Crystal Structures and Cytotoxicity of ent-Kaurane-Type Diterpenoids from Two Aspilia Species

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/ Crystal Structures and Cytotoxicity of ent-Kaurane-Type Diterpenoids from Two Aspilia Species © the Authors, 2018. Published version Yaouba, Souaibou; Valkonen, Arto; Coghi, Paolo; Gao, Jiaying; Guantai, Eric M.; Derese, Solomon; Wong, Vincent K. W.; Erdélyi, Máté; Yenesew, Abiy Yaouba, S., Valkonen, A., Coghi, P., Gao, J., Guantai, E. M., Derese, S., Wong, V. K. W., Erdélyi, M., & Yenesew, A. (2018). Crystal Structures and Cytotoxicity of ent-Kaurane-Type Diterpenoids from Two Aspilia Species. Molecules, 23(12), Article 3199. https://doi.org/10.3390/molecules23123199 2018 molecules Article Crystal Structures and Cytotoxicity of ent-Kaurane-Type Diterpenoids from Two Aspilia Species Souaibou Yaouba 1, Arto Valkonen 2, Paolo Coghi 3, Jiaying Gao 3, Eric M. Guantai 4, Solomon Derese 1, Vincent K. W. Wong 3, MátéErdélyi 5,6,7,* and Abiy Yenesew 1,* 1Department of Chemistry, University of Nairobi, P. O. Box 30197, 00100 Nairobi, Kenya; [email protected] (S.Y.); [email protected] (S.D.) 2Department of Chemistry, University of Jyvaskyla, P.O. Box 35, 40014 Jyvaskyla, Finland; [email protected] 3State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078, China; [email protected] (P.C.); [email protected] (J.G.); [email protected] (V.K.W.W.) 4Department of Pharmacology and Pharmacognosy, School of Pharmacy, University of Nairobi, P. O. Box 19676, 00202 Nairobi, Kenya; [email protected] 5Department of Chemistry–BMC, Uppsala University, Husargatan 3, 75237 Uppsala, Sweden 6The Swedish NMR Centre, Medicinaregatan 5, 40530 Gothenburg, Sweden 7Department of Chemistry and Molecular Biology, University of Gothenburg, 40530 Gothenburg, Sweden *Correspondence: [email protected] (M.E.); [email protected] (A.Y.); Tel.: +46-72-999-9166 (M.E.); +254-73-383-2576 (A.Y.); Fax: +254-20-444-6138 (A.Y.) Academic Editors: Isabel C.F.R. Ferreira and Nancy D. Turner Received: 11 November 2018; Accepted: 30 November 2018; Published: 4 December 2018   Abstract: A phytochemical investigation of the roots of Aspilia pluriseta led to the isolation of ent-kaurane-type diterpenoids and additional phytochemicals ( 1 – 23 ). The structures of the isolated compounds were elucidated based on Nuclear Magnetic Resonance (NMR) spectroscopic and mass spectrometric analyses. The absolute configurations of seven of the ent-kaurane-type diterpenoids ( 3 – 6 , 6b, 7 and 8 ) were determined by single crystal X-ray diffraction studies. Eleven of the compounds were also isolated from the roots and the aerial parts of Aspilia mossambicensis. The literature NMR assignments for compounds 1 and 5 were revised. In a cytotoxicity assay, 12 α -methoxy-ent-kaur-9(11),16-dien-19-oic acid ( 1 ) (IC 50 = 27.3 ± 1.9 µ M) and 9 β -hydroxy-15 α -angeloyloxy-ent-kaur-16-en-19-oic acid ( 3 ) (IC 50 = 24.7 ±2.8 µM ) were the most cytotoxic against the hepatocellular carcinoma (Hep-G2) cell line, while 15 α -angeloyloxy-16 β ,17-epoxy-ent-kauran-19-oic acid ( 5 ) (IC 50 = 30.7 ± 1.7 µ M) was the most cytotoxic against adenocarcinomic human alveolar basal epithelial (A549) cells. Keywords: Asteraceae; Aspilia pluriseta;Aspilia mossambicensis;ent-kaurane diterpenoid; X-ray crystal structure; cytotoxicity 1. Introduction The genus Aspilia belongs to the family Asteraceae. The majority of plants in this family are herbaceous, while trees and shrubs are rare [ 1 ]. Plants belonging to the Asteraceae family are found worldwide, except Antarctica [ 2 ]. They are found in cooler montane habitats or temperate areas in tropical regions, and are not common dwellers of hot lowland tropical rain forests [ 1 , 2 ]. The family of Asteraceae is one of the largest plant families and the richest in vascular plants in the world. The family has about 1,600–1,700 genera and 24,000–30,000 species [ 1 , 3 , 4 ]. Plants from the genus Aspilia (Asteraceae) occur widely in South, South-West, and West Kenya, from the coast to Lake Molecules 2018,23, 3199; doi:10.3390/molecules23123199 www.mdpi.com/journal/molecules Molecules 2018,23, 3199 2 of 13 Victoria. The genus Aspilia exhibits biological activities, including antibacterial and antifungal effects, mainly attributed to the presence of kaurane-type diterpenoids [3,5] and sesquiterpene lactones [5,6]. Aspilia pluriseta Schweinf has been used in traditional medicine to treat lacerations, bruises and burns, and it is reputed to aid in the healing of cutaneous lesions [ 7 ]. The plant is found in Kenya and is commonly known as ‘Dwarf Aspilia’ [ 7 ]. The presence of diterpenoids from aerial parts of A. pluriseta has been reported previously [ 8 ], and four of these diterpenoids exhibited moderate activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. The aqueous extract of the plant was also reported by the same authors to exhibit hypoglycemic properties in alloxanized mice. A. pluriseta is locally known in Kenya as Muuti (Kikuyu), Wuti (Kamba), Ol-oiyabase (Maasai), and Shilambila (Luhya). Many communities in Kenya, as well as some in the rest of Eastern and Southern Africa, use the plant ethnomedically to treat wounds [7]. Aspilia mossambicensis (Oliv.) Wild is a shrub native to central and Eastern tropical Africa. The plant is found in the Democratic Republic of Congo, Ethiopia, Kenya, Malawi, Mozambique, Tanzania, Uganda, Zambia, and Zimbabwe [ 9 ]. In Eastern Africa, the plant is well known for the treatment of cystitis, gonorrhea, abdominal pain, intestinal worms, and skin infections [ 9 – 12 ]. The thiophene derivatives, thiarubrines A and B, have previously been isolated from Aspilia mossambicensis [ 9 ]. The roots of this plant exhibited antibacterial activity, which was suggested to explain its use by wild chimpanzees [ 9 , 11 ]. Herein, we report the phytochemical investigation and the cytotoxicity study of the constituents of Aspilia pluriseta Schweinf and Aspilia mossambicensis (Oliv.) Wild (Asteraceae). 2. Results and Discussion Compound 1 , [ α]20 D− 88 ◦ , was isolated as colorless crystals (m.p. 184–186 ◦ C) from the CH 2 Cl 2 /MeOH (1:1) extract of the roots of Aspilia pluriseta. HRMS (Figure S7, Supplementary Material) showed a [M–H] − ion peak at m/z = 329.2191, which is in agreement with the molecular formula C 21 H 30 O 3 . The NMR spectra (Table 1) indicated that this compound is a kaurene diterpenoid (Figure 1). The 1 H–NMR spectrum further revealed the presence of three olefinic protons, namely H-11 ( δH 5.30), H-17a ( δH 4.84) and H-17b ( δH 4.94), suggesting two double bonds. The 13 C–NMR chemical shifts of C-16 (δC152.9) and C-17 (δC108.1) are typical of a terminal double bond in an ent-kaurene skeleton. Molecules 2018, 23, x 2 of 13 The genus Aspilia exhibits biological activities, including antibacterial and antifungal effects, mainly attributed to the presence of kaurane-type diterpenoids [3,5] and sesquiterpene lactones [5,6]. Aspilia pluriseta Schweinf has been used in traditional medicine to treat lacerations, bruises and burns, and it is reputed to aid in the healing of cutaneous lesions [7]. The plant is found in Kenya and is commonly known as ‘Dwarf Aspilia’ [7]. The presence of diterpenoids from aerial parts of A. pluriseta has been reported previously [8], and four of these diterpenoids exhibited moderate activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. The aqueous extract of the plant was also reported by the same authors to exhibit hypoglycemic properties in alloxanized mice. A. pluriseta is locally known in Kenya as Muuti (Kikuyu), Wuti (Kamba), Ol-oiyabase (Maasai), and Shilambila (Luhya). Many communities in Kenya, as well as some in the rest of Eastern and Southern Africa, use the plant ethnomedically to treat wounds [7]. Aspilia mossambicensis (Oliv.) Wild is a shrub native to central and Eastern tropical Africa. The plant is found in the Democratic Republic of Congo, Ethiopia, Kenya, Malawi, Mozambique, Tanzania, Uganda, Zambia, and Zimbabwe [9]. In Eastern Africa, the plant is well known for the treatment of cystitis, gonorrhea, abdominal pain, intestinal worms, and skin infections [9–12]. The thiophene derivatives, thiarubrines A and B, have previously been isolated from Aspilia mossambicensis [9]. The roots of this plant exhibited antibacterial activity, which was suggested to explain its use by wild chimpanzees [9,11]. Herein, we report the phytochemical investigation and the cytotoxicity study of the constituents of Aspilia pluriseta Schweinf and Aspilia mossambicensis (Oliv.) Wild (Asteraceae). 2. Results and Discussion Figure 1. Structures of compounds 1–8. Compound 1, [α]20 D−88°, was isolated as colorless crystals (m.p. 184–186 °C) from the CH2Cl2/MeOH (1:1) extract of the roots of Aspilia pluriseta. HRMS (Figure S7, Supplementary Material) showed a [M–H]− ion peak at m/z = 329.2191, which is in agreement with the molecular formula C21H30O3. The NMR spectra (Table 1) indicated that this compound is a kaurene diterpenoid (Figure 1). The 1H–NMR spectrum further revealed the presence of three olefinic protons, namely HH H 3 CO H 1 1 35 7 9 12 13 15 17 20 OHO H 2 H OO OH 3 OOH OH OO H OCH 3 O 4 OHO OO H H O 5 H OHO 6 6a OHO H 6b OO H OHO 7 OHO H 8 OH H H H OHO H OCH 3 OH O 18 19 OHO OO H H O OHO OO H H O 5a 5b 5' 4' 1' 1' 4' 5' 1' 4' 5' Figure 1. Structures of compounds 1–8. Molecules 2018,23, 3199 3 of 13 Table 1. The 1H (800 MHz) and 13C-NMR (200 MHz) data for compound 1acquired in CDCl3. Position δCLit. [13] * δCδH, mult. (Jin Hz) HMBC (2J,3J) 138.17 40.6 1.14 ddd (13.5, 9.5, 4.2) C-2, C-3, C-10, C-20 1.90 ddd (13.5, 3.5, 1.4) C-2, C-3, C-10, C-20 218.35 20.0 1.43 dddd (14.2, 9.5, 3.9, 3.5) C-1, C-3, C-4, C-5, C-10 1.79 ddddd (14.2, 11.1, 4.2, 3.5, 1.4) C-1, C-4, C-5 329.03 38.1 0.93 ddd (13.4, 11.1, 3.9) C-1, C-2, C-4, C-18, C-19 2.08 ddd (13.4, 3.5, 3.5) C-1, C-4, C-5, C-7 4 43.43 44.6 5 43.81 46.1 1.56 dd (11.1, 8.5) C-4, C-7, C-9, C-10, C-18, C-19, C-20 6 20.07 18.3 1.82 dddd (14.2, 10.0, 8.5, 2.5) C-3, C-4, C-5, C-7, C-10 2.43 dddd (14.2, 11.1, 9.5, 3.5) C-4, C-5, C-8 7 40.60 28.9 1.42 ddd (13.8, 3.5, 2.5) C-5, C-6, C-8, C-9, C-15 1.95 ddd (13.8, 10.0, 9.5) C-6, C-8, C-9, C-14, C-15 8 44.66 43.4 9 160.28 160.2 10 38.94 38.9 11 115.42 115.3 5.30 dd (4.3, 1.4) C-8, C-9, C-10, C-12, C-13, C-15, C-20 12 81.79 81.7 3.38 dd (4.3, 2.9) C-9, C-11, C-13, C-16, C-20, OCH3-12 13 46.17 43.7 2.89 dd (2.9, 1.4) C-10, C-11, C-12, C-15, C-16 14 40.60 40.5 1.31 dd (10.8, 4.3) C-7, C-8, C-9, C-12, C-13, C-15 1.58 dd (10.8, 2.5) C-9, C-12, C-13, C-15, C-16 15 47.17 47.1 2.08 dd (15.4, 4.3) C-7, C-8, C-9, C-16, C-17 2.35 dd (15.4, 2.5) C-7, C-9, C-13, C-14, C-16, C-17 16 153.00 152.9 17 108.12 108.1 4.84 dd (3.0, 1.6) C-12, C-13, C-15, C-16 4.94 dd (3.0, 1.6) C-12, C-13, C-15, C-16 18 28.22 28.2 1.17 sC-3, C-4, C-5, C-8, C-19 19 182.98 183.2 20 23.41 23.4 1.01 sC-1, C-5, C-9, C-10 OCH3-12 56.53 56.5 3.34 sC-12 * CDCl3at 100 MHz [13]. The second double bond was placed between C-9 ( δC 160.2) and C-11 ( δH 5.30; δC 115.3) by comparison of the NMR data with that found in the literature [ 13 – 15 ]. Signals indicating the presence of a methoxy ( δH 3.34, δC 56.5) and a carboxylic acid ( δC 183.2) substituent were observed. The Heteronuclear Multiple Bond Correlations (HMBCs) of CH 3 -18 ( δH 1.17), H-3 ( δH0.93 ), and H-5 ( δH 1.56) with the carboxy resonance C-19 ( δC 183.2) suggested the location of the carboxy group (C-19) at C-4. Out of the three methyl groups expected in kaurene diterpenoid, only two, i.e., CH 3 -18 ( δH 1.17, δC 28.2) and CH 3 -20 ( δH 1.01, δC 23.4), were observed. This corroborated the suggestion of the third methyl group being oxidized to a carboxylic acid (C-19, δC 183.2). The methoxy group OCH 3 -12 ( δH3.34 ) showed HMBC correlation with C-12 ( δC 81.7), whereas H-12 ( δH 3.38) showed HMBC correlation with C-9 ( δC 160.2), C-11 ( δC 115.3), C-13 ( δC 43.7), C-16 ( δC 152.9), and OCH 3 -12 ( δC 56.5). Furthermore, CH 3 -20 ( δH 1.01) showed HMBC correlation with C-1 ( δC40.6 ), C-5 ( δC 46.1), the olefinic carbon C-9 ( δC 160.2), and C-10 ( δC 38.9). This confirmed that the second double bond in the molecule is located at C-9. Moreover, the HMBC correlation of CH 2 -14 ( δH 1.31, 1.58) with a deshielded carbon C-12 ( δC 81.7) is in agreement with OCH 3 being connected to C-12. The above findings confirmed the identity of compound 1 as a C-12-methoxy substituted ent-kaur-9(11),16-dienoic acid derivative. The relative configuration at C-12 was deduced from the Nuclear Overhauser Effect (NOE) of OCH 3 -12 ( δH 3.34) to H-13 ( δH 2.89) (Figure 2), indicating them to be syn-oriented, and hence OCH 3 -12 to be α -oriented. It should be noted that H-12 (δH3.38 ) also showed a weak NOE to H-13 ( δH 2.89), which is expected in a strained ring system. The proposed configuration at C-12 is further corroborated by the NOE of H-12 ( δH3.38 ) with H-14b ( δH 1.58). The NOE of H-12 ( δH 3.38 ppm) with H-17b ( δH 4.94 ppm) supported H-12 to be β -oriented, and hence OCH 3 -12 to be α -oriented. Based on the above spectroscopic evidence, compound 1 , 12 α -methoxy-ent-kaur-9(11),16-dien-19-oic acid, (Figure 1) was identified as (4R,4aS,6aS,9R,10S,11bR)-10-methoxy-4,11b-dimethyl-8-methylene-1,2,3,4,4a,5,6,7,8,9,10,11bdodecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylic acid. This compound has previously been reported both as synthetic derivative [ 13 ] and as a natural product [ 14 , 15 ]. However, Molecules 2018,23, 3199 4 of 13 our NMR data assignment differs from that reported in the literature [ 13 ] for its C-3 and C-7. The accuracy of the corrected assignment, given in Table 1, is corroborated by the HMBC correlations of H-3 and H-7 (Table 1), by the HSQC crosspeaks of CH 2 -3 ( δH 0.93/2.08) to C-3 ( δC 38.1) along with the TOCSY(Total Correlation Spectroscopy) correlations of CH 2 -3 to CH 2 -1 ( δH 1.14/1.90) and CH 2 -2 ( δH 1.43/1.79), and by the HSQC (Heteronuclear Single Quantum Correlation) crosspeaks of CH 2 -7 ( δH 1.42/1.95) to C-7 ( δC 28.9) along with the TOCSY correlations of CH 2 -7 to CH 2 -6 ( δH1.82/2.43 ) and H-5 ( δH 1.56) (Figures S4–S6, Supplementary Material). Besides the compound having been reported earlier, it is unlikely to be an extraction artifact as the extraction (with CH 2 Cl 2 /MeOH, 1:1) has been performed at low temperature at neutral pH that does not promote formation of methyl ethers. The compound has been detected in the crude extract indicating that the compound is a natural product and has not formed during the chromatographic isolation. Molecules 2018, 23, x 4 of 13 expected in a strained ring system. The proposed configuration at C-12 is further corroborated by the NOE of H-12 (δH 3.38) with H-14b (δH 1.58). The NOE of H-12 (δH 3.38 ppm) with H-17b (δH 4.94 ppm) supported H-12 to be β-oriented, and hence OCH3-12 to be α-oriented. Based on the above spectroscopic evidence, compound 1, 12α-methoxy-ent-kaur-9(11),16-dien-19-oic acid, (Figure 1) was identified as (4R,4aS,6aS,9R,10S,11bR)-10-methoxy-4,11b-dimethyl-8-methylene1,2,3,4,4a,5,6,7,8,9,10,11b-dodecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylic acid. This compound has previously been reported both as synthetic derivative [13] and as a natural product [14,15]. However, our NMR data assignment differs from that reported in the literature [13] for its C-3 and C-7. The accuracy of the corrected assignment, given in Table 1, is corroborated by the HMBC correlations of H-3 and H-7 (Table 1), by the HSQC crosspeaks of CH2-3 (δH 0.93/2.08) to C-3 (δC 38.1) along with the TOCSY(Total Correlation Spectroscopy) correlations of CH2-3 to CH2-1 (δH 1.14/1.90) and CH2-2 (δH 1.43/1.79), and by the HSQC (Heteronuclear Single Quantum Correlation) crosspeaks of CH2-7 (δH 1.42/1.95) to C-7 (δC 28.9) along with the TOCSY correlations of CH2-7 to CH26 (δH 1.82/2.43) and H-5 (δH 1.56) (Figures S4–6, Supplementary Material). Besides the compound having been reported earlier, it is unlikely to be an extraction artifact as the extraction (with CH2Cl2/MeOH, 1:1) has been performed at low temperature at neutral pH that does not promote formation of methyl ethers. The compound has been detected in the crude extract indicating that the compound is a natural product and has not formed during the chromatographic isolation. Figure 2. Some of the key NOE correlations observed for compound 1. The NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum (800 MHz, CDCl3, 25 °C, 700 ms mixing time) is shown in the Supplementary Materials. Additional compounds (Figures 1 and 3) isolated from the roots of Aspilia pluriseta include (16R)- hydroxy-ent-kauran-19-oic acid (2) [16,17], 9β-hydroxy-15α-angeloyloxy-ent-kaur-16-en-19-oic acid (3) [18], methyl-9β-hydroxy-15α-angeloyloxy-ent-kaur-16-en-19-oate (4) [19], 15α-angeloyloxy16β,17-epoxy-ent-kauran-19-oic acid (5) [20], ent-kaur-9(11),16-dien-19-oic acid (6) [21], 15αangeloyloxy-ent-kaur-16-en-19-oic acid (7) [22], ent-kaur-9(11),16-dien-12-one (9) [23] and methyl-entkaur-16-en-19-oate (10) [24]. The aerial part of Aspilia pluriseta contained ent-kaur-16-en-19-oic acid (11) [21,25], ent-kaur-16-en-19-ol (12) [26], lanosterol (13) [27], stigmasta-5,22(E)-dien-3β-ol (14) [28], 3β-hydroxy-olean-12-en-29-oic acid (15) [29], and carissone (16) [30]. Similar phytochemical investigation of the roots of Aspilia mossambicensis resulted in the isolation of methyl-15α-angeloyloxy-ent-kaur-16-en-19-oate (17) [24,31], 12-oxo-ent-kaur-9(11),16-dien-19-oic acid (18) [32], (16S)-ent-kauran-19-oic acid (8) [33], oleanolic acid (19) [34] (Figure 3), and compounds 3–5. The aerial part of A. mossambicensis afforded compound 6 [21,26], 3β-acetyloxy-olean-12-ene (20) [35,36] ent-kaur-9(11),16-diene (21) [37], 15a-hydroxy-kaur-9(11),16-diene (22) [38], and methyl cinnamate (23) [39]. The crystal structures for compounds 3–8 (Figure 4) are also reported here, where the identities of the compounds were confirmed, and the absolute configurations established. In the crystal state, compound 3 exhibited a continuous network, involving intermolecular O(9)-H···O(19a) and O(19b)- H···O(21) hydrogen bonds. Similar O(9)-H···O(19a) hydrogen bonding motif was found in compound 4, which also showed static disorder exhibiting two different spatial orientations of (Z)-2-methylbutFigure 2. Some of the key NOE correlations observed for compound 1 . The NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum (800 MHz, CDCl3, 25 ◦C, 700 ms mixing time) is shown in the Supplementary Materials. Additional compounds (Figures 1and 3) isolated from the roots of Aspilia pluriseta include (16R)-hydroxy-ent-kauran-19-oic acid ( 2 ) [ 16 , 17 ], 9 β -hydroxy-15 α -angeloyloxy-ent-kaur-16-en-19-oic acid ( 3 ) [ 18 ], methyl-9 β -hydroxy-15 α -angeloyloxy-ent-kaur-16-en-19-oate ( 4 ) [ 19 ], 15 α -angeloyloxy-16 β ,17-epoxy-ent-kauran-19-oic acid ( 5 ) [ 20 ], ent-kaur-9(11),16-dien-19-oic acid ( 6 ) [ 21 ], 15 α -angeloyloxy-ent-kaur-16-en-19-oic acid ( 7 ) [ 22 ], ent-kaur-9(11),16-dien-12-one ( 9 ) [ 23 ] and methyl-ent-kaur-16-en-19-oate ( 10 ) [ 24 ]. The aerial part of Aspilia pluriseta contained ent-kaur-16-en-19-oic acid ( 11 ) [ 21 , 25 ], ent-kaur-16-en-19-ol ( 12 ) [ 26 ], lanosterol ( 13 ) [ 27 ], stigmasta-5,22(E)-dien-3 β -ol ( 14 ) [ 28 ], 3 β -hydroxy-olean-12-en-29-oic acid ( 15 ) [ 29 ], and carissone (16) [30]. Similar phytochemical investigation of the roots of Aspilia mossambicensis resulted in the isolation of methyl-15 α -angeloyloxy-ent-kaur-16-en-19-oate ( 17 ) [ 24 , 31 ], 12-oxo-ent-kaur-9(11),16-dien-19-oic acid ( 18 ) [ 32 ], (16S)-ent-kauran-19-oic acid ( 8 ) [ 33 ], oleanolic acid ( 19 ) [ 34 ] (Figure 3), and compounds 3 – 5 . The aerial part of A. mossambicensis afforded compound 6 [ 21 , 26 ], 3 β -acetyloxy-olean-12-ene ( 20 ) [ 35 , 36 ]ent-kaur-9(11),16-diene (21) [ 37 ], 15a-hydroxy-kaur-9(11),16-diene ( 22 ) [ 38 ], and methyl cinnamate (23) [39]. The crystal structures for compounds 3 – 8 (Figure 4) are also reported here, where the identities of the compounds were confirmed, and the absolute configurations established. In the crystal state, compound 3 exhibited a continuous network, involving intermolecular O(9)-H ··· O(19a) and O(19b)-H ··· O(21) hydrogen bonds. Similar O(9)-H ··· O(19a) hydrogen bonding motif was found in compound 4 , which also showed static disorder exhibiting two different spatial orientations of (Z)-2-methylbut-2-enoyl group in an approximately 1:1 ratio. Compounds 5 – 8 showed common double hydrogen bonding motifs for carboxylic acids leading to the formation of hydrogen-bonded pairs. Molecules 2018,23, 3199 5 of 13 Molecules 2018, 23, x 5 of 13 2-enoyl group in an approximately 1:1 ratio. Compounds 5–8 showed common double hydrogen bonding motifs for carboxylic acids leading to the formation of hydrogen-bonded pairs. Figure 3. Structures of compounds 9–23. Crystal structure of compound 6 was determined at 120 K, which corresponds to the previously reported structure at room temperature [40]. Crystal structure analysis of a synthetic derivative of compound 6 led to the identification of ent-kaur-9(11)-en-19-oic acid (6b). In the structure 6b, there is a slight (~10%) disorder in the main ring system, where carbons C7–C17 have different positions giving a shade of different conformations for the corresponding aliphatic rings. The data quality for compound 7 is slightly deficient and the absolute structure of it could not be justified on the basis of data. The X-ray diffraction data parameters, thermal ellipsoid diagrams, and hydrogen bonding geometries are presented in the Supplementary Information section of this article. In most of the ent-kaurane-type diterpenoids (except for compounds 9, 21, and 22) isolated from the two Aspilia species, A. pluriseta and A. mossambicensis, the α-methyl group at C-4 (C-19) is oxidized into carboxylic acid or methyl ester groups, which could be a characteristic feature of the genus Aspilia [3,8]. There are also examples where oxidation has occurred at C-12 (compounds 1, 9 and 18), C-16 (compound 2), C-9 (compounds 3 and 4), and C-15 (compounds 4, 5, 6, and 18). In agreement with the literature [36], oxidation has not been observed at other carbon atoms in ent-kaurane-type diterpenoids of Aspilia species. Some ent-kaurane-type diterpenoids, including 16,17-epoxy-15β-tigloyloxy-ent-kauran-18-oic acid (5a) and 16,17-epoxy-15β-senecioyloxy-ent-kauran-18-oic acid (5b), were reported earlier from Aspilia pluriseta [8]. These compounds were reported to have the 15β-tigloyloxy and 15β-senecioyloxy groups, respectively, occupying the less favorable orientation [8]; however, the authors have not provided evidence for these proposals. Our single crystal X-ray analyses have shown that the C-15 substituent of compounds 3, 4, 5, and 7 is an angeloyloxy group occupying the more favourable-15αposition (Figure 4). In fact, the proposed stereochemical assignment of ent-kaurane-type diterpenoids reported from this genus, particularly in highly functionalized compounds, lacks evidence. We have Figure 3. Structures of compounds 9–23. Crystal structure of compound 6 was determined at 120 K, which corresponds to the previously reported structure at room temperature [ 40 ]. Crystal structure analysis of a synthetic derivative of compound 6 led to the identification of ent-kaur-9(11)-en-19-oic acid ( 6b ). In the structure 6b , there is a slight (~10%) disorder in the main ring system, where carbons C7–C17 have different positions giving a shade of different conformations for the corresponding aliphatic rings. The data quality for compound 7 is slightly deficient and the absolute structure of it could not be justified on the basis of data. The X-ray diffraction data parameters, thermal ellipsoid diagrams, and hydrogen bonding geometries are presented in the Supplementary Information section of this article. In most of the ent-kaurane-type diterpenoids (except for compounds 9 , 21, and 22 ) isolated from the two Aspilia species, A. pluriseta and A.mossambicensis, the α -methyl group at C-4 (C-19) is oxidized into carboxylic acid or methyl ester groups, which could be a characteristic feature of the genus Aspilia [ 3 , 8 ]. There are also examples where oxidation has occurred at C-12 (compounds 1, 9 and 18 ), C-16 (compound 2 ), C-9 (compounds 3 and 4 ), and C-15 (compounds 4 , 5 , 6, and 18 ). In agreement with the literature [ 36 ], oxidation has not been observed at other carbon atoms in ent-kaurane-type diterpenoids of Aspilia species. Some ent-kaurane-type diterpenoids, including 16,17-epoxy-15 β -tigloyloxy-ent-kauran-18-oic acid ( 5a ) and 16,17-epoxy-15 β -senecioyloxy-ent-kauran-18-oic acid ( 5b ), were reported earlier from Aspilia pluriseta [ 8 ]. These compounds were reported to have the 15 β -tigloyloxy and 15 β -senecioyloxy groups, respectively, occupying the less favorable orientation [ 8 ]; however, the authors have not provided evidence for these proposals. Our single crystal X-ray analyses have shown that the C-15 substituent of compounds 3 , 4 , 5, and 7 is an angeloyloxy group occupying the more favourable-15 α -position (Figure 4). In fact, the proposed stereochemical assignment of ent-kaurane-type diterpenoids reported from this genus, particularly in highly functionalized compounds, lacks evidence. We have filled this knowledge gap by determining the absolute configuration of seven ent-kaurane-type diterpenoids, as shown in Figure 4, using single crystal X-ray analyses. Molecules 2018,23, 3199 6 of 13 Molecules 2018, 23, x 6 of 13 filled this knowledge gap by determining the absolute configuration of seven ent-kaurane-type diterpenoids, as shown in Figure 4, using single crystal X-ray analyses. Figure 4. Crystal structure representations of compounds 3–8. The structures were deposited with the following CCDC (Cambridge Crystallographic Data Centre) codes: 3 (1868318), 4 (1868319), 5 (1868321), 6 (1868320), 6b (1868324), 7 (1868323), and 8 (1868322). The 13C-NMR data of compound 5 (Table 2) is in close agreement to that previously reported in the literature [20], except for the chemical shift assignment of C-2 and C-12 (Table 2). Related structures, 5a and 5b, have been proposed for two compounds earlier reported [8] from Aspilia pluriseta. The 13C-NMR assignment (Table 2) for these compounds differs from our assignment, which is based on 2D NMR correlations, despite the common 16,17-epoxy-15-oxy-ent-kauran-18-oic acid skeleton (Figure 1). The 13C-NMR chemical shifts of C-4’ (δC 27.4) and C-5’ (δC 20.8) in compound 5a do not support a tigloyloxy group at C-15, as proposed in Reference [8]; methyl carbon atoms in such group are expected to resonate at ~14 ppm (for C-4’) and at ~11 ppm (for C-5’), based on chemical shift prediction [41] and previous literature [14]. The NMR spectra of compound 5b that are given in Figure 4. Crystal structure representations of compounds 3 – 8 . The structures were deposited with the following CCDC (Cambridge Crystallographic Data Centre) codes: 3 (1868318), 4 (1868319), 5 (1868321), 6(1868320), 6b (1868324), 7(1868323), and 8(1868322). The 13 C-NMR data of compound 5 (Table 2) is in close agreement to that previously reported in the literature [ 20 ], except for the chemical shift assignment of C-2 and C-12 (Table 2). Related structures, 5a and 5b , have been proposed for two compounds earlier reported [ 8 ] from Aspilia pluriseta. The 13 C-NMR assignment (Table 2) for these compounds differs from our assignment, which is based on 2D NMR correlations, despite the common 16,17-epoxy-15-oxy-ent-kauran-18-oic acid skeleton (Figure 1). The 13 C-NMR chemical shifts of C-4’ ( δC 27.4) and C-5’ ( δC 20.8) in compound 5a do not support a tigloyloxy group at C-15, as proposed in Reference [ 8 ]; methyl carbon atoms in such group are expected to resonate at ~14 ppm (for C-4’) and at ~11 ppm (for C-5’), based on chemical shift prediction [ 41 ] and previous literature [ 14 ]. The NMR spectra of compound 5b that are given in the supporting information of [ 8 ] are of low quality and do not allow confirmation of the proposed Molecules 2018,23, 3199 7 of 13 assignment. It should be noted that the numbering used in this paper does not follow the literature convention [ 3 ]. Hence, the carboxylic group of 5b and of its structural analogues should not be assigned as C-18, but rather as C-19, following reference [ 3 ]. Overall, several details reported [ 8 ] for these compounds appear debatable, and consequently so are the proposed structures. To avoid such uncertainties, the NMR assignments of all compounds discussed in this paper are presented in Tables S1 and S2 in the Supplementary Material. Table 2. The literature reported NMR data for 5 , 5a, and 5b and the 1 H (800 MHz) and 13 C-NMR (200 MHz) data for compound 5acquired in CDCl3. Position 5 [20] 5a [8] 5b [8] 5 5 δCδCδCδCδH, mult. (Jin Hz) 141.2 40.6 40.6 40.6 0.80 ddd (7.2, 7.1, 1.3) 1.86* dd (2.9, 1.4) 228.9 19.8 19.0 19.7 1.55 ddd (7.3, 3.6, 2.4) 1.75 dd (3.7, 3.6) 337.7 36.7 36.4 37.6 0.96 ddd (13.7, 13.6, 4.3) 2.11 dd (13.7, 3.1) 4 43.6 46.9 47.8 43.5 5 56.7 20.3 56.6 56.5 1.16 dd (9.1, 7.1) 6 19.0 41.2 20.3 20.8 1.76 ddd (5.7, 3.4, 2.1) 1.86* ddd (3.4, 3.4, 2.7) 7 35.4 47.8 41.2 35.3 1.25 ddd (14.4, 13.9, 4.4) 1.79 ddd (13.8, 13.2, 4.3) 8 47.9 52.9 43.6 47.8 9 52.9 43.6 53.0 52.8 1.28 dd (13.8, 3.8) 10 39.8 56.6 39.8 39.7 11 19.8 20.8 19.8 18.9 1.40 ddd (13.8, 3.4, 3.4, 3.1) 1.81 dd (13.8, 4.3) 12 20.8 28.9 28.9 28.8 1.50 ddd (13.5, 7.8, 7.2) 13 41.2 36.4 35.1 41.1 1.82 dd (13.8, 4.4) 14 36.5 37.7 37.7 36.4 1.68 dd (14.5, 3.3) 1.97 dd (13.1, 3.4) 15 81.9 81.2 81.2 81.9 4.73 br s 16 66.3 66.4 66.4 66.3 17 49.6 49.6 49.6 49.6 2.78 dd (5.6, 1.3) 3.09 dd (5.8, 1.3) 18 28.8 28.9 28.9 28.7 1.28 s 19 182.3 182.6 182.6 182.7 20 15.7 15.8 16.0 15.9 1.03 s 1’ 167.9 166.5 166.5 167.8 2’ 128.1 129.0 115.9 128.0 3’ 137.3 137.1 156.8 137.3 5.96 q(7.1) 4’ 15.9 27.4 20.8 15.7 1.96 d(1.9) 5’ 20.6 20.8 27.4 20.6 s Most of the compounds isolated in this study were assayed against two normal (BEAS-2B and LO 2 ) and two cancer (A549 and Hep-G2, Table 3) cell lines. Of the fifteen compounds tested, 1 , 3 , 5 , 9, and 18 showed cytotoxicity towards some of the cell lines. Compound 1 showed selective activity against the human hepatoma (HepG2) cancer cell line without significant toxicity to other cell lines (IC 50 above 100 µM ). Compound 3 was moderately cytotoxic against the Hep-G2 cancer cell line ( IC50 = 24.7 ±2.8 ), but also showed weak cytotoxicity towards the normal cell lines LO 2 (IC 50 = 57.2 ± 1.2) and BEAS-2B (IC 50 = 89.9 ± 2.0 µ M), indicating low selectivity. Compound 5 ( IC50 = 30.7 ±1.7 µM ) was the most active against the A549 cell line, followed by compound 18 ( IC50 = 80.5 ±1.8 µM ). The other compounds tested, i.e., 2 , 4 , 6 , 7, 10 , 11 , 14, or 17 , did not exhibit significant cytotoxicity (IC 50 above 100 µ M). The lack of cytotoxicity against the two normal cell lines of these diterpenoids is valuable information, considering that some kaurane-type diterpenoids from Aspilia species have showed antibacterial and antifungal effects [5]. Molecules 2018,23, 3199 8 of 13 Table 3. Cytotoxicity (IC 50 , µ M) of compounds isolated from Aspilia species against various cell lines. Compound Normal Cell Lines Cancer Cell Lines BEAS-2B LO2A549 Hep-G2 1>100 >100 >100 27.3 ±1.9 2>100 >100 >100 >100 389.9 ±2.0 57.2 ±1.2 >100 24.7 ±2.8 4>100 >100 >100 >100 5>100 >100 30.7 ±1.7 >100 6>100 >100 >100 >100 6a >100 >100 >100 >100 6b >100 >100 >100 >100 7>100 >100 >100 >100 9>100 75.3 ±2.8 >100 >100 10 >100 >100 >100 >100 11 >100 >100 >100 >100 14 >100 >100 >100 >100 17 >100 >100 >100 >100 18 38.6 ±2.5 30.0 ±1.7 80.5 ±1.8 81.3 ±0.3 Paclitaxel <0.1 <0.1 0.0033 0.19 3. Materials and Methods 3.1. General Experimental Procedures NMR spectra were acquired on a Bruker Avance II 600 MHz, a Bruker Avance III HD 800 MHz (Bruker BioSpin AG, Fällanden, Switzerland) or a Varian Unity 500 MHz (Varian Inc, Palo Alto, CA, USA) NMR spectrometer, using the residual solvent peaks as a reference. The spectra were processed using the software MestReNova (version 10.0, Mestrelab Research S.L., Santiago de Compostela, Spain) Coupling constants (J) are given in Hz. EI-MS and LC-MS were carried out using 70 eV ionization electron voltage on a Micromass GC-TOF spectrometer (Micromass, Wythenshawe, Waters Inc., UK). TLC (Thin Layer Chromatography) was carried out on Merck pre-coated silica gel 60 F 254 plates (Merck, Darmstadt, Germany). Preparative TLC was performed on 20 × 20 cm glass plates, pre-coated with silica gel 60F 254 with thicknesses of 0.25 to 1 mm. Column chromatography was run on silica gel 60 Å (70–230 mesh). Gel filtration was performed on Sephadex LH-20(Merck, Darmstadt, Germany). 3.2. X-ray Diffraction Analyses The single crystal X-ray diffraction data were collected using Agilent Super-Nova (Agilent Technologies, Wrocław, Poland) dual wavelength diffractometer with a micro-focus X-ray source and multilayer optics monochromatized Cu-K α ( λ = 1.54184 Å) radiation. Program CrysAlisPro [ 42 ] was used for the data collection and reduction. The intensities were corrected for absorption using analytical face index absorption correction method. The structures were solved with intrinsic phasing method (SHELXT [ 43 ]) and refined by full-matrix least squares on F 2 with SHELXL-2018/3 [ 44 ]. Anisotropic displacement parameters were assigned to non-H atoms. All C-H hydrogen atoms were refined using riding models. Hydroxy hydrogens were found from electron density maps and restrained to the proper distance from oxygen atom (0.84 Å). All hydrogen atoms were refined with U eq (H) of 1.5 × U eq (C,O) for hydroxy and terminal methyl groups or 1.2 × U eq (C) for other C-H groups. Further geometric least-squares restraints (s = 0.02) were applied to structures 4 , 6b, and 7 to obtain more chemically reasonable bond distances between disordered atoms. Anisotropic displacement parameters of few disordered or terminal atoms were restrained (s = 0.01, st = 0.02) to be more equal in structures 3 , 4 , 6b, and 7 . CCDC 1868318-1868324 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected]).