, s C-3′′, C-2′′, C-5′ 5′′ 25.8, CH31.65, s C-3′′, C-2′′, C-4′ OH-5 12.61, s C-5, C-6, C-4a, C-4, C-7 OH-7 6.80, s C-6, C-8, C-8a, C-7, C-4a 2′′′ 75.4, CH 5.06, dd (2.0, 11.5) C-4′′′, C-1′′′′, C-2′′′′/6′′′′, C-8a 3′′′ 36.9, CH22.18, dt (2.0, 6.0, 14.0) 2.31, dt (2.0, 11.5, 14.0) C-1′′′′, C-4′′′, C-6, C-4′′′, C-4′′′a, C-6 4′′′ 26.8, CH 4.66, dd (2.0, 6.0) C-2′′′, C-3′′′, C-4′′′a, C-5′′′, C-8′′′a, C-5, C-6, C-7 4′′′a 102.8, C 5′′′ 157.5, C−O 6′′′ 88.4, CH 6.17, s C-4′′′, C-4′′′a, C-5′′′, C-7′′′, C-8′′′, C-1′′′′′ (w) 7′′′ 158.6, C−O 8′′′ 110.5, C 8′′′a 154.3, C−O 1′′′′ 141.4, C 2′′′′/6′′′′ 126.0, CH 7.38, m C-2′′′, C-2′′′′/6′′′′, C-3′′′′/5′′′′ 3′′′′/5′′′′ 128.3, CH 7.34, m C-3′′′′/5′′′′, C-1′′′′ 4′′′′ 127.6, CH 7.29, m C-2′′′′/6′′′′ 1′′′′′ 21.9, CH23.35, br t C-8′′′, C-8a′′′, C-7′′′, C-3′′′′′, C-2′′′′′ 2′′′′′ 123.2, CH 5.24, br t C-1′′′′′, C-4′′′′′, C-5′′′′′ 3′′′′′ 130.8, C 4′′′′′ 17.8, CH31.66, s C-2′′′′′, C-3′′′′′, C-5′′′′′ 5′′′′′ 25.9, CH31.68, s C-2′′′′′, C-3′′′′′, C-4′′′′′ OMe-5′′′ 55.9, C−O 3.73, s C-5′′′ OMe-7′′′ 55.8, C−O 3.87, s C-7′′′ Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX B E) [δH7.38 (H-2′′′′/6′′′′), 7.34 (H-3′′′′/5′′′′), 7.29 (H4′′′′)], two methoxy groups at δH3.73 (OMe-5′′′) and 3.87 (OMe-7′′′) and a prenyl side chain [δH3.35 (H-1′′′′′), 5.24 (H-2′′′′′), 1.66 (H-4′′′′′), and 1.68 (H-5′′′′′)], and a singlet at δH6.17 (H-6′′′). The placement of the two methoxy groups was established based on the HMBC correlation of H-6′′′ (δH 6.17) with C-4′′′ (δC102.8), C-5′′′ (δC157.5), and C-7′′′ (δC 158.6). The prenyl group was positioned at C-8′′′ (δC110.5) based on the HMBC cross-peaks of H2-1′′′′′ (δH3.35) to C8′′′ (δC110.5), C-8a′′′ (δC154.3), and C-7′′′ (δC158.6). Methoxy groups were placed at C-5′′′ (δC157.5) and C-7′′′ (δC158.6) upon biogenetic considerations, 24 as well as based on the HMBC correlations of MeO-5′′′ (δH3.73) to C-5′′′ (δC157.5), and of MeO-7′′′ (δH3.87) to C-7′′′ (δC158.6). This assignment was corroborated by the NOEs of MeO-5′′′ (δH3.73) and MeO-7′′′ (δH3.87) with H-6′′′ (δH6.17). The C-6 (δC109.8) to C-4′′′ (δC26.8) linkage of the flavanone and the flavan moieties was confirmed by the HMBC correlations of H-4′′′ (δH4.66) with C-5 (δC159.3), C-6 (δC109.8), and C-7 (δC162.8), and of H2-3′′′ (δH2.18 and 2.31) with C-6 (δC 109.8). Because of its three stereocenters at C-2, C-2′′′, and C4′′′,1has eight possible stereoisomers. The large 3J2,3 = 13.1 Hz indicated H-2 to be axial, and thereby ring B to be equatorial. Based on the available literature on the flavanones of this genus, C-2 is expected to be S-configured. 1,25 Similarly, the large 3J2′′′,3′′′ = 11.5 Hz suggests H-2′′′ to be axial, and accordingly ring B of the flavan moiety to be equatorially oriented. Furthermore, based on the literature, 25 C-2′′′ is also expected to be S-configured as in the flavanones and flavans of this genus. The 3J3a′′′,4′′′ = 2.0 suggests the axial−equatorial or equatorial−equatorial orientation of the involved protons, whereas 3J3b′′′,4′′′ = 6.0 Hz gauche orientation, suggesting H4′′′ to be axially oriented and, consequently, the flavan moiety to occupy the more favorable equatorial orientation with C-4′′′ being R-configured. 25 The NOE correlation of H-2′′′ (δH5.06) and HO-7 (δH6.80) reveals the flavanone and H-2′′′ to be oriented in the same direction. This absolute configuration (2S,2′′′S,4′′′R), proposed on the basis of biogenetic considerations and NMR analyses was subsequently confirmed by single-crystal X-ray crystallography (Figure 1). Thus, based on the above spectroscopic data, this new compound, rhodimer (1), was characterized as (2S,2′′′S,4R)-5,7-dihydroxy-5′′′,7′′′-dimethoxy-8,8′′′-bis(3-methylbut-2-en-1-yl)- 2′,2′′′-diphenyl-[4′′′,6-bichroman]-4-one. Compound 2was isolated as an oily paste and was assigned the molecular formula C30H38O5, based on HRESIMS ([M + H]+m/z479.2493, calcd 479.2753) and NMR data analyses (see Table 2, as well as Figures S9−S14 in the Supporting Information). The 1H NMR signals at δH5.22 (H-2), 1.80 (H3ax), 2.31 (H-3eq), and 4.99 (H-4), along with the 13C NMR signals at δC76.1 (C-2), 37.0 (C-3), and 57.4 (C-4), suggested it to be a flavan-4-ol derivative, which was corroborated by the UV absorptions λmax = 240 and 290 nm. The characteristic flavan-4-ol ring C of 2was confirmed by the HMBC correlations of H-4 (δH4.99) with C-2 (δC76.1), C-3 (δC 37.0), C-5 (δC194.7), C-4a (δC114.9), and C-8a (δC171.0), and by the COSY correlations of H-2 (δH5.22) with CH2−3 (δH2.31 and 1.80), and of the latter with H-4 (δH4.99). The six methyl (H3-4′′,H 3-4′′′,H 3-4′′′′,H 3-5′′,H 3-5′′′, and H35′′′), along with the three methine signals (H-2′′, H-2′′′, and H-2′′′′) were diagnostic for three prenyl groups. The NMR signals of H-2′/6′, H-3′/5′, and H-4′(Table 2) further suggested ring B to be unsubstituted and, hence, all three prenyl groups to be connected to ring A. This ring is oxygenated at C-5 (δC194.7) and C-7 (δC206.4), with their placement having been derived from biogenetic considerations. 24 An additional oxygenation is presented at C-6 (δC 83.3), which uncharacteristically is an sp3hybridized carbon. Prenyl substitution at this carbon was confirmed by the HMBC correlation of H-2′′′′ (δH4.99) with C-6 (δC83.3). The location of the other two prenyl groups at C-8 (δC58.3), also sp3hybridized, was established from the 3JHMBC correlations of H-2′′ (δH5.03, m) and H-2′′′ (δH4.66, m) with C-8 (δC 58.3). Moreover, CH2−1′′ (δH2.60 and 2.81) and CH2−1′′′ (δH2.46 and 2.77) showed HMBC cross-peaks with C-7 (δC 206.4) and C-8a (δC171.0), whereas CH2−1′′′′ (δH2.71 and 2.25) with C-5 (δC194.7), confirming that ring A is fully substituted. Based on the above data, compound 2was characterized as 4,6-dihydroxy-6,8,8-tris(3-methylbut-2-en-1yl)-2-phenyl-2,3,4,8-tetrahydro-5H-chromene-5,7(6H)-dione. Its configurational assignment is discussed below, together with that of the structurally closely related compound 3. Compound 3was isolated as an oily paste and was assigned the molecular formula C30H38O5based on HRESIMS ([M + H]+m/z479.2493, calcd 479.2753) and NMR data (see Table 2, as well as Figures S16−S21 in the Supporting Information). The 1H NMR signals at δH5.24 (H-2), 2.00 and 2.30 (CH2− 3), and δH4.55 (H-4) and 13C NMR signals at δC76.2 (C-2), 36.5 (C-3), and 59.5 (C-4) suggested 3to be a flavan derivative with identical core structure, 4,6-dihydroxy-6,8,8tris(3-methylbut-2-en-1-yl)-2-phenyl-2,3,4,8-tetrahydro-5Hchromene-5,7(6H)-dione to compound 2. The highly similar NMR data of 2and 3(Table 2) indicated these compounds to be stereoisomers. Compounds 2([α]D+66.1) and 3([α]D+17.5) are both dextrorotatory (for details, see the Experimental Section) and, hence, are diastereomers (as enantiomers would be expected to rotate plan-polarized light in opposite directions). The large coupling constant 3J2,3ax (12.6 Hz in both 2and 3) is consistent with H-2 being axial and ring B of both compounds to occupy the more favorable equatorial orientation. 25 The 3J3ax,4 (4.2 Hz for 2, and 3.7 Hz for 3) and 3J3eq,4 (1.9 Hz in 2, 2.3 Hz in 3) are small, suggesting H-4 to be equatorial and hence HO-4 to be axial in both compounds, according to Pouget et al. 26 This is further corroborated by the 3.4 Å H-2−H-4 interatomic distance of 3, estimated based on the NOE cross-peak intensities using H-3a/b as an internal reference (1.8 Å; see Figure 1. Single crystal X-ray structure of compound 1with the thermal displacement parameters at 40% probability level. Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX C Figure S23 in the Supporting Information). This distance was computed to 2.3 Å for the (2S*,4S*) diastereomer, and 3.7 Å for the (2S*,4R*)configured C-ring, using Boltzmann averaging of the H-2−H-3 distances of the output conformers of Monte Carlo conformational searches (see Experimental Section for details; this distance was not measured for 2, because of signal overlap). Based on biogenetic considerations discussed for compound 1and the NMR data, compounds 2 and 3are C-6 epimers and possess a (2S,4R) absolute configuration. In order to determine their absolute configuration, their electronic circular dichroism (ECD) spectra (Figure 2) were recorded. The observed Cotton effects supported the ORD-based suggestion of the diastereomeric relationship of 2and 3as enantiomers are expected to show mirror-image ECD spectra. The comparison of the experimental spectra with those reported for flavan-4-ols 11 did not allow configurational assignment, because of the dissimilarity of rings A of 2and 3, compared to those of the reported compounds. Consequently, the Boltzmann-weighted ECD spectra of the possible diastereomers were calculated using Table 2. NMR Spectroscopic Data (800 MHz, CDCl3) for Rhodiflavan A (2) and Rhodiflavan B (3) 23 position δC, type δH, m(Jin Hz) HMBC δC, type δH, m(Jin Hz) HMBC 2 76.1, CH 5.22, dd (12.6, 2.3) C-3, C-4, C-1′, C-2′, C-6′76.2, CH 5.24, dd (12.6, 2.3) C-3, C-4, C-1′, C-2′, C-6′ 3 37.0, CH22.31, ddd (14.6, 2.3, 1.9) C-4, C-4a 36.5, CH22.30, dt (14.6, 2.3, 2.0) C-4, C-4a 1.80, ddd (14.6, 12.6, 4.2) C-1′, C-2 2.00, ddd (14.6, 12.6, 3.6) C-2, C-1′ 4 57.4, CH 4.99, m C-2, C-3, C-5, C-4a, C-8a 59.5, CH 4.55, dd (2.0, 3.6) C-2, C-4a, C-8a, C-5 4a 114.9, C 114.2, C 5 194.7, C−O 197.0, C−O 6 83.3, C−OH 83.4, C−OH 7 206.4, CO207.2, CO 8 58.3, C 58.3, C 8a 171.0, C−O 171.0, C−O 1′138.9, C 139.2, C 2′,6′125.9, CH 7.40, m C-2, C2′, C-4′, C-6′125.9, CH 7.40, m C-2, C-4′, C-2′, C-6′ 3′,5′128.7, CH 7.43, m C-1′, C-3′, C-5′128.7, CH 7.43, m C-1′, C-3′, C-5′ 4′128.5, CH 7.39, m C-1′, C-2′, C-6′128.5, CH 7.39, m C-1′, C-2′, C-6′ 1′′ 35.0, CH22.81, dd (14.2, 9.2) C-7, C-8, C-8a, C-2′′, C-3′′, C-1′′′ 34.5, CH22.89, dd (14.2, 9.8) C-7, C-8, C-8a, C-2′′, C-3′′, C-1′′′ 2.60, dd (6.3, 14.2) C-7, C-8, C-8a, C-2′′, C-3′′, C-1′′′ 2.59, m C-7, C-8, C-8a, C-2′′, C-3′′, C-1′′′ 1′′′ 38.5, CH22.77, m C-7, C-8, C-8a, C-2′′′, C-3′′′, C-1′′ 38.5, CH22.69, dd (13.5, 7.3) C-7, C-8, C-8a, C-2′′′, C-3′′′, C-1′′ 2.46, dd (13.7, 8.2) C-7, C-8, C-8a, C-2′′′, C-3′′′, C-1′′ 2.43, dd (13.5, 8.7) C-7, C-8, C-8a, C-2′′′, C-3′′′, C-1′′ 1′′′′ 38.5, CH22.71, dd (14.9, 6.3) C-6, C-5, C-2′′′′ 38.2, CH22.63, dd (14.7, 8.7) C-6, C-5, C-2′′′′ 2.25, dd (15.0, 5.7) C-6, C-5, C-2′′′′ 2.18, m C-6, C-5, C-2′′′′ 2′′ 118.8, CH 5.03, m C-1′′, C-4′′, C-5′′ 118.8, CH 5.13, m C-8, C-1′′, C-4′′, C-5′′ 2′′′ 117.4, CH 4.66, m C-8, C-1′′′, C-4′′′, C-5′′′ 117.4, CH 4.71, m C-8, C-1′′′, C-4′′′, C-5′′′ 2′′′′ 115.6, CH 4.99, m C-6, C-4′′′′, C-5′′′′ 115.5, CH 4.97, m C-6, C-4′′′′, C-5′′′′ 3′′ 136.0, C 136.0, C 3′′′ 136.9, C 136.9, C 3′′′′ 136.9, C 136.9, C 4′′ 26.0, CH31.64, s C-1′′, C-2′′, C-5′′ 18.0, CH31.61, s C-1′′, C-2′′, C-5′′ 4′′′ 18.1, CH31.52, s C-1′′′, C-2′′′, C-5′′′ 17.8, CH31.52, s C-1′′′, C-2′′′, C-5′′′ 4′′′′ 26.0, CH31.71, s C-2′′′′, C-3′′′′, C-5′′′′ 25.9, CH31.71, s C-2′′′′, C-3′′′′, C-5′′′′ 5′′ 18.1, CH31.59, s C-1′′, C-2′′, C-4′′′ 26.0, CH31.68, s C-1′′, C-2′′, C-4′′′ 5′′′ 25.8, CH31.55, s C-1′′, C-2′′, C-4′′′ 25.7, CH31.56, s C-1′′, C-2′′, C-4′′′ 5′′′′ 17.9, CH31.55, s C-2′′′′, C-3′′′′, C-4′′′′ 17.9, CH31.54, s C-2′′′′, C-3′′′′,C-4′′′′ Figure 2. ECD spectra of compounds 2(black) and 3(red) along with the calculated spectra of the stereoisomers [(2S,4R,6S) yellow, (2S,4R,6R) blue, (2S,4S,6R) green, (2S,4S,6S) lilac, (2R,4S,6R) pink, (2R,4S,6S) light blue]. See details of the ECD calculations in the Experimental Section. Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX D the TD-DFT method (see Experimental Section for details), and compared to those obtained experimentally for 2and 3 (Figure 2). The experimental ECD spectrum of 2showed a negative Cotton effect at ca. 300 nm, positive at 260, and negative at 230 nm, best matched with the calculated spectrum of the (2S,4R,6S) isomer. Nonetheless, the mirror imaged spectrum of the (2S,4R,6R) isomer, corresponding to a (2R,4S,6S)configuration, shows similar Cotton effects. Furthermore, the calculated spectra of the (2S,4R,6S) and (2S,4S,6S) isomers are remarkably similar, and the same goes for the other two calculated isomers. None of these show agreement with the experimentally obtained ECD spectrum of 3. Based on the above spectroscopic evidence, the new compounds rhodiflavan A (2) and B (3) are characterized as diastereomers of 4,6-dihydroxy-6,8,8-tris(3-methylbut-2-en-1yl)-2-phenyl-2,3,4,8-tetrahydro-5H-chromene-5,7(6H)-dione, with (2S*,4R*,6S*) and (2S*,4R*,6R*) relative configurations, respectively. Table 3. 1H and 13C NMR Spectroscopic Data (800 MHz, CDCl3) for Rhodiflavan C (4) position δC, type δH, m(Jin Hz) HMBC 2 78.8, CH 5.45, dd (12.3, 2.2) C-3, C-4, C-1′, C-2′/C-6′ 3 37.0, CH22.31, dt (14.8, 2.3) C-4, C-4a 2.02, ddd (14.9, 12.3, 3.5) C-2, C-1′ 4 55.6, CH 4.86, m C-2, C-5, C-4a, C-7a 4a 125.9, C 5 184.5, CO 6 203.4 CO 7 52.9, C, 7a 188.6, C−O 1′137.9, C 2′,6′126.3, CH 7.40, m C-2, C-2′, C-4′, C-6′ 3′,5′128.9, CH 7.46, m C-1′, C-2′, C-3′, C-5′, C-6′ 4′129.1, CH 7.43, m C-2′, C-6′, C-3′, C-5′ 1′′ 32.8, CH22.51, dd (14.3, 7.2) C-5, C-6, C-7, C-7a, C-2′′, C-3′′, C-1′′′ 2.42, dd (14.3, 7.9) C-5, C-6, C-7, C-7a, C-2′′, C-3′′, C-1′′′ 1′′′ 32.6, CH22.56, dd (14.2, 7.2) C-5, C-6, C-7, C-7a, C-2′′′, C-3′′′, C-1′′ 2.44, dd (14.2, 7.9) C-5, C-6, C-7, C-7a, C-1′′, C-2′′′, C-3′′′ 2′′ 116.9, CH 4.86, m C-8, C-1′′, C-4′′, C-5′′ 2′′′ 116.9, CH 4.86, m C-8, C-1′′′, C-4′′′, C-5′′′ 3′′ 136.6, C 3′′′ 136.8, C 4′′ 25.9, CH31.61, s C-1′′, C-2′′, C-5′′ 4′′′ 25.9, CH31.61, s C-1′′, C-2′′, C-4′′′ 5′′ 17.9, CH31.57, s C-1′′′, C-2′′′, C-5′′′ 5′′′ 17.8, CH31.56, s C-1′′, C-2′′, C-4′′′ Table 4. 1H and 13C NMR Spectroscopic Data (800 MHz, CDCl3) of Compound 5 position δC, type δH,m(Jin Hz) HMBC 1 131.8, CH 7.20, s C-3, C-11a, C-4a, C-1′ 2 121.8, C 3 155.8, C−O 4 104.0, CH 6.40 C-2, C-3, C-11b, C-4a 4a 154.1, C−O 6 69.6, CH23.95, d (11.5) C-4a, C-6b, C-11a 4.15, d (11.5) C-4a, C-6a, C-6b, C-11a 6a 77.2, C−H 6b 119.0, C 7 103.0, CH 6.80, s C-6a, C-8, C-9, C-10a 8 142.4, C−O 9 149.7, C−O 10 94.2, CH 6.81, s C-8, C-9, C-6b, C-10a 10a 149.7, C 11a 84.8, C−H 5.25, s C-1, C-4a, C-6, C-6a, C-11b, C-10a 11b 112.1, C OCH2O-8, 9 101.5, CH25.91, d (1.4) 5.94, d (1.4) C-8, C-9 1′29.2, CH23.32, d (7.2) C-1, C-3, C-2′, C-3′ 2′121.8, CH 5.31, m C-4′, C-5′, C-1′ 3′135.8, C 4′17.9, CH31.78, s C-2′, C-3′, C-5′ 5′25.8, CH31.78, s C-2′, C-3′, C-4′ Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX E Compound 4was isolated as an oily paste. Its molecular formula was deduced as C24H28O4based on HREIMS (m/z [M]+381.2062, calcd 381.2066) and NMR data (see Table 3, as well as Figures S23−S28 in the Supporting Information) analyses. The 1H NMR signals at δH5.45 (H-2), 2.02 (H-3ax), 2.31 (H-3eq), and 4.86 (H-4), the 13C NMR signals at δC78.8 (C-2), 37.0 (C-3), and 55.6 (C-4), along with the UV λmax at 230 and 270 nm suggested compound 4to have an analogous ring C to those of 2and 3. Its 1H NMR signals at δH7.4 (H2′/6′), 7.46 (H-3′/5′), and 7.43 (H-4’) indicated ring B to be unsubstituted. The lack of aromatic protons for ring A suggested it to be fully substituted. As rings B and C were fully assigned, the carbonyl groups at δC184.5 (C-5) and 203.4 (C-6), and the two prenyl groups (see Table 3 for signals 1′′− 5′′, and 1′′′−5′′) could only be related to ring A. The HMBC correlations of H-2′′ (δH4.86) and H-2′′′ (δH4.86) with C-7 (δC52.9) revealed the two prenyl groups to be both attached to C-7. Both methylene groups CH2−2′′ (δH4.86) and CH2− 2′′′ (δH4.86) showed HMBC correlation to δC188.6 (C-7a), δC203.4 (C-6), and δC52.9 (C-7), indicating the placement of the two carbonyl groups at C-5 and C-6, which, being a fivemembered ring, is unique for the genus Tephrosia. The difference in the chemical shifts of C-5 (δC184.5) and C-6 (δC 203.4) is rationalized by the strong intramolecular hydrogen bond of HO-4 and the oxygen of the C-5 carbonyl group, and C-5 has strong conjugation with C-7a through the 4a (7a) double bond. The observed mass and NMR data (vide supra), the UV spectrum only showing a benzenoid band, and the HMBC of H-4 (δH4.86) with C-5 (δC184.5) corroborate the proposed structure. It is optically active ([α]D+90 (c0.001, CH2Cl2), and its ECD spectrum (see Figure S30 in the Supporting Information) shows a positive Cotton effect at λ 300−340 nm and a negative one at 275 nm. Based on the similarity of the NMR data (δ,J,Table 3) of H-2, H-3, and H4 (ring C) of 4to those of compounds 2and 3(Table 2), compound 4was assigned the (2S*,4R*) relative configuration. Therefore, based on the above spectroscopic data, this unique compound, rhodiflavan C (4), was characterized as (2S*,4R*)-4-hydroxy-7,7-bis(3-methylbut-2-en-1-yl)-2-phenyl2,3,4,7-tetrahydrocyclopenta[b]pyran-5,6-dione. Compound 5was isolated as an amorphous solid and was assigned the molecular formula C21H20O6, based on HRESIMS ([M + H]+m/zobs 369.1367) and NMR data (see Table 4,as well as Figures S31−S36 in the Supporting Information) analyses. It showed characteristic UV (λmax 230, 190, 310, and 350 nm), 1H NMR [δH3.95 and 4.15 (CH2−6), 5.25 (H11a)], and 13C NMR data [δC69.6 (C-6), 77.2 (C-6a), 84.8 (C-11a) for a 6a-hydroxypterocarpan skeleton. The NMR data further indicated the presence of a prenyl group [δH3.32 (H21′), 5.31 (H-2′), 1.78 (H3-4′and H3-5′), and δC29.2 (C-1′) 121.8 (C-2′), 135.8 (C-3′), 17.9 (C-4′), 25.8 (C-5′)] connected to C-2, as revealed by HMBC cross-peaks of H-1 (δH7.20) with C-1′(δC29.2), and of CH2−1′(δH3.32) with C-1 (δC131.8) and C-3 (δC155.8). The position of the prenyl group was confirmed by the NOE correlation between H-1 (δH 7.20) and CH2−1′(δH3.32). Hence, ring A is substituted with a prenyl group at C-2 and a hydroxy group at C-3. Its C-1 and C-4 are unsubstituted as evidenced by the two singlets in its 1H NMR data. In ring D, the NMR data (Table 4) indicated the presence of an 8, 9-methylenedioxy group (δH5.91 and 5.94; δC101.5) and two p-substituted aromatic protons at δH6.80 (H-7) and δH6.81 (H-10). The NMR spectra of this compound showed resemblance to those of previously published pterocarpans. 7,27−29 The absolute configuration at the B/C-ring junction was determined as (6aS,12aS), based on the ECD spectrum (see Figure S34 in the Supporting Information), which exhibited a positive and negative Cotton effect at 309 nm and 241 nm, respectively. 30 Based on the above spectroscopic data, this new compound, rhodacarpin (5), was characterized as (6aS,12aS)-2-(3-methylbut-2-en-1yl)-6H-[1,3]dioxolo[4′,5′:5,6]benzofuro[3,2-c]chromene-3,6a- (12aH)-diol. The crude extract of the roots of T. rhodesica and the isolated compounds were tested for antiplasmodial activity using a previously established protocol. 31 The crude extract gave 100% growth inhibition of the chloroquine-sensitive (3D7) strain of Plasmodium falciparum at 10 μg/mL concentration, whereas the isolated compounds 2,3,4,5,9, and 15 showed moderate activities (Table 5). Among the tested compounds, 3showed the highest activity (IC50 = 5.7 ± 1.9 μM). 32 In conclusion, five new compoundsrhodimer (1), rhodiflavan A (2), rhodiflavan B (3), rhodiflavan C (4), and rhodacarpin (5)were isolated from the roots of Tephrosia rhodesica, along with 16 known natural products. Rohodimer is an unusual flavanone-flavan dimer, whereas rhodiflavan C has a five-membered A-ring, which is unprecedented for this genus. The crude root extract and several of its isolated constituents showed activities against the chloroquine-sensitive (3D7) strain of Plasmodium falciparum. ■EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341-LC system, whereas ECD experiments were performed on a Jasco Model J-715 spectropolarimeter. UV spectra were recorded on a Specord S600 (Analytik Jena AG) spectrophotometer. Melting points were obtained on a Buchi Melting Point B-545 Switzerland apparatus; NMR spectra were acquired on a Bruker Avance III HD 800 MHz NMR spectrometer equipped with a TCI cryogenic probe and were processed with the MestReNova 10.0 software, using the solvent residual signal (CDCl3δH7.26; δC77.16) as chemical shift reference. LC-ESIMS data were obtained on a Micromass GC-TOF micro mass spectrometer (Micromass, Wythenshawe, Waters, Inc., U.K.), using direct inlet, and 70 eV ionization voltage. TLC was performed on Merck precoated silica gel 60 F254 plates. Column chromatography was run on silica gel 60 (70−230 mesh). Gel filtration was done on Sephadex LH-20. Preparative HPLC was performed on a Waters 600E instrument using the Table 5. In Vitro Antiplasmodial Activities (IC50)of Isolated Compounds against 3D7 Strains of P. falciparum a sample IC50,μM(Pf 3D7) LD50 (HEK-293) T. rhodesica (roots) crude extract 100% active at 10 μg/mL Rhodiflavan A (2) 7.3 ±1.8 71% at 40 μM Rhodiflavan B (3) 5.7 ±1.9 34% at 40 μM Rhodiflavan C (4) 7.0 ±2.4 101% at 40 μM Rhodacarpin (5) 10.2 ±0.2 27% at 40 μM Quercetol B (9) 7.4 ±0.3 87% at 40 μM 6-Hydroxyrotenone (15) 8.6 ±2.6 10.9 μM Tephrowatsin A (21) 14.5 ±0.7 Chloroquine 0.0047 Artesunate 0.00067 a Compounds were tested independently either two or three times against Pf 3D7. All compounds were tested in a single experiment against HEK 293 mammalian cells (LD50). Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX F Chromulan (Pikron, Ltd.) software and an RP C8Kromasil (250 mm ×55 mm) column eluting with repeated CH3OH-H2O (5 to 95) gradients. Single-crystal X-ray data of 1were collected at 120 K on an Agilent SuperNova dual wavelength diffractometer with a microfocus X-ray source and multilayer optics monochromatized Cu Kα(λ= 1.54184 Å) radiation. Plant Material. The roots of Tephrosia rhodesica were collected in April 2015 from Kilungu Hills in Makueni County, Kenya. The plant specimen was identified by Mr. Patrick C. Mutiso of the School of Biological Sciences, the University of Nairobi. A voucher specimen (Mutiso-842/April 2015) was deposited at the University Herbarium of the University of Nairobi, Kenya. Extraction and Isolation. The air-dried roots (2 kg) of T. rhodesica were ground and extracted with CH2Cl2−CH3OH (1:1) (3 ×2 L) by percolation at room temperature to yield 70 g of a dark brown paste after evaporation of the solvent. A portion of the extract (37 g) was subjected to column chromatography over silica gel (400 g) eluting with a mixture of iso-hexane containing increasing amounts of EtOAc. The fraction eluted with 1% EtOAc in iso-hexane was purified by preparative HPLC (CH3OH-H2O gradient elution) to give tephrowatsin B (6, 20 mg). 8 The fraction eluted with 3% EtOAc in iso-hexane was purified by column chromatography on a Sephadex LH-20 column (CH2Cl2−CH3OH; 1:1) to give tephrinone (7, 200 mg) 9 and glabranin (8, 100 mg). 10 The fractions eluted with 5% EtOAc in iso-hexane were purified by column chromatography on a Sephadex LH-20 column (CH2Cl2−CH3OH; 1:1), followed by recrystallization from CH2Cl2−CH3OH (1:1) to afford rhodimer (1, 15 mg). The fractions eluted with 6% EtOAc in iso-hexane were subjected to column chromatography on a Sephadex LH-20 (CH2Cl2−CH3OH (1:1)) system to give quercetol B (9, 300 mg). 11 The fractions eluted with 7% EtOAc in iso-hexane were purified by column chromatography on a Sephadex LH-20 column (CH2Cl2−CH3OH (1:1)) to give maackiain (10, 20 mg), 12 6ahydroxymaackiain (11, 10 mg), 13 and pisatin (12, 5 mg). 14 The fractions eluted with 8% EtOAc in iso-hexane were subjected to column chromatography on a silica gel (200 g) with 3% EtOAc in isohexane to give tephrowatsin A (21, 10 mg), 8 with 5% EtOAc in isohexane to give rhodiflavan A (2, 50 mg), and with 10% EtOAc in isohexane to provide rhodiflavan B (3, 20 mg) and rhodiflavan C (4,15 mg), which were further purified by preparative HPLC (CH3OH− H2O gradient elution). The fractions eluted with 9%−10% EtOAc in iso-hexane were combined and purified by column chromatography on a Sephadex LH-20 column (CH2Cl2−CH3OH; 1:1), and subsequently by preparative HPLC (CH3OH−H2O gradient elution) to give tephrosin (13, 10 mg), 15 rotenone (14, 15 mg), 15 6hydroxyrotenone (15, 10 mg), 16 12a-hydroxyrotenone (15, 10 mg), 17 rhodacarpin (5, 10 mg), hildecarpin (17, 10 mg), 18 and 3-hydroxy-2methoxy-8−9-methylenedioxypterocarpene (18, 15 mg). 19 The fractions eluted with 12% EtOAc in iso-hexane gave isoliquirtigenin (19, 15 mg), 20 and those eluted with 20% EtOAc in iso-hexane gave D-pinitol (20, 900 mg). 21 Rhodimer (1). White crystals (CH2Cl2−CH3OH; 1:1); [α]D20 +13.4 (c0.001, CH3OH); UV (CH2Cl2)λmax (log ε) 230 (4.05), 290 (4.25), and 350 (4.30) nm; ECD (c0.05, CH3OH) λmax (Δε) 314 (10.16),292 (−40.36), 249 (−23.33), 235 (50.08), 220 (−56.36), 212 (62.23);1H and 13C NMR, see Table 1; HREIMS m/z660.3095 [M]+(calcd for C42H44O7, 660.3087). Rhodiflavan A (2). Yellow oily paste; [α]D20 +17.5 (c0.001, CH2Cl2); UV (CH2Cl2)λmax (log ε) 240 (3.05) and 290 (3.40) nm; ECD (c0.05, CH3OH) λmax (Δε) 300 (−92.25), 257 (85.45), 243 (−119.45); 1H and 13C NMR, see Table 2; HRESIMS m/z479.2493 [M + H]+(calcd for C30H38O5, 479.2753). Rhodiflavan B (3). Yellow oily paste; [α]D20 +66.1 (c0.001, CH2Cl2); UV (CH2Cl2)λmax (log ε) 230 (3.08) and 280 (3.20) nm; ECD (c0.05, CH3OH) λmax (Δε) 301 (199.73), 257 (−130.29), 225 (65.0); 1H and 13C NMR, see Table 2; HRESIMS m/z479.2493 [M +H] +(calcd for C30H38O5, 479.2753). Rhodiflavan C (4). Yellow oily paste. [α]D20 +90.0 (c0.001, CH2Cl2); UV (CH2Cl2)λmax (log ε) 230 (3.20) and 270 (3.08) nm; ECD (c0.05, CH3OH) λmax (Δε) 316 (89.0), 284 (−32.21), 245 (91.80), 226 (−26.26), 218 (70.0), 210 (80.5); 1H and 13C NMR, see Table 3; HREIMS m/z381.2062 [M]+(calcd for C24H28O4, 381.2066). Rhodacarpin (5). White amorphous solid; UV (CH2Cl2)λmax (log ε) 230 (3.04), 290 (3.45), 310 (3.42) and 350 (3.10) nm; ECD (c 0.05, CH3OH) λmax (Δε) 309 (12.0), 241 (−82.1), 220 (7.0), 205 (−80); 1H and 13C NMR, see Table 4; HRESIMS m/z369.1367 [M +H] +(calcd for C21H20O6, 369.1338). Plasmodium falciparum Culture. In vitro parasite culture of the P. falciparum strain 3D7 was maintained in RPMI with 10 mM Hepes (Life Technologies), 50 μg/mL hypoxanthine (Sigma) and 5% human serum from male AB plasma and 2.5 mg/mL AlbuMAX II (Life Technologies). Human 0+erythrocytes were obtained from the Australian Red Cross Blood Service (Agreement No. 13-04QLD-09; 17-06QLD-16). The parasites were maintained at 2%−8% parasitaemia (% P) at 5% hematocrit (% H), and incubated at 37 °C, 5% CO2,5%O 2, 90% N2and 95% humidity. Plasmodium falciparum Growth Inhibition Assay. A wellestablished asexual P. falciparum imaging assay was used to determine parasite growth inhibition according to the procedure described by Duffy and Avery. 31 Computation. A low-energy conformation library of postulated compounds 2and 3were generated using Macromodel as implemented in the Macromodel v12.1 Schrodinger suite by performing careful Monte Carlo conformational analysis using MMFF force fields, each with the GB/SA solvation models CHCl3 and H2O. Elimination of redundant conformations was performed by comparison of heavy atom coordinates applying an RMSD cutoffset to 2.0 Å. Next, all unique conformers were optimized at the B3LYP/6311++G(3df,2pd) level of theory. The conformers having a Boltzmann weight, calculated using ΔH°, above 1% were selected for subsequent TD-DFT calculations at the same level of theory using 50 singlet excited states. To obtain the Boltzmann-weighted ECD spectrum, the individual spectra were line-broadened using a Gaussian band shape (σ= 0.3 eV). All (TD-)DFT calculations were performed using Gaussian 16, Revision A.03. 33 The solvent was taken into account by using the integral equation formalism model (IEFPCM) as implemented in Gaussian 16 and the dielectric constant for methanol (ε= 32.613). To compensate for the typical underestimation of the transition energies, a 20 nm blue shift was applied on the computed ECD spectra for comparison with the experimental one. 34 ■ASSOCIATED CONTENT * sıSupporting Information CCDC 1987379 has been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: +44- (0)1223-336033 or e-mail: [email protected]). The Supporting Information is available free of charge at https:// pubs.acs.org/doi/10.1021/acs.jnatprod.0c00245. Original MS and NMR spectra for all compounds, along with the corresponding NMReDATA 35 for the new compounds 1−5; the X-ray structure of 1(details are freely available on Zenodo at https://10.5281/zenodo. 3679345); NMR and MS data for compounds (1−21) (PDF) Crystallographic data for 1(CIF) ■AUTHOR INFORMATION Corresponding Authors MateErde lyi −Department of ChemistryBMC, Uppsala University, SE-751 23 Uppsala, Sweden; Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden; orcid.org/0000-0003Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX G 0359-5970; Phone: +46-72-9999166; Email: mate.erdelyi@ kemi.uu.se Abiy Yenesew −Department of Chemistry, University of Nairobi, 30197-00100 Nairobi, Kenya; Phone: +254 733 832 576; Email: [email protected] Authors Yoseph Atilaw −Department of Chemistry, University of Nairobi, 30197-00100 Nairobi, Kenya; Department of ChemistryBMC, Uppsala University, SE-751 23 Uppsala, Sweden Lois Muiva-Mutisya −Department of Chemistry, University of Nairobi, 30197-00100 Nairobi, Kenya Jonathan Bogaerts −Department of Chemistry, University of Antwerp, B-2020 Antwerp, Belgium Sandra Duffy−Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Nathan, Qld 4111, Australia Arto Valkonen −University of Jyvaskyla, Department of Chemistry, FI-40014 Jyvaskyla, Finland; orcid.org/00000003-2806-3807 Matthias Heydenreich −Institut fur Chemie, Universitat Potsdam, D-14476 Potsdam, Germany Vicky M. Avery −Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Nathan, Qld 4111, Australia Kari Rissanen −University of Jyvaskyla, Department of Chemistry, FI-40014 Jyvaskyla, Finland; orcid.org/00000002-7282-8419 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jnatprod.0c00245 Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS Y.A. is grateful to the German Academic Exchange Services (DAAD) for a scholarship that was offered through the Natural Products Research Network for Eastern and Central Africa (NAPRECA). The Swedish Research Council (Swedish Research Links, NOs. 2012-6124 and 2019-03715), the International Science Program (ISP Sweden, Grant No. KEN-02), and the Australian Research Council (Grant No. LP120200557 to V.M.A.) are gratefully acknowledged for financial support. We thank the Australian Red Cross Blood Service for the provision of human blood. Jonathan Bogaerts thanks the Research foundation Flanders (FWO-Vlaanderen) for the appointment of a predoctoral scholarship (No. 1198318N) and acknowledges the Flemisch Supercomputing Centre (VSC) for providing computational resources and support. The Academy of Finland (Grant No. 314343 to A.V.) is also gratefully acknowledged for funding. The Swedish NMR Centre is acknowledged for access to an 800 MHz spectrometer. ■REFERENCES (1) Chen, Y.; Yan, T.; Gao, C.; Cao, W.; Huang, R. Molecules 2014, 19, 1432−1458. (2) Muiva, L. M.; Yenesew, A.; Derese, S.; Heydenreich, M.; Peter, M. G.; Akala, H. M.; Eyase, F.; Waters, N. C.; Mutai, C.; Keriko, J. M.; Walsh, D. Phytochem. Lett. 2009,2,99−102. (3) Juma, W. P.; Akala, H. M.; Eyase, F. L.; Muiva, L. M.; Heydenreich, M.; Okalebo, F. A.; Gitu, P. M.; Peter, M. G.; Walsh, D. S.; Imbuga, M.; Yenesew, A. Phytochem. Lett. 2011,4, 176−178. (4) Touqeer, S.; Saeed, M. A.; Ajaib, M. Phytopharmacology 2013,4, 598−637. (5) Muiva-Mutisya, L.; Macharia, B.; Heydenreich, M.; Koch, A.; Akala, H. M.; Derese, S.; Omosa, L. K.; Yusuf, A. O.; Kamau, E.; Yenesew, A. Phytochem. Lett. 2014,10, 179−183. (6) Atilaw, Y.; Muiva-Mutisya, L.; Ndakala, A.; Akala, H. M.; Yeda, R.; Wu, Y. J.; Coghi, P.; Wong, V. K. W.; Erdelyi, M.; Yenesew, A. Molecules 2017,22, 1514. (7) Atilaw, Y.; Duffy, S.; Heydenreich, M.; Muiva-Mutisya, L.; Avery, V. M.; Erdelyi, M.; Yenesew, A. Molecules 2017,22, 318. (8) Gomez, F.; Quijano, L.; Calderon, J. S.; Rodriquez, C.; Rios, T. Phytochemistry 1985,24, 1057−1059. (9) Dagne, E.; Mammo, W.; Sterner, O. Phytochemistry 1992,31, 3662−3663. (10) Cespedes, C. L.; Achnine, L.; Lotina-Hennsen, B.; Salazar, J. R.; Gomez-Garibay, F.; Calderon, J. S. Pestic. Biochem. Physiol. 2001,69, 63−76. (11) Gomez-Garibay, F.; Quijano, L.; Calderon, J. S.; Morales, S.; Rios, T. Phytochemistry 1988,27, 2971−2973. (12) Mizuno, M.; Tanaka, T.; Katsuragawa, M.; Saito, H.; Iinuma, M. J. Nat. Prod. 1990,53, 498−499. (13) Bilton, J. N.; Debnam, J. R.; Smith, I. M. Phytochemistry 1976, 15, 1411−1412. (14) Kobayashi, A.; Akiyama, K.; Kawazu, K. Phytochemistry 1992, 32,77−78. (15) Stevenson, P. C.; Kite, G. C.; Lewis, G. P.; Forest, F.; Nyirenda, S. P.; Belmain, S. R.; Sileshi, G. W.; Veitch, N. C. Phytochemistry 2012,78, 135−146. (16) Krupadanam, G. L. D.; Srimannarayana, G.; Rao, N. V. S. Indian J. Chem., Sect. B 1978,16B, 770−772. (17) Oberholzer, M. E.; Rall, G. J. H.; Roux, D. G. Phytochemistry 1976,15, 1283−1284. (18) Lwande, W.; Bentley, M. D.; Macfoy, C.; Lugemwa, F. N.; Hassanali, A.; Nyandat, E. Phytochemistry 1987,26, 2425−2426. (19) Lwande, W.; Bentley, M. D.; Hassanali, A. Int. J. Trop. Insect Sci. 1986,7, 501−503. (20) Takahashi, T.; Takasuka, N.; Iigo, M.; Baba, M.; Nishino, H.; Tsuda, H.; Okuyama, T. Cancer Sci. 2004,95, 448−453. (21) Raya-Gonzalez, D.; Pamatz-Bolanos, T.; del Rio-Torres, Rosa E. d.; Martinez-Munoz, R. E.; Ron-Echeverria, O.; Martinez-Pacheco, M. M. Z. Naturforsch., C: J. Biosci. 2008,63, 922−924. (22) Yan, X.-T.; Li, W.; Sun, Y.-N.; Yang, S.-Y.; Lee, S.-H.; Chen, J.- B.; Jang, H.-D.; Kim, Y.-H. Bioorg. Med. Chem. Lett. 2014,24, 1397− 1402. (23) Asada, Y.; Sukemori, A.; Watanabe, T.; Malla, K. J.; Yoshikawa, T.; Li, W.; Kuang, X.; Koike, K.; Chen, C.-H.; Akiyama, T.; Qian, K.; Nakagawa-Goto, K.; Morris-Natschke, S. L.; Lu, Y.; Lee, K.-H. J. Nat. Prod. 2013,76, 852−857. (24) Bogorad, L. Annu. Rev. Plant Physiol. 1958,9, 417−448. (25) Slade, D.; Ferreira, D.; Marais, J. P. J. Phytochemistry 2005,66, 2177−2215. (26) Pouget, C.; Fagnere, C.; Basly, J.-P.; Leveque, H.; Chulia, A.-J. Tetrahedron 2000,56, 6047−6052. (27) Mai, H. D.; Nguyen, T. T.; Pham, V. C.; Litaudon, M.; Gueritte, F.; Tran, D. T.; Nguyen, V. H. Planta Med. 2010,76, 1739− 1742. (28) Soby, S.; Bates, R.; van Etten, H. Phytochemistry 1997,45, 925− 929. (29) Soby, S.; Caldera, S.; Bates, R.; VanEtten, H. Phytochemistry 1996,41, 759−765. (30) Goel, A.; Kumar, A.; Raghuvanshi, A. Chem. Rev. 2013,113, 1614−1640. (31) Duffy, S.; Avery, V. M. Am. J. Trop. Med. Hyg. 2012,86,84−92. (32) Mishra, L. C.; Bhattacharya, A.; Bhasin, V. K. Acta Trop. 2009, 109, 194−198. (33) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. Journal of Natural Products pubs.acs.org/jnp Article https://dx.doi.org/10.1021/acs.jnatprod.0c00245 J. Nat. Prod. XXXX, XXX, XXX−XXX H