Antiprotozoal Activity-Based Profiling of a ... - ACS Publications

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Antiprotozoal Activity-Based Profiling of a Dichloromethane Extract from Anthemis nobilis Flowers Maria De Mieri,† Giannicola Monteleone,† Isidor Ismajili,† Marcel Kaiser,‡,§ and Matthias Hamburger*,† †

Department of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, 4000 Basel, Switzerland § University of Basel, Petersplatz 1, 4001 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: A dichlomethane extract of Anthemis nobilis flower cones showed promising in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense and Leishmania donovani, with IC50 values of 1.43 ± 0.50 and 1.40 ± 0.07 μg/mL, respectively. A comprehensive profiling of the most active fractions afforded 19 sesquiterpene lactones, including 15 germacranolides, two seco-sesquiterpenes, one guaianolide sesquiterpene lactone, and one cadinane acid. Of these, 13 compounds were found to be new natural products. The compounds were characterized by extensive spectroscopic data analysis (1D and 2D NMR, HRMS, circular dichroism) and computational methods, and their in vitro antiprotozoal activity was evaluated. The furanoheliangolide derivative 15 showed high potency and selectivity in vitro against T. b. rhodesiense bloodstream forms (IC50 0.08 ± 0.01 μM; SI 63). In silico calculations were consistent with the drug-like properties of 15.

T

In the present work, an activity-based profiling was performed on the dichloromethane extract of Anthemis nobilis (Roman chamomile), on the basis of the promising in vitro protoicidal activity of the extract (IC50 1.43 ± 0.50 and 1.40 ± 0.07 μg/mL against T. b. rhodesiense and Leishmania donovani, respectively) and of the chemotaxonomy of the plant (Asteraceae). Anthemis nobilis is an indigenous plant of Western Europe but also grows in all of Europe, North Africa, and Southwest Asia. The plant, in particular its dried flower cones, has been used in traditional medicine for antiphlogistic, antibacterial, and antidiabetic properties.16 Detailed phytochemical studies on the plant have mostly been performed on its essential oil17 and methanolic extracts,18 revealing as main components α-bisabolol, α-pyrene, angelic and tiglic acid esters, phenolic compounds, and flavonoid glycosides, having apigenin, luteolin, and quercetin as major aglycones. Largescale isolation of the ethanolic extract has allowed the isolation of nobilin (1),19 the major sesquiterpene lactone, and of a few derivatives of nobilin with a similar germacranolide skeleton.20 We herein report on the identification of new sesquiterpene lactones from the plant and on their in vitro antiprotozoal activity.

ropical parasitic diseases such as malaria, leishmaniasis, and trypanosomiasis are a diverse group of infectious diseases that affect more than one billion people worldwide in tropical and subtropical regions. Malaria, caused by five different Plasmodium species, is the most virulent, with more than 214 million new reported cases and about 438 000 deaths in 2015. Although artemisinin derivatives are relatively safe and effective antimalarial remedies, artemisinin-resistant Plasmodium falciparum strains have already emerged in Asia.1 On the other hand, African sleeping sickness2 and Chagas’ disease,3,4 caused by Trypanosoma species, and cutaneous and visceral leishmaniasis5 (“Kala-Azar”) are currently classified as “neglected diseases”. Only a few drugs exist for their treatment, and they are all associated with severe side effects and increasing resistance, thus illustrating the urgent need for new drugs or lead structures.6,7 Natural products have in many instances been instrumental in providing interesting leads for such diseases.8−10 Among many other examples, the class of sesquiterpene lactones has attracted a great deal of attention in the past few years.11,12 Helenalin13 and cynaropicrin14 have so far been found as the most active and selective against Trypanosoma brucei rhodesiense, the most virulent causative agent of sleeping sickness. The antitrypanosomal activities of many other sesquiterpene lactones have been attributed to the Michael addition of the α-methylene-γ-lactone to biological thiols,15 even though other structural features such as the stereochemical rearrangement of the compounds seem to have a pivotal role in determining the bioactivity and are still worthy of further investigations. © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The antiplasmodial activity of the dichloromethane extract of A. nobilis flower cones was investigated by fractionating the extract into three major fractions, A−C, according to normalReceived: October 25, 2016 Published: January 24, 2017 459

DOI: 10.1021/acs.jnatprod.6b00980 J. Nat. Prod. 2017, 80, 459−470

Journal of Natural Products

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configuration of 4 was established by electronic circular dichroism (ECD). The ECD spectrum of 4 was superimposable to the calculated values (Figure 2), thus confirming compound 4 as 8α-angeloxy-3β-hydroxy-1-oxo-germacra-Z4(5),10(14),11(13)-trien-6α,12-olide. This new natural product was named nobilinon A. Compound 5 had the same molecular formula as 4, as deduced from HRMS data of the sodium adduct ion at m/z 383.1474 [M + Na]+ (calcd for C20H24NaO6 383.1465). The NMR data of 5 were almost superimposable to those of 4. The main differences in the 1H NMR spectra were the downfield NMR shift of H-18 (δH = 6.13 in 4 vs δH = 6.90 in 5) and the upfield shifts of H3-19 and H3-20 (δH = 1.93 and 1.88 in 4 vs δH = 1.82 and 1.82 in 5). The data indicated a geometrical rearrangement where the olefinic proton was periplanar to the carboxylic function, indicative of a tigloyl group. The ECD spectrum of 5 was comparable to that of 4 (Figure 2). Thus, compound 5 (nobilinon B) was identified as 3β-hydroxy-1-oxo8α-tigloxy-germacra-Z4(5),10(14),11(13)-trien-6α,12-olide. Compound 6 also had a molecular formula of C20H24O6 (HRESIMS m/z 383.1476 [M + Na]+). Analysis of the NMR data confirmed the same planar structure as in 4, but indicated significant differences in NMR shifts and J couplings of H-3 (δH = 4.52, dd, J = 4.2 and 4.2 Hz in 4 vs δH = 5.14, dd, J = 11.0 and 4.5 Hz in 6) due to its β-axial orientation. The α-orientation of the C-3 hydroxy function strongly affected the 1H shift of H-6 (Table 1), as observed in the case of epi-nobilin (2) and nobilin (1).20 The ECD spectrum of 6 was similar to that of 4 (Figure 2). Hence, compound 6 (nobilinon C) was established as 8αangeloxy-3α-hydroxy-1-oxo-germacra-Z4(5),10(14),11(13)trien-6α,12-olide. A molecular formula of C20H24O6 (HRESIMS m/z 383.1473 [M + Na]+) was assigned to compound 7, which, similar to 1, showed a germacradienolide skeleton bearing an angelate moiety at C-8. Diagnostic differences were the presence of an aldehyde proton (δH = 9.90, br s) attached to a carbonyl carbon resonating at δC 191.2 and the absence of one methyl group attached to the cyclodecadiene ring. Key HMBC correlations from the aldehyde proton to C-9 and C-10 and from H-1 to the aldehyde carbon (Figure 1) located this carbonyl group at C14. The absolute configuration of 7 was established by comparison of calculated and experimental ECD spectra (Figure 3). Thus, compound 7 was identified as 8α-angeloxy3β-hydroxy-14-oxo-germacra-Z4(5),Z1(10),11(13)-trien6α,12-olide and named formylnobilin. The 8-epi-tiglate derivative of compound 7 has been reported from Eupatorium chinense and named eupatochinilide I.23 Compound 8 gave a molecular formula of C20H26O6 (HRESIMS m/z 385.1641 [M + Na]+, calcd for C20H26NaO6 385.1622), which differed by 18 amu from that of 1. The NMR data of 8 were close to those of nobilin (1), but the compound was found to lack a C-9 methylene group. The presence of an additional oxygenated methine (δH 4.95, br d, J = 11.0 Hz) indicated that the compound is a hydroxylated analogue of 1. COSY correlations of H-1/H2-2 and H-9/H-8, as well as HMBC correlations (Figure 1), established the fragment C-1− C-9 as depicted. The β-orientation of H-1 was inferred through its dipolar couplings with H-8 and H-6. Compound 8 was designated as 8-epi-desacetyleupacunin, since its epimer at C-8 has been isolated from Eupatorium lancifolium.24 The ECD spectra of 8 showed similar Cotton effects to those reported for desacetyleupacunin (Figure S63, Supporting Information).

phase TLC analysis. On the basis of the in vitro activity data, fractions B and C were selected for further phytochemical profiling. Further separation of fractions B and C by normaland reversed-phase chromatography afforded compounds 1−4, 13, 14, 16−18, and 5−12, 15, and 19, respectively. UV spectra with an absorption maximum at about 220 nm and a molecular weight in the range of m/z 346−378 were indicative of sesquiterpene lactones. Finally, compounds were identified by 1D and 2D NMR spectroscopic data and HRMS analysis. Several known sesquiterpene lactones were identified as nobilin (1),19 3-epi-nobilin (2),20 8-methacrylate nobilin (3),11 hydroxyisonobilin (9),21 1,10-epoxynobilin (13),20 and cadinane acid 19.11 Compound 4 exhibited a molecular formula of C20H24O6, as determined by its 13C NMR and HRESIMS data (molecular ion at m/z 383.1481 [M + Na]+; calcd for C20H24NaO6 383.1465). Analysis of 1H and 13C NMR spectra (Table 1) indicated a germacranolide scaffold similar to that of nobilin (1), but with the E-1(10) unsaturation replaced by an α-methylene carbonyl group (C-1, δC = 202.4; C-10, δC = 146.0; CH2-14, δH = 6.39 and 6.28, δC = 134.0), as confirmed by key HMBC correlations (Figure 1). The relative configurations at the asymmetric carbons C-3, C-6, C-7, and C-8 were as in 1 and inferred as follows. The magnitude of the 3J(H-7,H-8) = 9.7 Hz was indicative of H-7 and H-8 being in an axial position on opposite sides of the cyclodecadiene ring. Consequently, strong dipolar couplings between H-6/H-8 and H-7/H-5 supported their reciprocal cofacial orientation and confirmed the trans junction of the α-methylene-γ-lactone ring, in agreement with a 3J(H6,H-7) of 2.1 Hz. The α-equatorial orientation of H-3 (δH = 4.53, dd, J = 4.5 and 4.0 Hz) confirmed the carbocyclic ring in a UD conformation, as in nobilin (1). 22 The absolute 460


a

461

(dd, 1.9, 0.8) (br d, 1.9) (br s) (br s) (d, 1.4)

(dq, 10.7, 1.4) (dd, 10.7, 2.1) (dddd, 9.7, 2.1, 1.9, 1.9) (ddd, 10.8, 9.7, 4.5,) (dd, 12.6, 10.8) (br dd, 12.6, 4.5)

6.13 (qq, 7.0, 1.5) 1.93 (dq 7.0, 1.5) 1.88 (quint, 1.5)

5.67 6.16 6.39 6.28 1.75

5.13 6.04 2.91 4.91 3.04 2.70

2.83 (dd, 12.7, 4.0) 3.49 (dd, 12.7, 4.5) 4.53 (dd, 4.5, 4.0)

C C C CH2

CH C CH CH CH CH CH2

23.4, 168.1, 128.7, 140.2, 16.0, 20.5,

CH3 C C CH CH3 CH3

134.0, CH2

146.0, 137.0, 171.9, 127.0,

73.7, 142.8, 126.6, 77.6, 50.9, 74.0, 36.6,

202.4, C 44.2, CH2

(m) (d, 1.8) (br s) (br s) (d, 1.5)

(dq, 10.8, 1.5) (dd, 10.8, 2.3) (dddd, 9.8, 2.3, 1.8, 1.8) (ddd, 10.7, 9.8, 4.5) (dd, 12.3, 10.7) (dd, 12.3, 4.5)

6.90 (m) 1.82a 1.82a

5.70 6.14 6.39 6.27 1.75

5.13 6.04 2.93 4.88 3.02 2.66

2.80 (dd, 12.7, 4.2) 3.49 (dd, 12.7, 4.2) 4.52 (dd, 4.2, 4.2)

δH (mult. J in Hz)

δC

δH (mult. J in Hz)

C C C CH2

CH2 CH C CH CH CH CH CH2

22.5, 168.0, 129.0, 138.3, 11.0, 13.6,

CH3 C C CH CH3 CH3

133.0, CH2

145.2, 136.2, 171.5, 126.0,

43.6, 73.2, 142.5, 125.7, 76.5, 50.1, 74.0, 35.8,

202.1, C

δC b

(d, 1.7) (d, 2.0) (m) (br s) (d, 1.5)

6.15 1.94 (dq 7.0, 1.5) 1.87 (quint, 1.5)

5.70 6.19 6.50 6.36 1.77

5.13a 5.08 (dd, 10.5, 2.0) 2.85 (dddd, 10.0, 2.0, 2.0, 1.8) 5.00 (ddd, 11.0, 10.0, 4.3) 2.60a 3.06 (dd, 12.0, 11.0) C C C CH2


15.7, 166.3, 127.5, 138.6, 15.0, 19.0,

CH3 C C CH CH3 CH3

130.3, CH2

143.0, 135.0, 169.7, 125.8,

45.5, 66.8, 141.7, 123.0, 75.0, 50.0, 73.0, 34.3,

199.5, C

(br d, 10.4) (br d, 10.4) (br d, 8.8) (ddd, 11.0, 8.8, 4.0) (m) (m)

(dd, 9.5, 8.6) (m) (m) (br m)

6.10 (br q 7.2) 1.88 (br d, 7.2) 1.83 (br m)

1.73 (br s)

5.59 (br s) 6.07 (br s) 9.90 (br s)

5.06 5.66 3.00 4.89 2.34 2.89

6.73 2.61 3.06 4.39

δH (mult. J in Hz)

δH (mult. J in Hz)

δC

7 b

6

3.60 (dd, 11.6, 4.5) 2.60a 5.14 (dd, 11.0, 4.5)

Overlapping signals. bExtracted from 1H−13C 2D inverse-detected experiments due to a low amount of sample.

15 16 17 18 19 20

14

10 11 12 13

3 4 5 6 7 8 9

1 2

position

5

4

C C C CH2


22.1, 165.7, 126.7, 137.5, 14.4, 19.4,

CH3 C C CH CH3 CH3

191.2, CH

137.2, 134.8, 168.7, 125.3,

30.0, 72.3, 139.8, 124.4, 75.9, 48.7, 69.4, 36.4,

147.2, CH

δ Cb

Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 4−7 (CD3OD for 4−6, DMSO-d6 for 7; 500.13 MHz for 1H, and 125.77 MHz for 13C NMR; δ in ppm)

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Figure 1. Key HMBC and NOESY correlations for selected compounds.

Figure 2. Experimental ECD spectra in MeOH of compounds 4−6 and calculated ECD spectrum of compound 4.

Figure 3. Experimental and calculated ECD spectra in MeOH of compound 7.

Compounds 9−12 all are based on a germacranolide skeleton that showed as characteristic features an exomethylene group located at C-10(14) and a hydroxy group at C-1, reminiscent of hydroxyisonobilin (9). This compound had been previously reported from A. nobilis, but without reporting the relative configuration at C-1.21 The main structural differences among compounds 9−12 were in the configuration at the diol sites and the side chain at C-8. Compound 10 showed a molecular formula of C20H26O6 (HRESIMS m/z 385.1635 [M + Na]+; calcd for C20H26NaO6 385.1622). Chemical shifts and J couplings of the fragment C3−C-8 were similar to those reported for nobilin (1) and, thus, suggested the same relative configuration. NMR shifts and J coupling values of H-1 (δH 4.03, dd, J = 11.2 and 3.3 Hz) were similar to those reported for hydroxyisonobilin,21 and the key NOESY correlation of H-1 with H-2α/H-7/H-9α proved its α-

orientation, thus completing the relative configuration of 9 and 10 as represented. As in compound 5, the resonances of the ester side chain indicated a tigloyl group (Table 2). Compound 10 was thus identified as 8-tigloylhydroxyisonobilin. Compound 11 showed a molecular formula of C19H24O6 (HRESIMS m/z 371.1469 [M + Na]+; calcd for C19H24NaO6 m/z 371.1465) that differed by 14 amu from that of 9, due to the replacement of the tiglate ester moiety with an acrylate (Table 2). Compound 11 was thus 8-acryloylhydroxyisonobilin. Compound 12, with a molecular formula of C20H26O6 according to the HRESIMS m/z 385.1630 [M + Na]+, was found to be a diastereoisomer of 9. As in 6, the β-axial orientation of H-3 was inferred through its J couplings (Table 2) and was also supported by the key NOESY correlations of H-1/H-7 and H-3/H-6. 462


a

463

1.99 (dq, 7.0, 1.5) 1.92 (quint, 1.5)

19 20

C C C CH2

CH3 C C CH

16.2, CH3 20.7, CH3

23.6, 168.1, 128.4, 141.0,

17.4, CH3

142.6, 138.3, 171.8, 123.7,

CH2 CH C CH CH CH CH CH

(d, 1.4) (d, 1.4) (br s) (br s) (d, 1.2)

1.82 (dq, 7.0, 1.5) 1.83 (quint, 1.5)

6.90 (qq, 7.0, 1.5)

5.72 6.17 5.54 5.37 1.78

5.27 (dq, 9.2, 1.2) 6.17a 3.20 (dq, 10.5, 1.4) 4.98 (ddd, 10.5, 9.0, 3.7) 2.46 (br dd, 14.2, 9.0) 2.85 (br dd, 14.2, 3.7)

(dd, 11.2, 3.3) (ddd, 15.2, 5.5, 3.3) (ddd, 15.2, 11.2, 2.6) (dd, 5.5, 2.6)

δC

C C C CH2

CH3 C C CH

14.5, CH3 12.1, CH3

23.3, 168.2, 129.1, 139.5,

118.6, CH2

145.6, 137.2, 171.8, 127.0,

39.0, CH2 72.84, CH 144.5, C 125.7, CH 76.9, CH 50.2, CH 75.2, CH 39.6, CH

72.91, CH

δH (mult. J in Hz)

11

(dq 9.3, 1.5) (dd, 9.3, 2.0) (dddd, 10.8, 2.0, 2.0, 1.5) (ddd, 10.8, 9.2, 3.8) (br dd, 14.0, 3.8) (dd, 14.0, 9.0)

(dd, 11.2, 3.2) (ddd, 15.0, 11.2, 2.6) (ddd, 15.0, 5.7, 3.4) (dd, 5.7, 2.6)

6.13 (quint, 1.0) 5.67 (quint,1.5) 1.93 (m)

5.75b (d, 1.5) 6.20a (d, 2.0) 5.38 (br s) 5.55 (br s) 1.79 (d, 1.5)

5.28 6.19 3.23 4.98 2.87 2.49

4.04 2.32 2.04 4.46

Overlapping signals. bExtracted from 1H−13C 2D inverse-detected experiments due to the low amount of sample.

6.24 (qq, 7.0, 1.5)

1.72 (d, 1.5)

5.75 (d, 2.6) 6.16 (d, 3.0) 1.71 (br s)

5.21 (dd 10.4, 1.5) 6.24a 2.89 (dddd, 10.0, 9.0, 3.0, 2.6) 5.91 (dd, 11.0, 9.0) 4.99 (br d, 11.0)

36.0, 73.5, 144.1, 125.5, 78.0, 47.5, 72.3, 127.3,

4.03 2.02 2.32 4.44

δH (mult. J in Hz)

δC 64.9, CH

δH (mult. J in Hz)

4.95 (br d 11.0) 1.94a 2.17 (ddd, 14.0, 11.0, 2.0) 4.42 (dd, 5.0, 2.0)

15 16 17 18

14

10 11 12 13

3 4 5 6 7 8 9

1 2

position

10

8

CH3 C C CH2

C C C CH2


18.4, CH3

118.7, CH2 23.3, 167.5, 137.2, 127.0,

145.6, 137.6, 172.0, 126.8,

39.0, 72.9, 144.6, 125.6, 76.9, 50.2, 75.4, 40.4,

72.9, CH

δC

12 (dd, 11.5, 3.6) (ddd, 13.0, 11.5, 3.6) (ddd, 13.0, 11.5, 3.6) (dd, 11.5, 3.5)

(d, 1.7) (d, 1.7) (br s) (br s) (br s)

1.96 (dq, 7.0, 1.5) 1.88 (quint,1.5)

6.15 (qq, 7.0, 1.5)

6.22 5.73 5.56 5.51 1.83

5.28a 5.29a 3.12 (dq 10.0, 1.7) 5.10 (ddd, 11.0, 10.0, 3.0) 2.88 (br dd, 13.0, 3.0) 2.38 (br dd, 13.0, 11.0)

3.66 2.24 2.00 4.78

δH (mult. J in Hz)

Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 8 and 10−12 (CD3OD; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) δC

C C C CH2


CH3 C C CH 16.1, CH3 20.6, CH3

17.3, 167.9, 128.6, 140.2,

117.1, CH2

145.5, 136.7, 171.3, 127.0,

40.1, 67.3, 144.2, 124.7, 76.0, 51.5, 75.4, 42.0,

71.8, CH

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revealed the typical spin system of the 3-hydroxy germacranolide nobilin (1) at positions C-2−C-9, as well as an angelate ester side chain. However, the unsaturation at C-1−C-10 was lacking. Distinct features of the NMR spectra of 15 were the presence of an oxygenated methine (δH 3.90 br d, J = 4.2 Hz) and an oxygenated methylene group (δH 3.68, and 3.55 d, J = 11.0 Hz), as well as an oxygenated quaternary carbon (δC 87.4). COSY correlations of H2-2 with the oxygenated methine were used to locate the latter at C-1, while HMBC correlations of H1, H2-2, H-8, H2-9, and H2-14 (Figure 1) helped to establish C10 as a quaternary carbon bearing a methylene group and an oxygenated function. According to the 13C NMR spectrum, eight oxygen-bearing carbons were detected, but only seven oxygen atoms accounted for the molecular formula. Since eight degrees of hydrogen deficiency were needed, an epoxy bridge had to be present in the molecule and attached with one bridgehead at C-10 (δC 87.4, C). The 13C NMR shift of C-1 and C-14 (δC 74.2 and 61.1 ppm, respectively) ruled out the attachment of an oxirane group at these positions. On the other hand, the downfield shift of C-3 (δC 80.3) with respect to that of other germacranolides suggested C-3 as a bridgehead for the epoxy group. The configuration at C-1 and C-3 was established through J couplings (Table 3) and corroborated through key NOEs of H-3/H-6 and H-1/H2-14 (Figure 1). Finally, NOESY correlations of H2-14/H-8 and H2-14/H-9β confirmed the βorientation of the methylene group at C-10 and the αorientation of the furano-epoxy group. The ECD spectrum of compound 15 showed a negative Cotton effect at 220 nm, similar to nobilin (1) (Figure S63, Supporting Information). Thus, the same stereochemistry was assumed at the lactone ring of 15, and the compound was named furanonobilin. A molecular formula of C20H24O5 was assigned to compound 16 on the basis of a sodiated molecular ion peak (m/z 367.1534 [M + Na]+, calcd for C20H24NaO5 367.1516) in the HRESIMS and 13C NMR data. Close inspection of the 1D and 2D NMR spectra revealed the presence of a C-5−C-7 bicyclic ring indicative of a guaianolide scaffold. Analysis of the 1H−1HCOSY spectrum revealed a nine-proton spin system (C-3 to C9) that included two vicinal sp2 protons, five methines, two of which were oxygenated, and a methylene group. The connection of C-3 and C-5 through an oxygenated quaternary carbon (C-4, δC 84.6 ppm) was inferred through key HMBC correlations of H3-15/C-3 and H3-15/C-5. Likewise, the attachment of a terminal double bound at C-10 was supported by HMBC correlations of H2-14/C-1 and H2-14/C-9. The relative configuration was established via analysis of J couplings and from the NOESY spectrum. The 3J coupling values of 3 J(H-5,H-6) = 11.3 Hz, 3J(H-6,H-7) = 8.5 Hz, and 3J(H-7,H-8) = 11.1 Hz suggested an antidiaxial orientation of H-5, H-6, H-7, and H-8 and a chair-like conformation of the cycloheptane ring. On the other hand, the 3J(H-1,H-5) = 9.6 Hz, compatible with a dihedral angle of about 0°, and the strong NOESY correlations of H-1/H-5 were in favor of a cis junction of the octahydroazulene rings. Key NOESY correlations of H-1/H-5, H-5/H-7, and H-6/H-8 supported the postulated configuration and confirmed the trans junction of the α-methylene-γ-lactone ring (Figure 1). Finally, the β-orientation of H3-15 was inferred through NOEs of H3-15/H-6 and H3-15/H-14b. The absolute configuration of compound 16 was assigned on the basis of a good match of the experimental and calculated ECD spectra (Figure 6). Hence, compound 16 was established as 8αangeloxy-4α-hydroxyguaian-2(3),10(14),11(13)-trien-6α,12olide and named guaianonobilin.

Compounds 9−12 showed similar ECD spectra, with a negative Cotton effect at about 220 nm (Figure 4). The calculated ECD spectrum of 12 was in good accord with the experimental values, and the stereochemistry of 9−12 was confirmed as depicted.

Figure 4. Experimental ECD spectra in MeOH of compounds 9−12 and calculated spectrum of compound 12.

Compound 14 showed the same molecular formula as 1,10epoxynobilin (13) (HRESIMS m/z 383.1479 [M + Na]+) and similar NMR data. Differences were seen only in resonances arising from the side chain at C-8 that were indicative of a tigloyl group. Thus, compound 14 was identified as 8tigloylepoxynobilin. The C-8 epimer of compound 14 has been previously isolated from other Asteraceae and named heliangine.25 The calculated ECD spectrum of compound 13 showed a positive Cotton effect around 230 nm, arising from the α-methylene-γ-lactone, as in the calculated spectrum for the 6S,7R,8S stereoisomer. In the ECD spectrum of 14, the positive Cotton effect was shifted to lower wavelength (219 nm) due to the presence of the tigloyl group (Figure 5). Compound 15 showed a sodium adduct ion at m/z 401.1566 [M + Na]+ in the HRESIMS, corresponding to a molecular formula of C20H26O7 (calcd for C20H26NaO7, m/z 401.1571) and eight degrees of hydrogen deficiency. The NMR data

Figure 5. Experimental ECD spectra in MeOH of compounds 13 and 14 and calculated spectrum of compound 13. 464



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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 14−16 (CD3OD for 14 and 16, DMSO-d6 for 15; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 14

15

position

δH (mult. J in Hz)

δ Cb

1 2

2.93 (dd, 10.0, 4.6) 2.47a) 1.63 (ddd, 12.8, 10.0, 2.6) 4.40 (dd, 4.2, 2.6)

61.0, CH 32.7, CH2

3 4 5 6 7 8 9 10 11 12 13

5.27 (dq, 10.6, 1.5) 6.36 (dd 10.6, 1.0) 3.05 (dq, 10.2, 1.5) 5.05 (ddd, 11.6, 10.2, 2.8) 2.46a 1.46 (dd, 13.0, 11.6)

5.73 (br s)

71.3, 142.2, 124.3, 76.9, 50.6, 69.8, 46.7,


57.5, 135.2, 170.2, 126.3,

C C C CH2

δH (mult. J in Hz) 3.90 (br d, 4.2) 2.18−2.08 (m)

16 δ Cb 74.2, CH 41.8, CH2

4.65 (dd, 9.3, 6.8) 5.26 (dq, 5.8 1.5) 5.88 (dd, 5.8, 5.5) 3.05 (dddd, 9.0, 5.5, 2.8, 2.0) 5.16 (br dd, 9.0, 9.0) 1.83a 2.21, (br d, 14.0)

5.50 (br d 2.0) 6.09 (br d 2.8)

80.3, 139.3, 123.5, 78.1, 53.0, 72.2, 39.8,


87.4, 137.0, 170.0, 123.9,

C C C CH2

6.19 (br s) 14 15 16 17 18 19 20 a

1.52 (br s) 1.81a

6.87 (m) 1.80a 1.80a

16.9, 21.9, 166.0, 127.7, 137.9, 10.5, 12.9,

CH3 CH3 C C CH CH3 CH3

3.68 (d, 11.0) 3.55 (d, 11.0) 1.60 (d, 1.5)

6.13 (dq, 7.0, 1.5) 1.88 (qq, 7.0, 1.5) 1.82 (quint, 1.5)

61.1, CH2 19.7, CH3 166.8, C 127.2, C 137.8 CH 15.2, CH3 19.8, CH3

δH (mult. J in Hz)

δC

3.69 (br d, 9.6) 5.63 (dd, 5.6, 2.6)

53.0, CH 131.7, CH

5.82 (dd, 5.6, 1.6)

141.5, 84.6, 56.9, 80.2, 48.3, 75.4, 45.6,


143.4, 138.2, 171.6, 124.6,

C C C CH2

2.66 4.32 3.44 5.04 2.23 3.02

(dd, 11.3, 9.6) (dd, 11.3, 8.5) (dddd, 11.1, 8.5, 3.1, 2.8) (ddd, 11.1, 10.1, 5.6) (dd, 12.3, 10.1) (dd, 12.3, 5.6)

6.21 (br d, 3.1) 5.79 5.08 4.86 1.37

(br d, 2.8) (br s) (br s) (s)

6.23 (dq, 7.0, 1.5) 2.00 (qq, 7.0, 1.5) 1.95 (quint, 1.5)

116.8, CH2 25.2, 168.0, 128.5, 140.6, 16.1, 20.7,

CH3 C C CH CH3 CH3

Overlapping signals. bExtracted from 1H−13C 2D inverse-detected experiments due to the low amount of sample.

H-3), respectively. These NMR data were consistent with compound 17 being a 2,3-seco-sesquiterpene lactone. The E4(5) configuration was assigned by NOESY correlations of H3/H-5 (Figure 1). As seen from the conformational analysis and in agreement with previous reports,26 the 3J(H-6,H-7) = 3.4 Hz could not be strictly correlated to the stereochemistry at the lactone ring due to the flexibility of the molecule. However, a cis configuration with a dihedral angle ΦH6−C6−C7‑H7 of ca. 30° could be excluded. To solve this configurational issue, a more rigorous investigation (e.g., by chemical derivatization or crystallography) would have been required, but this was precluded by the limited amount of material isolated. Once again, the absolute configuration was proposed on the basis of ECD data. Calculated ECD spectra for the two possible diastereoisomeric combinations bearing the trans-lactone are shown in Figure 7. The calculated ECD spectrum for the 6R,7R,8R stereoisomer was very similar but enantiomeric to the experimental one. Thus, it was concluded that compound 17 was (6S,7S,8S)-2,3-seco-germacra-E4(5),10(14),11(13)-trien6α,12-olide, and the compound was named seconobilin A. Compound 18 was assigned to the same molecular formula as 17 (C20H24O6; HRESIMS m/z 383.1479 [M + Na]+; calcd for C20H24NaO6 383.1465) and was a positional isomer of the latter. The olefinic and aliphatic methylene groups of 17 were absent and replaced by an olefinic methine (δH 5.64, H-9) and a methyl group (δH 2.06, H3-14) due to the presence of a C-9− C-10 double bond. As in 17, the key NOESY correlations of H3/H-5 and H-9/H3-14 confirmed the E4(5) and Z9(10) stereochemistry. On the basis of biogenetic considerations and the close similarity of the NMR shifts, J coupling, and ECD spectra of compounds 17 and 18, the same absolute


Compound 17 exhibited a molecular formula of C20H24O6, as deduced from its 13C NMR and HRESIMS data (molecular ion at m/z 383.1477 [M + Na]+; calcd for C20H24NaO6 383.1471). The 1D and 2D NMR data showed the presence of characteristic signals for α-methylene-γ-lactone and angelate ester groups. Additional resonances were attributed to an aliphatic methylene group (δH 2.83 and 2.50), an oxygenated methine (δH 5.40), a terminal double bond (δH 6.18 and 5.95), a trisubstituted double bond (δH 6.49), and two carbonyl groups (δC 201.3, C-1 and δC 196.0, C-3) attributable to a methyl ketone (δH 2.32, s, H3-2) and to an aldehyde (δH 9.44, s, 465



Article

cyclodecadiene ring. J(H-6,H-7) couplings in the range 1.0− 5.0 Hz were observed for the trans lactone, while the allylic coupling J(H-7,H-13) was always less than 3 Hz (1.0−3.0 Hz), in agreement with the modified lactone rule for heliangolides.20 The only exception was compound 8, which showed a J(H-6, H-7) of 9 Hz, arising from a conformation of the cyclodecadiene ring with UU orientation of H3-14 and H3-15 and parallel endocyclic double bonds. However, the configuration at the lactone ring, due to the influence of substituents on the J couplings value of the bridgehead protons, requires a more rigorous assignment. 26 Chiroptical methods have been historically used for the stereochemical analysis of sesquiterpene lactones.28 With respect to the ECD spectra, Geismann et al.29 correlated the sign of Cotton effects at ca. 220 and 260 nm, arising from π → π and n → π transitions, respectively, to the cis or trans junction of the α-methylene-γ-lactone ring. In nobilin (1), the n → π transitions could not be clearly detected, possibly due to the E−Z diene chromophore. Application of the empirical rule was here precluded since other chromophores were overlapping with the π → π transition of the lactone ring and, thus, required a comparison with calculated ECD spectra. Compounds 1, 3, 4, 7−10, 12, and 14−18 were tested for their in vitro antiprotozoal activity against T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum. In parallel, cytotoxicity of the compounds in L6 cells was evaluated, in order to obtain an initial assessment of the selective/toxicity ratio of the compounds (Table 5). Among the parasites tested, T. b. rhodesiense was generally found to be the most sensitive against the sesquiterpene lactones evaluated. For the other parasites, submicromolar activity was observed only against L. donovani after treatment with 4, 16, and 17, but without selectivity (IC50 0.38−0.81 μM, SI 3−7). In line with the reported activity of nobilin (1) and structurally related


configuration may be proposed (Figure S64, Supporting Information). Thus, compound 18 was identified as (6S,7R,8S)-2,3-seco-germacra-E4(5),Z9(10),11(13)-trien-6α,12olide and named seconobilin B. seco-Sesquiterpene lactones are quite rare in Nature. Pycnolide, with a similar framework to that of 17 and 18, has been reported from Liatris pycnostachya.27 It is worthy of note that compounds 3 and 19 were isolated for the first time as minor natural products of A. nobilis, but have been previously reported during acid-catalyzed degradation studies on nobilin 1.11 Structural analysis of germacranolides 1−15 revealed a preferred disposition of the cyclodecane ring, with a preferred UD orientation of the methyl groups attached to the

Table 4. 1H and 13C NMR Spectroscopic Data for Compounds 17 and 18 (CD3OD; 500.13 MHz for 1H and 125.77 MHz for 13 C NMR; δ in ppm) 17 δH (mult. J in Hz)

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2.32, s 9.44, s 6.49 5.48 3.37 5.40 2.83 2.50

6.43 5.99 6.18 5.95 1.87

(dq, 8.4, 1.0) (dd 8.4, 3.4) (dddd, 4.4, 3.4, 2.2, 2.0) (ddd, 10.2, 4.4, 3.1) (ddd, 13.7, 3.1, 1.0) (dd, 13.7, 10.2)

(d, 2.2) (d, 2.0) (br s) (br s) (d, 1.0)

6.11 (qq, 7.0, 1.5) 1.90 (dq, 7.0, 1.5) 1.80 (quint, 1.5)

18 δC 201.3, 25.7, 196.0, 142.6, 148.5, 77.1, 50.1, 73.1, 34.3,

C CH3 CH C CH CH CH CH CH

145.6, 135.5, 171.1, 126.7,

C C C CH2

129.8, CH2 9.8, CH3 168.4, C 128.4, C 140.5, CH 16.0, CH3 20.58, CH3 466

δH (mult. J in Hz) 2.32, s 9.45, s 6.52 5.52 3.42 5.88 5.64

(dq, 8.5, 1.2) (dd, 8.5, 3.6) (dddd, 5.4, 3.6, 2.2, 2.0) (dd, 8.6, 5.4) (dq, 8.6, 1.5)

6.36 (d, 2.2) 5.86 (d, 2.0) 2.06 (d, 1.5) 1.86 (d, 1.2)

6.16 (qq, 7.0, 1.5) 1.93 (dq, 7.0, 1.5) 1.85 (quint, 1.5)

δC 204.4, C 29.00, CH3 196.0, CH 142.4, C 148.9, CH 77.1, CH 50.1, CH 73.1, CH 133.5, CH 141.7, 135.5, 171.3, 126.5,

C C C CH2

20.54, CH3 9.8, CH3 168.1, C 128.3, C 140.9, CH 16.1, CH3 20.58, CH3 DOI: 10.1021/acs.jnatprod.6b00980 J. Nat. Prod. 2017, 80, 459−470


Article

Table 5. In Vitro Activity of Compounds 1, 3, 4, 7−10, 12, and 14−18 against T. b. rhodesiense (STIB 900), T. cruzi (Tulahuen C4 LacZ), L. donovani (MHOM-ET-67/L82), and P. falciparum (NF54) and Cytotoxicity in L6 Cells compound 30

1 3 4 7 8 9 10 12 14 15 16 17 18 positive controls a

T. b. rhodesiense IC50 (μM)a 2.1 4.6 0.4 0.4 1.4 0.61 0.36 0.88 1.2 0.08 1.3 0.42 1.2 0.01

± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.1 (2.4) 0.78 (6.1)b 0.1 (3.9)b 0.03 (3.9)b 0.1 (3.9)b 0.07 (8.3)b 0.05 (14.1)b 0.16 (8.3)b 0.2 (4.5)b 0.002 (63.1)b 0.2 (4.4)b 0.6 (4.3)b 0.2 (4.3)b 0.01c

T. cruzi IC50 (μM)a 11.3 4.2 2.8 9.8 26.1 29.3 26.7 31.1 7.30 37.3 10.9 5.0 11.3 2.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

L. donovani IC50 (μM)a

P. falciparum IC50 (μM)a

2.7 ± 1.1 (1.8)

3.1 ± 0.5 (1.6)

b

3.3 (0.4) 0.5 (6.1)b 0.2 (0.5)b 0.2 (0.2)b 3.0 (0.2)b 2.6 (0.2)b 0.2 (0.2)b 4.2 (0.2)b 1.2 (0.7)b 3.5 (0.1)b 0.4 (0.6)b 0.6 (0.3)b 0.2 (0.5)b 0.2d

0.5 1.6 8.4 13.2 5.3 230 2.7 9.8 0.8 0.38 0.5 0.13

± ± ± ± ± ± ± ± ± ± ± ±

b

0.1 (3.1)b 0.1 (1.1)b 0.2 (0.6)b 0.07 (0.4)b 0.3 (1.0)b 0.7(0.3)b 0.5 (1.9)b 0.2 (0.5)b 0.1 (7.2)b 0.05 (4.7)b 0.1 (11.2)b 0.01e

1.9 1.9 9.6 7.8 6.2 7.9 3.0 4.7 8.5 2.0 2.2 0.006

± ± ± ± ± ± ± ± ± ± ± ±

b

0.2(0.8)b 0.1 (0.9)b 0.36 (0.5)b 0.1 (0.6)b 0.7 (0.8)b 0.1(0.9)b 0.1 (1.8)b 0.1 (1.1)b 0.3 (0.68)b 0.2 (0.9)b 0.2 (2.3)b 0.01f

L6 cells IC50 (μM)a 4.9 28.2 1.5 1.8 5.3 5.1 5.1 7.3 5.3 5.1 5.8 1.8 5.2 0.016

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 1.2 0.2 0.1 0.41 0.2 0.1 0.2 0.5 0.3 0.3 0.1 0.2 0.01g

Average of three independent assays. bSelectivity index (SI): IC50 in L6 cells divided by IC50 in the titled parasitic strain. cMelarsoprol. Benznidazole. eMiltefosine. fChloroquine. gPodophyllotoxin.

d

germacranolides,11 change of stereochemistry at the side chain or at the hydroxy group function (e.g., in 3) did not improve the activity. On the other hand, decoration of the cyclodecadiene skeleton with more polar functional groups, such as a hydroxy group as in 8−12 and 14, increased activity up to 5fold. An additional α,β-unsaturated group, as the enone in 4, led to a significant increase of antitrypanosomal activity, but did not increase selectivity. A similar trend was observed for compound 7 and the two seco-sesquiterpene lactones 17 and 18 bearing formyl groups. In contrast to the reportedly high activity and selectivity for some guaianolides, such as cynaropicrin, compound 16 was only moderately active and highly toxic. Within the tested compound series, furanoheliangolide 15 showed the strongest inhibitory activity against T. b. rhodesiense (IC50 0.08 ± 0.002 μM) and was the only compound with good selectivity (SI 63). The present findings are in agreement with those reported by Schmidt et al.,30 who identified a furanoheliangolide as the most active sesquiterpene lactone against T. b. rhodesiense. Unfortunately, the limited amount of 15 available precluded an in vivo study. However, an in silico evaluation was performed by Percepta (ACDLabs).31 Optimal log p (1.74), good water solubility, and no violation of Lipinski’s rules were found. Also, good Caco-2 and BBB permeability were predicted for 15. As to drug safety profile, predictions indicated that 15 should not be a substrate for Pglycoprotein and should not be an inhibitor of cytochrome P450 1A2 and hERG channel.32 Antiprotozoal activity of many natural and synthetic sesquiterpene lactone derivatives has been reported, but none of them are currently in clinical development against parasitic diseases. Parthenolide and its dimethylamino derivative (DMAPT) are being evaluated for the treatment of leukemia.33 The main reason is the rather unspecific mode of action of sesquiterpene lactones that renders them relatively toxic. Attempts have been made to identify structural elements that confer selectivity to a given sesquiterpene lactone, but with no success.34 Our findings are in line with earlier observations that high selectivity is a function of potent activity (IC50 < 0.1 μM) rather than the presence of a particular structural feature in a molecule. The very high potency and selectivity of compound 15, along with its drug-like properties in silico, render

furanoheliangolides an attractive class of compounds for further lead optimization of antiprotozoal agents.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer 341 polarimeter using a 10 cm microcell and MeOH (1 mg/mL) as solvent. UV and ECD spectra were recorded in MeOH (150−250 μg/mL) on a Chirascan CD spectrometer, and data were analyzed with Pro-Data V2.4 software. NMR spectra were recorded on a Bruker Avance III NMR spectrometer operating at 500.13 MHz for 1H and 125.77 MHz for 13 C. 1H NMR, COSY, HSQC, HMBC, and NOESY spectra were measured at 18 °C in a 1 mm TXI probe with a z-gradient, using standard Bruker pulse sequences. 13C NMR/DEPTQ spectra were recorded at 23 °C in a 5 mm BBO probe with a z-gradient. Spectra were analyzed by Bruker TopSpin 3.0 software. High-resolution mass spectra (ESITOFMS) were recorded in the positive mode on a Bruker microTOF ESIMS system. Nitrogen was used as a nebulizing gas at a pressure of 2.0 bar and as a drying gas at a flow rate of 9.0 L/min (dry gas temperature 240 °C). The capillary voltage was set at 45 000 V and the hexapole at 230.0 Vpp. Instrument calibration was done with a reference solution of 0.1% sodium formate in 2-propanol−water (1:1) containing 5 mM NaOH. HPLC-PDA-ESIMS spectra were obtained in the positive mode on a Shimadzu LC-MS/MS 8030 triple quadrupole MS system, connected via T-splitter (1:10) to an Shimadzu HPLC system consisting of degasser, binary mixing pump, autosampler, column oven, and a diode array detector and to an Alltech 3300 ELSD detector. Data acquisition and processing was performed with LabSolution software. Semipreparative HPLC separations were carried out with an Agilent HP 1100 Series system consisting of a quaternary pump, an autosampler, a column oven, and a diode array detector (G1315B). Waters SunFire C18 (3.5 μm, 3.0 × 150 mm i.d.) and SunFire Prep C18 (5 μm, 10 × 150 mm i.d.) columns were used for reversed-phase analytical and semipreparative separations, respectively. HPLC-grade MeOH, acetonitrile (Scharlau Chemie S.A.), and water were used for HPLC separations. A Macherey-Nagel Nucleodur 105CN column (5.0 μm, 4.0 × 125 mm i.d.) was used for normal-phase HPLC separations. HPLC solvents contained 0.1% HCO2H for analytical and semipreparative separations. NMR spectra were recorded in methanol-d4 and DMSO-d6 (Armar Chemicals). Technical-grade solvents purified by distillation were used for extraction and open column chromatography. Silica gel (63−200 and 15−40 μm, Merck) was used for open column chromatography. 467



Article

Plant Material. Anthemis nobilis flower cones, batch number 11004258, were purchased from DIXA AG, St. Gallen, Switzerland, in March 2013. Extraction and Isolation. A. nobilis flower cones (2 kg) were milled and successively percolated with n-hexane (2 × 10 L, 24 h) and CH2Cl2 (2 × 10 L, 24 h). The dried CH2Cl2 extract (90 g) was fractionated by column chromatography (CC) on silica gel (40 × 5 cm, 70−230 mesh), using a gradient of n-hexane−EtOAc (100 to 60:40) as mobile phase. Fractions A−C were combined on the basis of TLC patterns (silica gel; n-hexane−AcOEt, 60:40, detection with 10% EtOH phosphomolybdic acid). The sesquiterpene lactone-enriched fraction B (22.9 g) was submitted to CC on silica gel (40 × 5 cm, 400−800 mesh), using a gradient of n-hexane−EtOAc (95:5 to 100) as mobile phase. Fractions B1−B28 were combined based on TLC analysis. Fraction B8 afforded nobilin (1) (1.5 g) without further purification. Fraction B9 (1.12 g) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 60% B (0−10 min), 60% B (10−20 min), 60 → 100% B (20−32 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 200 μL] to yield compounds 18 (13.5 mg, tR 23.9 min) and 3 (43.9 mg, tR 24.5 min). Fraction B11 (0.25 g) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 60% B (0−10 min), 60% B (10−27 min), 60 → 100% B (27−35 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 200 μL] to yield compounds 17 (7.5 mg, tR 21.1 min) and 2 (37.4 mg, tR 23.2 min). Fraction B13 (0.08 g) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 55% B (0−10 min), 55% B (10−27 min), 55 → 100% B (27−35 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 200 μL] to yield compound 16 (4.4 mg, tR 21.0 min). Fraction B14 (0.44 g) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 60% B (0−8 min), 60% B (8−25 min), 60 → 100% B (25− 35 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 200 μL] to afford compound 14 (10.6 mg, tR 16.2 min). Fraction B16 (0.09 g) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 60% B (0−10 min), 60% B (10−27 min), 60 → 100% B (27−35 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 200 μL] to yield compounds 13 (10.3 mg, tR 18.1 min) and 4 (1.8 mg, tR 19.0 min). Fraction C (19.3 g) was submitted to CC on silica gel (40 × 5 cm, 400−800 mesh), using a gradient of n-hexane−EtOAc (80:20 to 0:100) as mobile phase. Fractions C1−C19 were combined based on TLC analysis. Fraction C4 (91.8 mg) was purified by preparative RPHPLC [H2O (A), MeCN (B); 60% B (0−15 min), 60 → 100% B (10−25 min), 100% B (25−30 min); flow rate 15 mL/min; sample concentration 90 mg/mL in DMSO; injection volume 300 μL] to afford compound 19 (2.4 mg, tR 26.8 min). Fraction C9 (460 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 45% B (0−5 min), 45 → 50% B (5−30 min); 100% B (30−35 min); flow rate 15 mL/min; sample concentration 100 mg/mL in DMSO; injection volume 300 μL] to yield subfractions C9‑1−C9‑8. Subfraction C9‑6 (3.0 mg) was further purified by normal-phase HPLC (Nucleodur 105CN) [n-hexane (A), i-PrOH (B); 5 → 10% B (0−5 min), 10 → 15% B (5−25 min); 100% B (25−30 min); flow rate 0.8 mL/min; sample concentration 10 mg/mL in n-hexane−i-PrOH (90:10); injection volume 30 μL] to afford 6 (1.8 mg, tR 10.2 min). Subfraction C9‑7 (3.2 mg) was further purified by normal-phase HPLC (Nucleodur 105CN) [n-hexane (A), i-PrOH (B); 0 → 7% B (0−5 min), 7% B (5−15 min); 100% B (15−20 min); flow rate 0.8 mL/min; sample concentration 10 mg/mL in n-hexane−i-PrOH (90:10); injection volume 30 μL] to obtain 13 (0.9 mg, tR 9.1 min) and 5 (0.9 mg, tR 10.6 min). Fraction C11 (150 mg) was purified by preparative RPHPLC [H2O (A), MeCN (B); 30 → 60% B (0−20 min), 60 → 100% B (20−25 min), 100% B (25−30 min); flow rate 15 mL/min; sample concentration 100 mg/mL in DMSO; injection volume 300 μL] to yield compound 7 (7.1 mg, tR 24.1 min). Fraction C14 (260 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 40% B (0−5 min), 40 → 50% B (5−25 min), 50 →100% B (25−30 min), 100% B (30−35 min); flow rate 15 mL/min; sample concentration 100 mg/mL in DMSO; injection volume 300 μL] to afford compound

8 (4.9 mg, tR 23.8 min). Fraction C15 (60 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 20 → 45% B (0−5 min), 45 → 50% B (5−20 min), 50 →100% B (20−25 min), 100% B (25−30 min); flow rate 15 mL/min; sample concentration 100 mg/ mL in DMSO; injection volume 300 μL] to yield compound 15 (1.0 mg, tR 18.4 min). Fraction C17 (40 mg) was purified by semipreparative RP-HPLC [H2O (A), MeCN (B); 20 → 30% B (0−20 min), 30 →100% B (20−25 min), 100% B (25−30 min); flow rate 4 mL/min; sample concentration 20 mg/mL in DMSO; injection volume 100 μL] to yield compounds 11 (1.4 mg, tR 14.4 min), 10 (2.9 mg, tR 19.9 min), and 9 (23 mg, tR 21.6 min). Fraction C19 (200 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 30 → 40% B (0−5 min), 40 → 45% B (5−20 min), 45 → 100% B (20−25 min), 100% B (25−30 min); flow rate 15 mL/min; sample concentration 100 mg/mL in DMSO; injection volume 300 μL] to yield compound 12 (14.8 mg, tR 14.7 min). (+)-8α-Angeloxy-3β-hydroxy-1-oxogermacra-Z4(5),10(14),11(13)-trien-6α,12-olide (nobilinon A, 4): white solid; [α]25D +50.4 (c 0.4, CH3OH); UV λmax (MeOH) (log ε) 208 (2.86) nm; ECD (MeOH, c 0.4 mM, 0.1 cm); Δε +4.7 (203 nm, sh), +6.4 (212 nm), −2.7 (236); 1H and 13C NMR, see Table 1; HRESIMS m/z 383.1481 [M + Na]+ (calcd for C20H24O6Na, 383.1465). (+)-3β-Hydroxy-1-oxo-8α-tigloxygermacra-Z4(5),10(14),11(13)trien-6α,12-olide (nobilinon B, 5): white solid; [α]25D +10.6 (c 0.1, CH3OH); UV λmax (MeOH) (log ε) 208 (2.96) nm; ECD (MeOH, c 0.5 mM, 0.1 cm); Δε +2.0 (214 nm), −2.3 (236 nm); 1H and 13C NMR, see Table 1; HRESIMS m/z 383.1474 [M + Na]+ (calcd for C20H24O6Na, 383.1465). (+)-8α-Angeloxy-3α-hydroxy-1-oxo-germacra-Z4(5),10(14),11(13)-trien-6α,12-olide (nobilinon C, 6): white solid; [α]25D +64.24 (c 0.1, CH3OH); UV λmax (MeOH) (log ε) 213 (3.5) nm; ECD (MeOH, c 0.4 mM, 0.1 cm); Δε +6.0 (208 nm), −15.7 (228 nm); 1H and 13C NMR, see Table 1; HRESIMS m/z 383.1476 [M + Na]+ (calcd for C20H24O6Na, 383.1465). (−)-8α-Angeloxy-3β-hydroxy-14-oxogermacra-Z4(5),Z1(10),11(13)-trien-6α,12-olide (formylnobilin, 7): white solid; [α]25D −19.4 (c 0.6, CH3OH); UV λmax (MeOH) (log ε) 206 (2.38) nm; ECD (MeOH, c 0.4 mM, 0.1 cm); Δε +4.0 (215 nm), −2.0 (250 nm), −1.0 (335 nm); 1H and 13C NMR, see Table 1; HRESIMS m/z 383.1473 [M + Na]+ (calcd for C20H24O6Na, 383.1465). (+)-8α-Angeloxy-1α,3β-dihydroxygermacra-Z4(5),Z9(10),11(13)trien-6α,12-olide (8-epi-desacetyleupacunin, 8): white solid; [α]25D +67.1 (c 0.5, CH3OH); UV λmax (MeOH) (log ε) 211 (3.4) nm; ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −1.0 (213 nm), +7.0 (228 nm), −3.7 (266 nm); 1H and 13C NMR, see Table 2; HRESIMS m/z 385.1641 [M + Na]+ (calcd for C20H26O6Na, 385.1622). (+)-1β-Hydroxy-8α-tigloxygermacra-Z4(5),10(14),11(13)-trien6α,12-olide (8-tigloylhydroxyisonobilin, 10): white solid; [α]25D +23.4 (c 0.4, CH3OH); UV λmax (MeOH) (log ε) 209 (3.4) nm; ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −5.9 (212 nm); 1H and 13C NMR, see Table 2; HRESIMS m/z 385.1635 [M + Na]+ (calcd for C20H26O6Na, 385.1622). (+)-8α-Acryloxy-1β-hydroxygermacra-Z4(5),10(14),11(13)-trien6α,12-olide (8-acryloylhydroxyisonobilin, 11): white solid; [α]25D +4.8 (c 0.2, CH3OH); UV λmax (MeOH) nm (log ε) 211 (2.5); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −1.7 (211 nm); 1H and 13C NMR, see Table 2; HRESIMS m/z 371.1469 [M + Na]+ (calcd for C19H24O6Na, 371.1465). (+)-8α-Angeloxy-1β,3α-dihydroxygermacra-Z4(5),10(14),11(13)trien-6α,12-olide (3-epi-hydroxyisonobilin, 12): white solid; [α]25D +55.7 (c 0.25, CH3OH; UV λmax (MeOH) nm (log ε) 209 (3.3); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −5.4 (218 nm); 1H and 13C NMR, see Table 2; HRESIMS m/z 385.1630 [M + Na]+ (calcd for C20H26O6Na, 385.1622). (+)-1β,10α-Epoxy-3β-hydroxy-8α-tigloxygermacra-Z4(5),11(13)dien-6α,12-olide (8-tigloylepoxynobilin, 14): white solid; [α]25D +51.2 (c 0.25, CH3OH); UV λmax (MeOH) nm (log ε) 207 (2.26); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε +4.5 (195 nm), +1.3 (208 nm), +5.6 (219); 1H and 13C NMR, see Table 3; HRESIMS m/z 385.1659 [M + Na]+ (calcd for C20H26O6Na, 385.1622). 468



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(−)-8α-Angeloxy-3α,10α-epoxy-1α,14-dihydroxygermacra-Z4(5),11(13)-dien-6α,12-olide (furanonobilin, 15): white solid; [α]25D −3.1 (c 0.2, CH3OH); UV λmax (MeOH) nm (log ε) 206 (2.34); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −6.0 (208 nm); 1H and 13C NMR, see Table 3; HRESIMS m/z 401.1566 [M + Na]+ (calcd for C20H26O7Na, 401.1571). (−)-8α-Angeloxy-4α-hydroxyguaian-2(3),10(14),11(13)-trien6α,12-olide (guaianonobilin, 16): white solid; [α]25D −5.3 (c 0.4, CH3OH); UV λmax (MeOH) nm (log ε) 213 (2.63); ECD (MeOH, c 0.2 mM, 0.1 cm); Δε −3.6 (213 nm), +0.7 (239 nm); 1H and 13C NMR, see Table 3; HRESIMS m/z [M + Na]+ 367.1534 (calcd for C20H24O5Na, 367.1516). (+)-(6S,7S,8S)-2,3-seco-Germacra-E4(5),10(14),11(13)-trien6α,12-olide (seconobilin A, 17): white solid; [α]25D +4.4 (c 0.2, CH3OH); UV λmax (MeOH) nm (log ε) 206 (2.38); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −3.8 (216 nm), +4.5 (233 nm); 1H and 13C NMR, see Table 4; HRESIMS m/z 383.1477 [M + Na]+ (calcd for C20H24O6Na, 383.1465). (+)-(6S7R8S)-2,3-seco-Germacra-E4(5),Z9(10),11(13)-trien-6α,12olide (seconobilin B, 18): white solid; [α]25D +41 (c 0.4, CH3OH); UV λmax (MeOH) nm (log ε) 220 (2.52); ECD (MeOH, c 0.4 mM, 0.1 cm); Δε −8.3 (216 nm); 1H and 13C NMR, see Table 4; HRESIMS m/z 383.1479 [M + Na]+ (calcd for C20H24O6Na, 383.1465). Computational Methods. Conformational analysis was performed with Schrödinger MacroModel 9.8 (Schrödinger, LLC, New York) employing the OPLS2005 (optimized potential for liquid simulations) force field in H2O. Conformers within a 2 kcal/mol energy window from the global minimum were selected for geometrical optimization and energy calculation applying DFT with the Becke’s nonlocal three-parameter exchange and correlation functional and the Lee−Yang−Parr correlation functional level (B3LYP) using the B3LYP/6-31G** basis set in the gas phase with the Gaussian 09 program package.35 Vibrational evaluation was done at the same level to confirm minima. Excitation energy (denoted by wavelength in nm), rotator strength dipole velocity (Rvel), and dipole length (Rlen) were calculated in MeOH by TD-DFT/B3LYP/631G(d,p) or CAM-B3LYP/6-31G(d,p), using the SCRF method, with the CPCM model. ECD curves were obtained on the basis of rotator strengths with a half-band of 0.3 eV using SpecDis v1.61.36 ECD spectra were calculated from the spectra of individual conformers according to their contribution calculated by Boltzmann weighting. In Vitro Biological Testing. The in vitro activities against the protozoan parasites T. b. rhodesiense (STIB900) bloodstream forms, T. cruzi (Tulahuen C4 LacZ) intracellular amastigotes, L. donovani (MHOM-ET-67/L82) axenically grown amastigotes, and P. falciparum (NF54) erythrocytic stage and cytotoxicity in L6 cells (rat skeletal myoblasts) were determined as reported elsewhere.37

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS ECD spectra were measured at the Biophysics Facility, Biozentrum, University of Basel. Thanks are due to M. Cal, S. Keller-Märki, and R. Rocchetti for the in vitro assays. M..D.M. gratefully acknowledges F. Barbieri for proofreading the manuscript.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00980. 1D and 2D NMR spectra of new compounds 4−8, 10− 12, and 14−18; ECD spectra of compounds 1, 8, 15, 17, and 18; HPLC chromatogram of the dichloromethane extract of A. nobilis flower cones; 3D structures of compounds 15 and 16 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +41 61 267 14 25. Fax: +41 61 267 14 74. E-mail: [email protected]. ORCID

Maria De Mieri: 0000-0001-5567-2072 469



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