Alkamides from Anacyclus pyrethrum L. and Their in Vitro Antiprotozoal Activity

In our ongoing study to evaluate the antiprotozoal activity of alkamides from Asteraceae, a dichloromethane extract from the roots of Anacyclus pyrethrum L. showed a moderate in vitro activity against the NF54 strain of Plasmodium falciparum and against Leishmania donovani (amastigotes, MHOM/ET/67/L82 strain). Seven pure alkamides and a mixture of two further alkamides were isolated by column chromatography followed by preparative high performance liquid chromatography. The alkamides were identified by mass- and NMR-spectroscopic methods as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1), deca-2E,4E-dienoic acid isobutylamide (pellitorine, 2), deca-2E,4E,9-trienoic acid isobutylamide (3), deca-2E,4E-dienoic acid 2-phenylethylamide (4), undeca-2E,4E-dien-8,10-diynoic acid isopentylamide (5), tetradeca-2E,4E,12Z-trien-8,10-diynoic acid isobutylamide (6), and dodeca-2E,4E-dien acid 4-hydroxy-2-phenylethylamide (7). Two compounds—undeca-2E,4E-dien-8,10-diynoic acid 2-phenylethylamide (8) and deca-2E,4E-dienoic acid 4-hydroxy-2-phenylethylamide (9)—were isolated as an inseparable mixture (1:4). Compounds 3, 4, and 5 were isolated from Anacyclus pyrethrum L. for the first time. While compounds 4 and 5 were previously known from the genus Achillea, compound 3 is a new natural product, to the best of our knowledge. All isolated alkamides were tested in vitro for antiprotozoal activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani and for cytotoxicity against L6 rat skeletal myoblasts.


Introduction
Alkamides represent a class of natural products with considerable structural diversity and a broad spectrum of bioactivities [1].In a previous study, alkamides from Achillea ptarmica L. (sneezewort yarrow, Asteraceae) showed activity against Plasmodium falciparum (Pf ) and Trypanosoma brucei rhodesiense (Tbr), causative agents of tropical malaria (Pf ) and human African trypanosomiasis (HAT, "Sleeping Sickness"; Tbr), respectively.However, the activity of the isolated alkamides could not conclusively explain the high activity of the tested dichloromethane extract against T. b. rhodesiense (IC 50 0.67 µg/mL) [2].Recently, an alkamide structurally related to those found in A. ptarmica but with an endoperoxide structure was isolated from Acmella ciliata, (also Asteraceae) and showed considerable antiplasmodial activity against Pf [3].
These studies on alkamides from Asteraceae were extended to Anacyclus pyrethrum L. and its alkamide constituents.From a phylogenic point of view, Anacyclus pyrethrum is closely related to Achillea ptarmica L. within the tribe Anthemideae of Asteraceae [4][5][6], and both species are known to contain a structural pattern similar to alkamides [7]. A. pyrethrum has been known since ancient times for its use as medicinal plant used-among other ailments-against recurring fevers (De Materica Medica [8]), and it was mentioned in a review by Adams as one of the plants used against malaria during the Renaissance (16th and 17th century) [9].In a bioactivity study associated with the mentioned review, antiplasmodial activity was reported for an ethyl acetate extract of A. pyrethrum roots, but without mentioning the level of activity in terms of an IC 50 value and without specification of the active constituents [10].We recently reported on the antiprotozoal IC 50 values of a dichloromethane extract from this plant drug, including a value of 3 µg/mL for the activity against Plasmodium falciparum [2].
The aim of the present study was therefore to evaluate the antiprotozoal activity of the alkamides in the roots of A. pyrethrum.

Isolation and Structural Characterization of the Alkamides
The major alkamides of A. pyrethrum roots were isolated from the dichloromethane extract by column chromatography (CC) on silica with an n-hexane-ethylacetate gradient followed by preparative high performance liquid chromatography (HPLC) of representative CC-fractions (XV, XIX, XX and XXII) selected on the basis of their UHPLC/+ESI QqTOF MS chromatograms (see Figure 1).
These studies on alkamides from Asteraceae were extended to Anacyclus pyrethrum L. and its alkamide constituents.From a phylogenic point of view, Anacyclus pyrethrum is closely related to Achillea ptarmica L. within the tribe Anthemideae of Asteraceae [4][5][6], and both species are known to contain a structural pattern similar to alkamides [7]. A. pyrethrum has been known since ancient times for its use as medicinal plant used-among other ailments-against recurring fevers (De Materica Medica [8]), and it was mentioned in a review by Adams as one of the plants used against malaria during the Renaissance (16th and 17th century) [9].In a bioactivity study associated with the mentioned review, antiplasmodial activity was reported for an ethyl acetate extract of A. pyrethrum roots, but without mentioning the level of activity in terms of an IC50 value and without specification of the active constituents [10].We recently reported on the antiprotozoal IC50 values of a dichloromethane extract from this plant drug, including a value of 3 µg/mL for the activity against Plasmodium falciparum [2].
The aim of the present study was therefore to evaluate the antiprotozoal activity of the alkamides in the roots of A. pyrethrum.

Isolation and Structural Characterization of the Alkamides
The major alkamides of A. pyrethrum roots were isolated from the dichloromethane extract by column chromatography (CC) on silica with an n-hexane-ethylacetate gradient followed by preparative high performance liquid chromatography (HPLC) of representative CC-fractions (XV, XIX, XX and XXII) selected on the basis of their UHPLC/+ESI QqTOF MS chromatograms (see Figure 1).The alkamides were identified by their exact molecular masses determined by UHPLC/+ESI-QTOF-MS/MS and by NMR spectroscopy.
The main component of fraction XIX (Figure 1, chromatogram C) with m/z 272.2020 [M + H] + was identified by its mass and NMR data as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1) in direct comparison with the compound previously isolated from A. ptarmica and literature data [2,11].Three compounds were isolated from fraction XV (Figure 1, chromatogram B).A further compound with a molecular mass of 272 amu ([M + H] + at m/z 272.2025) was identified as deca-2E,4E-dienoic acid 2-phenylethylamide (4) by its 1 H-spectrum (Figure S8) in comparison with literature [13].The compound was known from Achillea wilhelmsii but is now described as a constituent of A. pyrethrum for the first time.
Fraction XX (Figure 1 S9), which were in full agreement with literature data [11] reported after isolation from Achillea ptarmica L. This is the first report on the occurrence of 5 in A. pyrethrum.In the case of compound 6, it was reported in the literature that the all-E-isomer (tetradeca-2E,4E,12E-trien-8,10diynoic acid isobutylamide) is a constituent of Anacyclus pyrethrum L. [1,14].In our investigation, the isolated compound was unambiguously found to be the Z-isomer with regard to the C-12-C-13 double bond, since the coupling constant of H-12 and H-13 was 11 Hz.Since no NMR data proving the double bond geometry were reported in the original literature [14], it cannot be excluded that the reported constituent of A. pyrethrum was identical with compound 6.It could be possible, however, that both isomers occur in the plant.A further compound with a molecular mass of 272 amu ([M + H] + at m/z 272.2025) was identified as deca-2E,4E-dienoic acid 2-phenylethylamide (4) by its 1 H-spectrum (Figure S8) in comparison with literature [13].The compound was known from Achillea wilhelmsii but is now described as a constituent of A. pyrethrum for the first time.
Fraction XX (Figure 1 S9), which were in full agreement with literature data [11] reported after isolation from Achillea ptarmica L. This is the first report on the occurrence of 5 in A. pyrethrum.In the case of compound 6, it was reported in the literature that the all-E-isomer (tetradeca-2E,4E,12E-trien-8,10-diynoic acid isobutylamide) is a constituent of Anacyclus pyrethrum L. [1,14].In our investigation, the isolated compound was unambiguously found to be the Z-isomer with regard to the C-12-C-13 double bond, since the coupling constant of H-12 and H-13 was 11 Hz.Since no NMR data proving the double bond geometry were reported in the original literature [14], it cannot be excluded that the reported constituent of A. pyrethrum was identical with compound 6.It could be possible, however, that both isomers occur in the plant.

Antiprotozoal Activity of the Isolated Alkamides
The IC 50 values for the in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense (Tbr), Trypanosoma cruzi (Tc), Leishmania donovani (Ld) and Plasmodium falciparum (Pf ), as well as the cytotoxicity against L6 rat skeletal myoblasts of the crude dichloromethane extract from A. pyrethrum roots and of anacycline and pellitorine (1 and 2) were tested earlier under identical conditions, and the results were reported in our earlier study [2].The respective data of the isolated compounds 3-7 as well as the 1:4 mixture of 8 + 9 were determined in this study and are reported in Table 1.
Table 1.In vitro antiprotozoal and cytotoxic activity (IC 50 values in µg/mL; values in µM of pure compounds are reported in brackets) of the dichloromethane (DCM) extract from Anacyclus pyrethrum roots and of the isolated alkamides (3)(4)(5).Selectivity indices (SIs) represent the ratio of cytotoxic over antiprotozoal IC 50 values.Data are means of two independent determinations ± deviation from mean value.The in vitro activity data of 1 (anacycline) and 2 (pellitorine) was previously reported by us [2].  Data for the crude extract were previously reported and are repeated here for comparison [2].

Sample
On the background of the reported medicinal use as an antimalarial [8,9], it appears noteworthy that the crude extract displayed its highest activity against Pf, even though the IC 50 value of 3 µg/mL is relatively moderate.The most active single constituent against this parasite is compound 7 with an IC 50 in the same range (3.2 µg/mL), and about equal to that of the pellitorine (2, 3.3 µg/mL) [2].Compound 7 was also found to be the most active compound against Tc.Unfortunately, however, the antiparasitic activity of 7 is not selective.The compound, as the only one in the series, displayed considerable cytotoxicity with an IC 50 of only 0.2 µg/mL, so further studies on its antiprotozoal activity do not appear to be useful.The mixture of 8 and 9 displayed some activity against Tbr, but with little selectivity.The major component in this mixture ( 9) is structurally very similar to 7, and hence is likely to dominate the observed antitrypanosomal as well as cytotoxic activity.The ratio of cytotoxic over antitrypanosomal activity being more favorable for the 8 + 9 mixture than for 7, it could be expected that 9 with its C 10 acyl moiety, as a pure compound, would be more active and selective against Tbr than its C 12 homologue 7.In terms of structure-activity relationships, the p-hydroxy-substituted phenethyl amide moiety of these two compounds appears to confer higher toxicity to trypanosomatid as well as mammalian cells than the unsubstituted phenethyl amide moiety or the aliphatic amide groups.The other compounds showed only moderate activity against all tested parasites, and also low cytotoxicity.No clear-cut structure-activity relationships became evident for these compounds.
Taken together, the two alkamides 2 and 7 are likely to be responsible for the major part of the antiplasmodial effect of the crude extract, and thus related to the ethnomedical use of A. pyrethrum as an antimalarial.It must remain an open question at present whether this use was justified and whether, possibly, other compounds from yet-untested fractions contribute to the overall effect.

UHPLC/+ESI-QTOF-MS/MS
The analytical profiling of samples were performed on the already-described system with the identical method [2].

Sohxlet Extraction
The powdered plant material (604 g) was exhaustively extracted in two equal portions with dichloromethane (1500 mL each) in a Soxhlet apparatus for 11 h.The extract was evaporated to dryness under reduced pressure, yielding 14 g of crude extract.

Gravity Flow Column Chromatography (CC)
The extract (12.95 g) was separated by CC on 1.2 kg silica (particle size 0.063 to 0.2 mm; Merck, (Darmstadt, Germany), column dimensions 108 cm × 6 cm).The column was covered with aluminum foil to prevent the light-induced polymerization of alkamides with a polyinic structure moiety [16].The silica was equilibrated at 90:10 n-hexane/EtOAc (2.4 L).The flow was adjusted to 1 mL/min, and 10 mL were collected per tube for the first 459 tubes.From tube 460 to tube 2023, 20 mL were collected per tube.A gradient with an increasing amount of EtOAc was used: n-hexane/EtOAc 90:10 Finally, 3 L of isopropanol was used to obtain the most polar fraction.Related eluates were combined after TLC control (silica gel 60 F 254 , Merck (10 cm × 20 cm); detection: anisaldehyde/sulfuric acid, UV 254 nm, 366 nm and daylight; elution: current solvent mixture of the CC column).The fractionation is summarized in Table 2.The preparative HPLC isolation of all alkamides was performed on a Jasco (Groß-Umstadt, Germany) prep.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 C-18 column (5 µm, 250 mm × 20 mm) column (precolumn: C-18, 5 µm, 30 mm × 20 mm) with a binary gradient (A: water (Millipore); B: acetonitrile (HPLC grade)) at 15 mL/min with: 0 to 5 min: linear from 40% B to 65% B; 5 to 30 min: isocratic 65% B; 30 min to 40 min: linear from 65% B to 100% B; 40 min to 50 min: isocratic 100% B. The subfractions XIX and XV were each dissolved in a concentration of 10 mg/mL acetonitrile and injected in portions of 400 µL.The subfractions XX and XXII were dissolved in a concentration of 10 mg/mL acetonitrile/water (70:30) each and injected in portions of 400 µL.Pellitorine (2), as well as compounds 3 and 4 were isolated as white powders in yields of 0.6 mg (2), 0.8 mg (3), and 0.8 mg (4)

Figure 1 .
Figure 1.UHPLC/+ESI QqTOF MS-MS chromatograms of the CH2Cl2-extract of Anacyclus pyrethrum L. (A) c = 10 mg/mL and the selected fractions (B-E) c = 5 mg/mL.(B) fraction XV, (C) fraction XIX, (D) fraction XX, and (E) fraction XXII.All peaks are labelled with the measured m/z values and compound numbers.Blue represents base peak chromatogram m/z 100-500, and magenta represents base peak UV-chromatogram at 260 nm.