Antiprotozoal Sesquiterpene Lactones and Other Constituents from

Article Cite This: J. Nat. Prod. 2018, 81, 124−130

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Antiprotozoal Sesquiterpene Lactones and Other Constituents from Tarchonanthus camphoratus and Schkuhria pinnata Njogu M. Kimani,† Josphat C. Matasyoh,‡ Marcel Kaiser,§,# Reto Brun,§,# and Thomas J. Schmidt*,† †

Institute of Pharmaceutical Biology and Phytochemistry (IPBP), University of Muenster, PharmaCampus Corrensstrasse 48, Muenster D-48149, Germany ‡ Department of Chemistry, Egerton University, P.O. Box 536, Egerton 20115, Kenya § Swiss Tropical and Public Health Institute (Swiss TPH), Socinstrasse 57, Basel CH-4051, Switzerland # University of Basel, Petersplatz 1, Basel CH-4003, Switzerland S Supporting Information *

ABSTRACT: In continuation of a search for new antiprotozoal agents from plants of the family Asteraceae, Tarchonanthus camphoratus and Schkuhria pinnata have been investigated. By following the promising in vitro activity of the dichloromethane extracts from their aerial parts, bioassay-guided chromatographic isolation yielded two known sesquiterpene lactones (1 and 2) from T. camphoratus and 20 known compounds of this type from S. pinnata. From the latter, a new eudesmanolide, (1R*,5S*,6R*,7R*,8R*,10R*)-1-hydroxy-8-[5″-hydroxy-4′-(2″-hydroxyisovaleroyloxy)tigloyloxy]-3-oxoeudesma-11(13)-en-6,12-olide (3), and two new germacranolides, 3β(2″-hydroxyisovaleroyloxy)-8β-(3-furoyloxy)costunolide (14) and 1(10)-epoxy-3β-hydroxy-8β-[5′-hydroxy-4′-(2″hydroxyisovaleroyloxy)tigloyloxy]costunolide (16), were obtained. Additionally, the flavonoid pectolinarigenin (24) and 3hydroxy-4,5-dimethoxybenzenepropanol (25) were also isolated from S. pinnata. The compounds were characterized by analysis of 1D and 2D NMR spectroscopic and HR/MS data. In vitro antitrypanosomal activity and cytotoxicity against mammalian cells (L6 cell line) were evaluated for all the compounds. Santhemoidin A (13) and 3β-(2″-hydroxyisovaleroyloxy)-8β-(3furoyloxy)costunolide (14) were the most active compounds found in this study, with IC50 values of 0.10 and 0.13 μM against Trypanosoma brucei rhodesiense trypomastigotes and selectivity indices of 20.5 and 29.7, respectively.

I

Somalia, South Africa, Tanzania, Uganda, and Zimbabwe, occurring in the savanna biome and woody grasslands.11 The plant is used in folk medicine to treat bronchitis and chest ailments, tired legs and sore feet, stomach ailments, asthma, overanxiety, and heartburn.11,12 The essential oil and solvent extracts have been reported to show antimicrobial activity.13,14 An aqueous extract has been shown to have analgesic and antipyretic activity.15 The sesquiterpene lactones costunolide, isocostic acid, 3β-hydroxy-1,2-dehydrocostic acid, and parthenolide have been reported from this plant as well as several sesquiterpene acid derivatives.16 Schkuhria pinnata Lam. (Asteraceae) is an annual herb that grows as a weed to about 70 cm tall. It has numerous hairy erect stems with yellow flowers. It is native to South America but has been introduced into other countries.17 It is reported to have been used to treat many ailments such as diabetes, malaria, diseases of the ear, nose, and throat, colds and influenza, wounds, kidney, liver, and renal problems, allergies, yeast infections, prostate inflammation, problems of the digestive tract, and rheumatism.17−21 Previous studies have reported sesquiterpene lactones of the germacranolide type and flavonoids as its chemical constituents.17,22

n the course of a continuing search for secondary metabolites from plants of the family Asteraceae with activity against protozoan parasites, a variety of natural products have been reported with interesting activity against Plasmodium falciparum and against species of the trypanosomatid genera Leishmania and Trypanosoma.1−9 Several sesquiterpene lactones, characteristic constituents of this family of plants, have been shown to have interesting activity particularly against Trypanosoma brucei, the causative agent of human African trypanosomiasis.1−4,10 Moreover, antiprotozoal flavonoids, alkamides, and chromene derivatives have been reported as well.5−7 In the present study, the crude extracts of the Tarchonanthus camphoratus L. (Asteraceae, subfamily Carduoideae, tribe Tarchonantheae) and Schkuhria pinnata Lam. (Asteraceae, subfamily Asteroideae, tribe Heliantheae) leaves and aerial parts, respectively, were screened for antiprotozoal activity. The dichloromethane extracts displayed significant activity against T. brucei rhodesiense (Tbr) with IC50 values of 0.61 and 0.64 μg/mL for T. camphoratus and S. pinnata, respectively. Furthermore, T. camphoratus was found to display considerable activity against L. donovani (Ldon), the etiological agent of visceral leishmaniasis, with an IC50 value of 0.49 μg/ mL. Tarchonanthus camphoratus L. (Asteraceae) is an evergreen highly branched narrow-crowned shrub that grows up to 9 m high. It is native to Angola, Ethiopia, Kenya, Lesotho, Namibia, © 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 31, 2017 Published: December 15, 2017 124

DOI: 10.1021/acs.jnatprod.7b00747 J. Nat. Prod. 2018, 81, 124−130

Journal of Natural Products

Article

Table 1. NMR Spectroscopic Data of Compounds 3, 14, and 16 (600 MHz, CDCl3) 3 position

■

δC

1 2

76.5, CH 46.2, CH2

3 4 5 6 7 8 9

207.5, C 44.2, CH 50.5, CH 78.0, CH 52.7, CH 66.9, CH 40.8, CH2

10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″

41.8, C 133.4, C 169.0, C 120.2, CH2 13.9, CH3 13.7, CH3 165.4, C 134.0, C 137.5, CH 61.0, CH2 57.2, CH2 174.6, C 75.1, CH 32.2, CH 18.7, CH3 16.0, CH3

14

δH (mult., J in Hz) 3.70, dd (11.3, 5.8) α 2.74, dd (15.6, 5.8) β 2.57, dd (14.9, 3.7) 2.60, m 1.62, t (11.4) 4.49, t (10.9) 2.87, dd (11.0, 2.9) 5.83, dt (7.5, 2.4) α 2.52, dd (15.3, 2.5) β 1.63, m

6.18, 5.47, 1.26, 1.32,

d (3.2) d (3.0) s d (6.6)

6.72, t (6.4) 4.98, dd (6.5, 2.7) 4.41, s 4.08 d (3.7) 2.09, dqq (6.9, 7.0, 3.6) 1.03, d (7.0) 0.86, d (6.8)

δC

16

δH (mult., J in Hz)

127.7, CH 32.1, CH2

5.22, q (1.5) α 2.74, q (7.1) β 2.35, m 5.35, dd (4.1, 2.6)

80.7, CH 138.7, C 129.6, CH 78.2, CH 51.2, CH 81.1, CH 46.2, CH2

5.24, dq (9.4, 1.5) 5.83, d (11.1) 2.98, br s 5.30, br s α 2.83, d (12.4) β 2.47, d (14.2)

138.4, C 139.8, C 172.0, C 127.5 CH2

6.36, 5.78, 1.78, 1.86,

22.0, CH3 25.7, CH3 164.6, C 121.4, C 146.8, CH 150.7, CH 112.2, CH 176.0, C 78.1, CH 34.3, CH 21.8, CH3 18.4, CH3

d (2.3) dd (2.0, 1.0) s d (1.5)

7.41, t (1.8) 8.01, dd (1.6, 0.8) 6.67, dd (1.9, 0.8) 4.08, 2.15, 1.04, 0.88,

RESULTS AND DISCUSSION Crude extracts were prepared from aerial parts of T. camphoratus and S. pinnata with solvents of increasing polarity [n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), methanol (MeOH), and water (H2O)]. They were tested for in vitro activity against Trypanosoma brucei rhodesiense (Tbr), T. cruzi (Tcr), Leishmania donovani (Ldon), and Plasmodium falciparum (Pf) and for cytotoxicity against mammalian cells. For these tests, Tbr (STIB 900 strain) trypomastigotes, Tcr (Tulahuen C4 strain) amastigotes, Ldon (MHOM-ET-67/L82 strain) axenic amastigotes, Pf (NF54 strain) intraerythrocytic forms, and the L6 rat-skeletal myoblast cell line were used. The results are summarized in Table S1 (Supporting Information). The CH2Cl2 extracts of both plants displayed considerable potency against Tbr with IC50 values of 0.61 and 0.64 μg/mL, in the case of T. camphoratus and S. pinnata, respectively. The CH2Cl2 extract of T. camphoratus also showed activity against Ldon with an IC50 value of 0.83 μg/mL. Therefore, these extracts were chosen for bioassay-guided fractionation, isolation, and characterization of their potentially active constituents. The crude CH2Cl2 extracts of the two plant materials were fractionated by silica gel column chromatography (CC), and representative fractions were subjected to bioactivity testing against Tbr (STIB 900 strain) trypomastigotes (Table S2, Supporting Information). The antiprotozoal activity of T. camphoratus against Tbr was highest in fractions F5 and F6 with IC50 values of 0.26 and 1.10 μg/mL, respectively. These fractions also displayed activity, in turn, against Ldon with IC50 values 0.73 and 0.20 μg/mL for F5 and F6. The selectivity index for F6 against Ldon was rather

d (3.5) dqq (6.9, 7.0, 3.5) d (6.9) d (6.8)

δC

δH (mult., J in Hz)

67.5, CH 35.9, CH2

2.67, dd (11.4, 2.4) α 2.41, ddd (13.3, 5.8, 2.4) β 1.53 m 4.61, dd (10.7, 5.7)

77.2, CH 150.6, C 123.7, CH 76.3, CH 55.6, CH 70.9, CH 45.2, CH2 62.2, C 138.3, C 171.6, C 124.3, CH2 22.3, CH3 15.2, CH3 167.6, C 136.6, C 140.4, CH 63.6, CH2 59.9, CH2 177.4, C 77.7, CH 34.9, CH 21.3, CH3 18.7, CH3

5.38, dd (9.9, 1.7) 5.22, t (9.5) 2.91, dd (8.7, 3.6) 5.76, dt (6.1, 1.7) α 2.83, dd (15.4, 6.0) β 1.31, m

6.31, 5.59, 1.17, 1.91,

d (3.4) d (3.0) s d (1.4)

6.75, t (6.5) 4.98, dd (7.7, 6.5) 4.52, s 4.08, 2.08, 2.02, 0.86,

d (3.9) m d (6.9) d (6.9)

promising at a value of 24.1. From the active fractions F5 and F6 of T. camphoratus, parthenolide (1)23 and 3-oxo-1,2dehydrocostic acid (2),24 respectively, were isolated and identified unambiguously by comparison of their 1D and 2D NMR (1H, 13C, COSY, HSQC, HMBC) and high-resolution mass spectrometric (HR/MS) data (obtained by UHPLC/ +ESIQTOFMS/MS analysis) with the values reported in the literature. In the case of S. pinnata, fraction F4 displayed the highest activity against Tbr with an IC50 value of 0.45 μg/mL followed by F5 with an IC50 value of 0.67 μg/mL. The selectivity indices of fractions from this plant were relatively low. By extensive chromatographic refractionation and purification of fractions F3−F7, compounds 3−25 were isolated. The structures of all the compounds were determined by analysis of their NMR (1D and 2D) and HR/MS data. For the previously known compounds, eupatoriopicrin (4),25 3β-hydroxy-8β-[5′-hydroxy-4′-(2″-hydroxyisovaleroyloxy)tigloyloxy]costunolide (5),26 3β-hydroxy-8β-[4′-hydroxytigloyloxy]costunolide (6),27 3β-hydroxy-8β-[5′-hydroxy-4′-(2″-hydroxy-3″methylvaleroyloxy)tigloyloxy]costunolide (7),26 eucannabinolide (8),28 schkuhrin II (9),29 2′(3′)-Z-eucannabinolide (10),29 2″-dehydroeucannabinolidesemiacetal (11),30 hiyodorilactone B (12),30 santhemoidin A (13),31 3-desacetyl-3-isovaleroyleucannabinolide (15),29 1β,10α-epoxyeucannabinolide (17),32 schkuripinnatolide A (18),26 schkuripinnatolide C (19),26 6α,14-dihydroxy-11βH,4E,1(10)E-germacradiene-8,12-olide (20),26 6α-hydroxy-14-oxo-11βH,4E,1(10)E-germacradiene8,12-olide (21),26 3-oxo-4β,15-dihydroliqustrin-[4′,5′-dihydroxytigloyloxy] (22), 3 3 8β-[4′,5′-dihydroxytigloyloxy]125



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Chart 1

preeupatundin (23),33 pectolinarigenin (24),34 and 3-hydroxy4,5-dimethoxybenzenepropanol (25),35 all analytical data obtained were in full agreement with those reported in the literature. Compound 3 was obtained as a colorless gum and determined to have the molecular formula C25H34O10 by (+)-HRESIMS. The 1H and 13C NMR spectroscopic data suggested that 3 has a eudesmanolide skeleton similar to known analogues.36,37 The 1H, 13C, and HSQC NMR data for 3 supported the presence of four carbonyl groups (δC 207.5, 174.6, 169.0, 165.4), four olefinic carbons (an exomethylene, δC 120.2/δH 6.18; 5.47 and a methine δC 137.5/δH 6.72 and the two respective quaternary carbons, δC 133.4 and 134.0), four oxymethines, two oxymethylenes, one sp3 quaternary carbon, four methines, two methylenes, and four methyl groups (Table 1). The core structure of the eudesmanolide skeleton was determined by analysis of the COSY and HMBC data. The C-3 ketone (δC 207.5) was assigned based on its HMBC correlations with H-2α, H-2β, and H-4. The 1H NMR and

13

C NMR spectra of 3 were partly similar to those of (1R*,5S*,6R*,7R*,8R*,10R*)-1-hydroxy-8-(4,5dihydroxytiglyloxy)eudesma-4(15),11(13)-dien-6,12-olide, previously isolated from Disynaphia multicrenulata.25 However, in 3 the 13C NMR signal of C-3, resonating at δC 207.5 ppm, was characteristic of a carbonyl group. Additionally, a high-field 13C NMR signal at δC 13.7 ppm, assigned to a methyl group with a proton resonance at δH 1.32, replaced the C-15 exomethylene downfield signals of the compound mentioned above.25 The ester side chain at C-8 of 3 differed from the known compound by the presence of an extra 2-hydroxyisovalerate ester group, bound to the C-4′-position of the dihydroxytiglate moiety. The α-orientation of H-5 was supported by the NOESY correlation between H-5 and H-8. The NOESY correlations between H-6 and H-14, H-4 and H-14, and H-3 and H-15 suggested the αorientation of H-3, the β-orientation of H-4, the β-orientation of C-14, and the α-orientation of H-6. Accordingly, compound 3 was assigned as the new eudesmanolide (1R*,5S*,6R*,7R*,8R*,10R*)-1-hydroxy-8-[5′-hydroxy-4′-(2″126



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Table 2. In Vitro Antitrypanosomal and Cytotoxic Activity IC50 Values of Compounds Isolated from T. camphoratus (1, 2) and S. pinnata (3−25)a Tbr

cytotoxicity

compd

IC50, μM

IC50, μM

SI

compd

IC50, μM

Tbr

IC50, μM

SI

1 2 3 4 5 6 7 8 9 10 11 12 13

0.393 2.8 ± 0.1 2.7 ± 0.9 1.4 ± 0.4 2.6 ± 1.0 5.4 ± 1.3 4.0 ± 0.1 1.1 ± 0.1 0.82 ± 0.17 1.7 ± 0.04 0.35 ± 0.08 1.7 ± 0.04 0.1 ± 0.02

7.2 17.3 ± 0.5 39.7 ± 4.7 15.5 ± 0.1 34.1 ± 3.0 51.7 ± 0.4 42.3 ± 0.4 7.8 ± 0.8 10.96 ± 0. 6 54.6 ± 10.7 4.1 ± 0.2 7.94 ± 0.2 2.1 ± 0.4

18. 6 6.2 14.6 11.1 13.0 9.6 10.5 6.9 13.4 31.1 11.5 4.6 20.5

14 15 16 17 18 19 20 21 22 23 24 25 PCb

0.13 ± 0.03 0.52 ± 0.07 0.91 ± 0.15 0.92 ± 0.15 1.4 ± 0.2 0.9 ± 0.2 198.2 ± 3.0 2.34 ± 0.1 0.6 ± 0.1 7.3 ± 1.1 6.1 ± 0.8 9.8 ± 0.7 0.003 ± 0.001

3.9 ±0.5 6.8 ± 1.6 14.4 ± 2.0 14.6 ±1.7 7.0 ± 1.1 3.8 ± 0.01 >375.7 26.4 ± 1.7 12.3 ± 2.3 42.8 ± 3.3 20.2 ± 2.0 149.0 ± 17.3 0.007 ± 0.001

29.7 13.0 15.8 15.8 5.0 4.1

cytotoxicity

11.0 19.2 5.9 3.3 15.2 2.3

Data are means of two independent determinations ± absolute deviation. bPC, positive control: melarsoprol (Tbr) and podophyllotoxin (cytotox. L6). a

(2), this is the first report of both its antiprotozoal activity and its isolation from T. camphoratus. It displayed only moderate activity against Tbr (IC50 2.8 μM), but showed a very promising level of activity against Ldon, with an IC50 value of 0.18 μM. The selectivity index of this compound toward Ldon was 95.4 and that for Tbr was 6.2. In vivo efficacy studies against Ldon are hence warranted. Several compounds from S. pinnata displayed promising activities toward Tbr trypomastigotes. The heliangolides santhemoidin A (13) and the new compound 14 were the most active, with IC50 values of 0.10 and 0.13 μM and SI values of 20.5 and 29.7, respectively. Compound 11 was the third most active compound, with an IC50 value of 0.35 μM and an SI value of 11.5. The potent activities of these substances, in comparison with the rest of the structurally related compounds obtained, can be attributed to the presence of the furoate ester unit at C-8 in compounds 13 and 14 and the hemiacetal (2′hydroxy-2,5-dihydrofuran-4′-oate) unit at C-8 in compound 11. The closely related heliangolides 16 and 17 also displayed considerable inhibitory activities with IC50 values of 0.91 and 0.92 μM, respectively. The heliangolide derivative schkuhripinnatolide C (19) was also significantly active, with an IC50 value of 0.92 μM and an SI value of 4.1. The activity of this compound was higher than that of schkuhripinnatolide A (18) (1.4 μM) by a factor of 2. This increase in activity may hence be attributed to the extra α,β-unsaturated carbonyl group in the dihydroxytiglate moiety at C-8 in schkuhripinnatolide C (19). The heliangolides eucannabinolide (8) and schkuhrin II (9) were the most abundant in the S. pinnata CH2Cl2 extract and displayed somewhat higher activity against Tbr than the crude extract with IC50 values of 1.1 and 0.82 μM and SI values of 6.9 and 13.4, respectively. Heliangolide 10, an isomer of eucannabinolide (8), had an IC50 value of 1.7 μM but a high SI value of 31.1. This activity was similar to that of the closely related heliangolide hiyodorilactone B (12), which had an IC50 value of 1.7 μM but a lower SI value of 4.7. Compound 15, closely related to schkuhrin II (9), had a high activity, with an IC50 value of 0.52 μM and an SI value of 13.0. The guaianolide 22 was also active, with an IC50 value of 0.64 μM and a moderate SI value of 19.2. This activity was more potent in comparison to that of its closely structurally related congener 23, which had an IC50 value of 7.3 and a low SI at 5.9.

hydroxyisovaleroyloxy)tigloyloxy]-3-oxoeudesma-11(13)-en6,12-olide. The molecular formula of compound 14, obtained as a colorless gum, was determined as C25H30O8 by (+)-HRESIMS. The 1H and 13C NMR spectroscopic data displayed the characteristic signals of a heliangolide.30,37,38 The data were partly similar to those of 9 and 13 and differed from compound 9 by the presence of signals for a 3-furoate moiety at C-8 instead of a 4,5-dihydroxytiglate group. The presence of this ester moiety was clearly proven by the 1H and 13C NMR shift values in addition to HMBC correlations. In comparison with compound 13, a 2-hydroxyisovalerate moiety was found to replace the acetate side chain at C-3 as apparent from the corresponding NMR data. Thus, the new compound 14 was assigned unambiguously the structure of 3β-(2″-hydroxyisovaleroyloxy)-8β-(3-furoyloxy)costunolide. Compound 16 was isolated as a colorless gum, and its molecular formula was determined to be C25H34O10 by (+)-HRESIMS. The 1H and 13C NMR spectroscopic data suggested that 16 has a heliangolide skeleton similar to the known heliangolide 17. The NMR data of the two compounds were closely comparable, differing significantly only in the ester moiety at C-8 and the substituent at C-3. In 16, the dihydroxytiglate moiety at C-8 was modified by esterification at the C-4′-position with 2-hydroxyisovalerate, as proven unambiguously by COSY and HMBC correlations. Additionally, in 16 a free hydroxy group replaced the acetate moiety at C-3 in 17. The ester moiety at C-8 in 16 was found to be identical with that in the known compound 5, and its structure was confirmed through all NMR signals, in particular COSY and HMBC correlations. The configuration was deduced from the observed proton couplings and chemical shifts that were the same as those of 17 and similar epoxides reported in the literature.39 Consequently, the new compound 16 was determined as 1(10)-epoxy-3β-hydroxy-8β-[5′-hydroxy-4′-(2″hydroxyisovaleroyloxy)tigloyloxy]costunolide. All the isolated compounds were subjected to antitrypanosomal activity and cytotoxicity tests (Table 2). Parthenolide (1), which has been previously reported from T. camphoratus and other species in the Asteraceae,40,41 has been tested previously for antitrypanosomal activity with an IC50 of 0.39 μM against Tbr.3 However, for 3-oxo-1,2-dehydrocostic acid 127



Article

(0°21′44.8″ S 35°55′26.9″ E), respectively, and identified by S. T. Kariuki, a taxonomist at the Biological Sciences Department, Egerton University. Voucher specimens have been deposited at the Institute of Pharmaceutical Biology and Phytochemistry, University of Muenster, Germany (voucher numbers Kimani, 02 and Kimani, 03). The plant materials were air-dried in the shade at ambient temperature to constant weight and then ground. Extraction and Isolation. For the initial bioassays, 100 g of each of the dried plant materials was macerated sequentially with solvents (350 mL each) of increasing polarity (hexane, CH2Cl2, EtOAc, and MeOH) for 48 h at room temperature. The extracts were tested for in vitro activity against Tbr, Tcr, Ldon, and Pf (Table S1, Supporting Information). For preparative separations, T. camphoratus (500 g) and S. pinnata (800 g) were extracted with 3 and 5.5 L of CH2Cl2, respectively, in a Soxhlet apparatus for approximately 13 h. Upon evaporation, 45.8 and 40.3 g of T. camphoratus and S. pinnata extracts were obtained, respectively. The T. camphoratus (25 g) and S. pinnata (30 g) extracts were each separated by CC on a silica gel 60 column (8.0 × 85 cm) by gradient elution with hexane−EtOAc mixtures (10:0, 7:3, 1:1, 3:7, 10:0, v/v) to yield fractions F1−F8 in each case. Representative fractions were tested for antiprotozoal activity (Table S2, Supporting Information). The T. camphoratus fraction F5 (20 mg) was chromatographed over silica gel 60 and eluted with a hexane−EtOAc mixture (1:1, v/v), yielding subfractions F51−F53. Fraction F52 (8 mg) was then purified by preparative TLC, with a hexane−EtOAc mixture (1:1), to obtain compound 1 (4.2 mg; Rf 0.49). Fraction F6 (35 mg) of T. camphoratus was also separated by CC on silica gel 60 and eluted with a hexane− EtOAc mixture (3:2, v/v) to give two subfractions (F61 and F62). Subfraction F61 (14.1 mg) was purified by preparative TLC, with a hexane−EtOAc mixture (3:2, v/v) as the mobile phase, to obtain compound 2 (5.3 mg; Rf 0.21). The S. pinnata fraction F3 (89 mg) was separated by CC on silica gel 60 with hexane−EtOAc (4:1, v/v) as mobile phase to yield subfractions F31−F34. Compounds 24 (6.7 mg; Rf 0.43) and 18 (7.1 mg; Rf 0.23) were obtained by purification of subfractions F32 (10 mg) and F34 (12.3 mg), respectively, by preparative TLC with hexane− EtOAc mixtures [(4:1, v/v) and (1:1, v/v), respectively]. Fraction F5 (5 g) yielded eight subfractions (F51−F58) after CC on silica gel 60 with hexane−EtOAc (3:2, v/v). Subfraction F58 consisted of pure compound 8 (70 mg). Subfraction F55 (1.2 g) was separated by CC on silica gel 60 with hexane−EtOAc (3:2, v/v) to yield six subfractions (F551−F556). Compounds 20 (4 mg; Rf 0.36) and 21 (0.5 mg; Rf 0.52) were obtained from F552 (7 mg) by preparative TLC with hexane− EtOAc (7:3, v/v). Separation of F556 by preparative HPLC yielded compounds 4 (2.0 mg; tR 36.00 min), 5 (3.8 mg; tR 29.03 min), 9 (45.2 mg; tR 33.53 min), and 19 (2.3 mg; tR 35.22 min). Subfraction F553 was also separated by preparative HPLC to yield compounds 13 (3.3 mg; tR 23.53 min) and 14 (3.2 mg; tR 28.75 min). Compounds 7 (1.3 mg; tR 30.84 min) and 15 (1.1 mg; tR 40.63 min) were obtained by separation of F554 (10 mg) by preparative HPLC. Subfractions F71−F78 were obtained after CC of fraction F7 on silica gel 60 with hexane−EtOAc (3:2, v/v). Further separation of F78 on a silica gel 60 column yielded four subfractions, F781−F784. Separation of F783 by preparative HPLC yielded compounds 23 (4.0 mg; tR 18.81 min) and 6 (4.1 mg; tR 28.63 min). Similarly, compounds 16 (3.6 mg; tR 21.15 min), 3 (1.1 mg; tR 26.67 min), 10 (10.5 mg; tR 34.45 min), and a mixture of compounds 22 and 17 (17.4 mg; tR 17.68 min) were obtained by preparative HPLC of F76. The mixture of compounds 22 and 17 was separated on an analytical Hypercarb HPLC column to yield compounds 22 (1.2 mg; tR 8.32 min) and 17 (2.2 mg; tR 4.15 min). Compounds 11 (2.1 mg; tR 33.08 min), 12 (3.5 mg; tR 35.28 min), and 25 (4.2 mg; tR 36.58 min) were obtained by separation of fraction F4 by preparative HPLC. Details of all preparative HPLC elution systems are described in the Supporting Information. (1R*,5S*,6R*,7R*,8R*,10R*)-1-Hydroxy-8-[5′-hydroxy-4′-(2″hydroxyisovaleroyloxy)tigloyloxy]-3-oxoeudesma-11(13)-en-6,12olide (3): colorless gum; [α]18D −29.8 (c 0.1, MeOH); 1H and 13C NMR (CDCl3), see Table 1; (+)HRESIMS m/z 495.2266 [M + H]+ (calcd for C25H35O10, 495.2230); 512.2535 [M + NH4]+ (calcd for

The melampolide aldehyde 21 displayed an IC50 value of 2.4 μM and an SI value of 11.0. The activity of this compound was quite high in comparison with the corresponding alcohol 20, which was the least active, with an IC50 value of 198.2 μM and an SI value of >100. This could be attributed to the presence of a Michael acceptor unit, i.e., the α,β-unsaturated formyl group3,10 in 21. The germacrolide eupatoriopicrin (4), which had previously been reported by Julianti et al. as having an IC50 value of 1.2 μM,40 showed an IC50 value of 1.4 μM and an SI value of 11.1. Compound 5 displayed moderate activity, with an IC50 value of 2.6 μM and an SI value of 13.0. The anti-Tbr activity of this compound was slightly higher than that of compound 7, which had an IC50 value of 4.0 μM and an SI value of 10.5. The germacrolide 6 was the least active of the germacrolides, with an IC50 value of 5.41 μM and an SI value of 9.6. The new eudesmanolide 3 showed only moderate potency, with an IC50 value 2.7 μM and an SI value of 14.6. The flavonoid pectolinarigenin (24) and 3-hydroxy-4,5dimethoxybenzenepropanol (25) only displayed rather low antitrypanosomal activities, with IC50 values of 6.1 and 9.8 μM and SI values of 3.3 and 15.2, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter (Groß-Umstadt, Germany) in MeOH. All 1D and 2D NMR spectra were recorded on a 600 MHz Agilent DD2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) at 298 K in CDCl3. The solvent signals (1H, 7.260 ppm; 13C, 77.000 ppm) were used to reference the spectra. MestReNOVA v. 11 (Mestrelab Research, Chemistry Software Solutions, Santiago de Compostela, Spain) software was used to process and evaluate the spectra. Column chromatography was performed on silica gel 60, 0.063−0.2 mm (Macherey-Nagel), in all cases. For analytical TLC, 60 F254 silica gel plates (Merck Chemicals GmbH, Darmstadt, Germany) were used with various solvent systems consisting of EtOAc and hexane as the mobile phases. The plates were visualized under UV light at 254/360 nm and then sprayed with anisaldehyde/sulfuric acid reagent and heated on a hot plate. Preparative TLC was performed on 20 × 20 cm glass silica gel 60 TLC plates, coated with fluorescent indicator F254 (Merck Chemicals GmbH, 1.05715, 0.25 mm, Darmstadt, Germany) with hexane−EtOAc mixtures as the mobile phases. Visualization was performed under UV light at 254 nm. Preparative HPLC isolations were performed on a JASCO (GroßUmstadt, Germany) preparative HPLC system (pump, PU-2087 plus; diode array detector, MD 2018 Plus; column thermostat, CO 2060 Plus; autosampler, AS 2055 Plus; LC Net II ADC Chromatography Data Solutions; sample injection loop, 2000 μL) on a Reprosil 100 C18 preparative reversed-phase column (5 μm, 250 mm × 20 mm, Macherey-Nagel, Düren, Germany) with binary gradients of the mobile phase consisting of water and MeOH (for details, see the Supporting Information). For UHPLC/+ESIQTOFMS/MS analyses, separation was performed on a Dionex Ultimate 3000 RS liquid chromatography system (Idstein, Germany) with a Dionex Acclaim RSLC 120 C18 column (2.1 × 100 mm, 2.2 μm) using a binary gradient of water and acetonitrile, both with 0.1% formic acid (Supporting Information). Detection was performed with a Dionex Ultimate DAD-3000 RS (wavelength range of 200−400 nm) and a Bruker Daltonics micrOTOF-QII quadrupole/ time-of-flight mass spectrometer (Bremen, Germany) with an Apollo electrospray ion source operated in positive ionization mode. For specific details on instrument settings and operation see the Supporting Information. Plant Material. The plant materials of T. camphoratus and S. pinnata were collected in May 2015 in Ngata, Nakuru, Kenya (0°16′0″ S 35°58′60″ E), and Egerton University Botanic Garden, Njoro, Kenya 128



Article

C 25 H 38 NO 10 , 512.2496); 517.2784 [M + Na] + (calcd for C25H34O10Na, 512.2050); UHPLC/+ESIQTOFMS tR 6.19 min. 3β-(2″-Hydroxyisovaleroyloxy)-8β-(3-furoyloxy)costunolide (14): colorless gum; [α]18D −83.5 (c 0.2, MeOH); 1H and 13C NMR (CDCl3), see Table 1; (+)HRESIMS m/z 476.2293 [M + NH4]+ (calcd for C25H34NO8, 476.2275); 481.1840 [M + Na]+ (calcd for C25H30O8Na, 481.1941); UHPLC/+ESIQTOFMS tR 9.01 min. 1(10)-Epoxy-3β-hydroxy-8β-[5′-hydroxy-4′-(2″-hydroxyisovaleroyloxy)tigloyloxy]costunolide (16): colorless gum; [α]18D +5.6 (c 0.2, MeOH); 1H and 13C NMR (CDCl3), see Table 1; (+)HRESIMS m/z 495.2266 [M + H]+ (calcd for C25H35O10, 495.2230); 512.2535 [M + NH 4 ] + (calcd for C 25 H38 NO 10 , 512.2496); UHPLC/+ESIQTOFMS tR 5.80 min. In Vitro Bioassays. In vitro assays for the bioactivity of crude extracts and all isolated compounds against Tbr (bloodstream trypomastigotes, STIB 900 strain), Tcr (amastigotes, Tulahuen C4 strain), Ldon (axenic amastigotes, MHOM-ET-67/L82 strain), and Pf (intraerythrocytic forms, NF54 strain) and cytotoxicity tests against mammalian cells (L6-cell line from rat-skeletal myoblasts) were performed at the Swiss Tropical and Public Health Institute (Swiss TPH, Basel, Switzerland), according to established protocols and as previously described.3

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(3) Schmidt, T. J.; Nour, A. M. M.; Khalid, S. A.; Kaiser, M.; Brun, R. Molecules 2009, 14, 2062−2076. (4) Nogueira, M.; Da Costa, F.; Brun, R.; Kaiser, M.; Schmidt, T. Molecules 2016, 21, 1237. (5) Althaus, J. B.; Kaiser, M.; Brun, R.; Schmidt, T. J. Molecules 2014, 19, 6428−6438. (6) Nour, A. M. M.; Khalid, S. A.; Kaiser, M.; Brun, R.; Abdalla, W. E.; Schmidt, T. J. J. Ethnopharmacol. 2010, 129, 127−130. (7) Harel, D.; Khalid, S. A.; Kaiser, M.; Brun, R.; Wünsch, B.; Schmidt, T. J. J. Ethnopharmacol. 2011, 137, 620−625. (8) Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M.; Biavatti, M. W.; Brun, R.; Da Costa, F. B.; de Castro, S. L.; Ferreira, V. F.; de Lacerda, M. V. Curr. Med. Chem. 2012, 19, 2176−2228. (9) Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M.; Biavatti, M. W.; Brun, R.; Da Costa, F. B.; de Castro, S. L.; Ferreira, V. F.; de Lacerda, M. V. Curr. Med. Chem. 2012, 19, 2128−2175. (10) Schmidt, T. J.; Da Costa, F. B.; Lopes, N. P.; Kaiser, M.; Brun, R. Antimicrob. Agents Chemother. 2014, 58, 325−332. (11) Tarchonanthus camphoratus (Camphor Bush) http://www.kew. org/science-conservation/plants-fungi/tarchonanthus-camphoratuscamphor-bush (accessed Jan 11, 2017). (12) Kiwanuka, N. S. Chemical Composition and Biological Potential of the Volatile and Non-volatile Constituents of Tarchonanthus camphoratus and Tarchonanthus trilobus var. galpinni of Kwazulu − Natal Province. Ph.D. Thesis, University of Zululand, Kwa-Dlangezwa, South Africa, 2009; p 27. (13) Matasyoh, J. C.; Kiplimo, J. J.; Karubiu, N. M.; Hailstorks, T. P. Food Chem. 2007, 101, 1183−1187. (14) Wetungu, M. W.; Matasyoh, J. C.; Kinyanjui, T.; Kinyanjui, J. C. J. Pharmacogn. Phytochem. 2014, 3, 123−127. (15) Amabeoku, G. J.; Green, I.; Eagles, P.; Benjeddou, M. Phytomedicine 2000, 7, 517−522. (16) Hegazy, M.-E. F.; Tawfik, W. A.; Hassan, E. M.; Mohamed, T. A.; Albar, H. A.; Debbab, A. Res. J. Pharm. Biol. Chem. Sci. 2015, 6, 659−663. (17) Tropical Plant Database File for: Canchalagua - Schkuhria pinnata http://www.rain-tree.com/canchalagua.htm#.WHZtLlPhDcs (accessed Jan 11, 2017). (18) Kareru, P. G.; Kenji, G. M.; Gachanja, A. N.; Keriko, J. M.; Mungai, G. Afr. J. Tradit., Complementary Altern. Med. 2007, 4, 75−86. (19) Muthaura, C. N.; Rukunga, G. M.; Chhabra, S. C.; Mungai, G. M.; Njagi, E. N. M. S. Afr. J. Bot. 2007, 73, 402−411. (20) Njoroge, G. N.; Bussmann, R. W. J. Ethnobiol. Ethnomed. 2006, 2, 54. (21) Njoroge, G. N.; Bussmann, R. W.; Gemmill, B.; Newton, E. L.; Ngumi, V. W. Lyonia 2004, 7, 71−87. (22) León, A.; Reyes, B. M.; Chávez, M. I.; Toscano, R. A.; Delgado, G. J. Mex. Chem. Soc. 2009, 53, 193−200. (23) Banthorpe, D. V.; Brown, G. D.; Janes, J. F.; Marr, I. M. Flavour Fragrance J. 1990, 5, 183−185. (24) Tsichritzis, F.; Jakupovic, J.; Bohlman, F. Phytochemistry 1990, 29, 195−203. (25) De Gutierrez, A. N.; Bardon, A.; Catalan, C. A. N.; Gedris, T. B.; Herz, W. Biochem. Syst. Ecol. 2001, 29, 633−647. (26) Ganzer, U.; Jakupovic, J. Phytochemistry 1990, 29, 535−539. (27) Jakupovic, J.; Sun, H.; Bohlman, F.; King, R. M. Planta Med. 1986, 53, 97−98. (28) Herz, W.; Govindan, S. V. Phytochemistry 1980, 19, 1234−1236. (29) Pacciaroni, A. D. V; Sosa, V. E.; Espinar, L. A.; Oberti, J. C. Phytochemistry 1995, 39, 127−131. (30) Bohlmann, F.; Schmeda-Hirschmann, G.; Jakupovic, J. Phytochemistry 1984, 23, 1435−1437. (31) Pérez, A. L.; Mendoza, J. S.; Romo de Vivar, A. Phytochemistry 1984, 23, 2911−2913. (32) Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H. Liebigs Annal. Chem. 1984, 1984, 250−258. (33) Boeker, R.; Jakupovic, J.; Bohlmann, F.; King, R. M.; Robinson, H. Phytochemistry 1986, 25, 1669−1672.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00747. Preparative HPLC elution systems, details on UHPLC/ +ESIQTOFMS/MS analyses, mass and NMR spectra of compounds 3, 14, and 16, as well as bioactivity data of crude extracts and fractions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-251-83-33378. Fax: +49-251-83-38341. E-mail: [email protected]. ORCID

Thomas J. Schmidt: 0000-0003-2634-9705 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Kenyan government, through the National Research Foundation, in cooperation with the German Academic Exchange Service (NRF-DAAD) for a doctoral fellowship to N.M.K. at the University of Muenster, Germany. The authors are grateful to S. T. Kariuki of Egerton University (Kenya) for the identification of the plant species. Thanks are due to J. Sendker and S. Brockmann, Institute of Pharmaceutical Biology and Phytochemistry, Muenster, for recording the LC-MS data and to J. Köhler and C. Thier, Institute of Pharmaceutical and Medicinal Chemistry, Muenster, for recording NMR spectra. This study formed part of a collaborative work within the Research Network Natural Products against Neglected Diseases (ResNetNPND, http:// www.resnetnpnd.org/).

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REFERENCES

(1) Kimani, N. M.; Matasyoh, J. C.; Kaiser, M.; Brun, R.; Schmidt, T. J. Molecules 2017, 22, 597. (2) Schmidt, T. J.; Brun, R.; Willuhn, G.; Khalid, S. A. Planta Med. 2002, 68, 750−751. 129



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