Synthesis and Natural Distribution of Anti-inflammatory Alkamides from Echinacea

The synthesis of the alkamides 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (1) and 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (5) was accomplished by organometallic coupling followed by introduction of the doubly unsaturated amide moiety. The distribution of these two amides in accessions of the nine species of Echinacea was determined.


Introduction
Echinacea angustifolia, Echinacea pallida and Echinacea purpurea are the main medicinal Echinacea species and have long been used to treat infections, to aid in wound healing and to enhance the immune system [1].In 2005, Echinacea products ranked among the top botanical supplements sold in the United States.In recent years, treatment of rhinoviruses has been the focus of several studies, a number of which have failed to show the efficacy of Echinacea [2].Commercial Echinacea products often are mixtures of the three main medicinal species and there is no regulation of the concentrations of the chemical constituents.Among the chemical constituents of Echinacea species, the alkamides, caffeic acid derivatives such as chicoric acid and the polyphenols are considered important for biological activity [3].
Alkamide levels differ significantly among roots, stems, and flowers of E. purpurea.The roots had higher levels of the dodeca-2,4-diene-8,10-diyne alkamides, whereas levels of the dodecatetraene alkamides and nonadeca-2,4-diene-8,10-diynes were highest in stems [4].Accessions from different geographical regions often show different chemical fingerprints.Additionally, preliminary studies on the stability of alkamide compounds in E. angustifolia revealed a 13% loss of alkamide levels over two months [5].The effects of storage time and temperature on alkamide levels in E. purpurea roots showed that levels of all alkamides fell by over 80% during storage at 24° C for 64 weeks [6].The ready availability of authentic standards of select alkamides would not only facilitate standardization for the purposes of medical studies, but would also permit biological evaluation of individual components.
The recent discovery that dodecadiendiynoic amide 1 from E. purpurea and E. pallida inhibited LPS-mediated activation of a murine macrophage line, RAW264.7,suggests that this alkamide may have anti-inflammatory activity [7].
Using male rats, an in vivo study examined the immunomodulatory effects of alkamides purified from Echinacea purpurea.These results suggest that the alkamides are among the active constituents of E. purpurea plants.At a dose level of approximately 12 µg/kg body weight/day they effectively stimulated alveolar macrophage function in healthy rats [8].Alkamides isolated from Echinacea angustifolia had inhibitory activity in in vitro cyclooxygenase (sheep microsomes) and 5-lipoxygenase (porcine leukocytes) assays [9].Hexane extracts of Echinacea variably inhibit growth of yeast strains of Saccharomyces cerevisiae, Candida shehata, C. albicans, and C. tropicalis under near UV irradiation and to a lesser extent without irradiation [10].Synergistic antioxidant effects were found when cichoric acid was combined with a natural mixture of alkamides [11].

Synthesis of alkamides
Despite the potential importance of the alkamides, few reports of synthesis of authentic standards have been reported.Crombie and co-workers have reported elegant syntheses of natural amides using organometallic coupling reactions [12].Bohlmann synthesized 1 and 5 in low overall yields, in part because the Wittig reactions that installed the cis-amide moiety produced the cis-isomers in only 13-14% yields [13].Kraus and Bae have reported syntheses of amides 2 and 3 and ketone 4 [14,15].We report herein the preparation of diacetylenic amides 1 and 5.The improved overall yields in our syntheses are the result of recent advances in synthetic methodology and the ready availability of larger quantities of these bioactive amides should accelerate the study of the scope of activity of these novel compounds.The general procedure for synthesis of amides 1 and 5 is shown in Scheme 1 below.Copper chloride-mediated coupling of 6 [16] followed by oxidation and a Wittig reaction provide 7a or 7b.Reduction of the ester to an aldehyde with DIBAL followed by Swern oxidation and a cis-selective Wittig reaction [17]

Characterization and distribution of the diacetylenic isobutylamides in Echinacea
The distribution of these two amides in accessions of the nine species of Echinacea (E.angustifolia, E. purpurea, E. pallida, E. sanguinea, E. simulata, E. tennesseensis, E. atrorubens, E. laevigata and E. paradoxa) was determined by HPLC.Amides 1 and 5 elute at 19.55 min and 23.28 min respectively.These two amides display very similar UV spectra, all with an absorption maximum at 260 nm, which agrees with that reported [3].The mean levels of amides 1 and 5 in roots, flowers and leaves from 6-month-old plants from nine Echinacea species are presented in Figure 2.These two amides are distributed widely in Echinacea.Our results show that of the nine Echinacea species examined, amide 1 is present in at least six species (E.purpurea, E. pallida, E. sanguinea, E. simulate, E. laevigata and E. paradoxa), while amide 5 is present in at least eight species (E.angustifolia, E. purpurea, E. pallida, E. sanguinea, E. simulata, E. tennesseensis, E. laevigata and E. paradoxa).The highest concentrations of both amide 1 (0.818 ± 0.06 mg g -1 fr.wt) and amide 5 (0.826 ± 0.1mg g -1 fr.wt) were found in E. purpurea roots, which have significantly higher amount of these two amides compared with all other species that contain these compounds.The species that is deficient in both amide 1 and amide 5 is E. atrorubens.Although not typically used for commercial medicinal preparations, some Echinacea species (e.g.E. paradoxa, E. laevigata and E. simulata) have fairly high contents of these amides.
We also found that the abundance of these two amides varied considerably with organ type.Generally speaking, they are present mainly in roots, displaying reduced abundance or even being not detectable in flowers and they are not detectable (the limit of HPLC detection for both amides is approximately 0.02 µg mL −1 ) in leaves.Interestingly, there is one exception, the E. paradoxa species, whose leaves have the highest quantity of amide 5 (0.303 ± 0.052 mg g -1 fr.wt), whereas flowers have less (0.147 ± 0.052 mg g -1 fr.wt ) and roots have the least (0.145 ± 0.019 mg g -1 fr.wt).This indicates that the biosyntheses of these amides may be regulated differently in different organs and species.

Conclusions
We have presented an improved synthesis of diacetylenic amides 1 and 5 which should pove useful for the preparation of appropriate standards.Investigation of their distribution in different Echinacea species revealed that both genetic source and organ type of the plant materials used in Echinacea preparations need to be considered for product standardization.The information reported in this work will be very useful for the standardization of Echinacea products.

General
Unless stated otherwise, all reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) using 0.25 mm precoated silica gel F254 plates (Sigma-Aldrich).Column or flash chromatography were performed with the indicated solvents using silica gel (230-400 mesh) purchased from Dynamic Adsorbents, LLC.All melting points were obtained on a Laboratory Devices capillary melting point apparatus and are uncorrected. 1H-and 13 C-NMR spectra were recorded on a Bruker VXR-300 (300 MHz) or a Bruker VXR-400 (400 MHz) spectrometer.Chemical shifts are reported relative to internal chloroform ( 1 H, 7.26 ppm; 13 C, 77.23 ppm).High resolution mass spectra were performed at the Iowa State University Mass Spectrometry Laboratory.

Plant material and extraction
Nine species of Echinacea, E. angustifolia (Accession 631267), E. purpurea (Accession 631307), E. pallida (Accession 631293), E. sanguinea (Accession A23878), E. simulata (Accession 631249), E. tennesseensis (Accession 631325), E. atrorubens (Accession 631262), E. laevigata (Accession 631312) and E. paradoxa (Accession 631301), provided by Dr. Mark P. Widrlechner at the USDA-ARS North Central Regional Plant Introduction Station, were studied to evaluate the natural distribution of amides 1 and 5 in Echinacea species.Six-month-old roots, flowers and leaves from each species/accessions were used.Specific plant growth conditions, plant material harvest and extraction method are the same as those in our previously published work [15].7-Hydroxy-(E)-Nisobutylundeca-2-ene-8,10-diynamide (C 15 H 21 O 2 ) was added as an internal standard prior to extraction for quantification purposes.All experiments were performed in triplicate on independently extracted plant samples from three individual plants.

HPLC analysis
Ethanol extract (15 µL) was injected into a YMC-Pack ODS-AM RP C18 (250 x 4.6 mm, 5 µm) column (Waters, MA) on a Beckman Coulter HPLC equipped with a 508 autosampler, 126 pump control and 168 UV-photodiode array detector (PDA) controlled by 32karat TM software (Version 5.0).The solvent system used was CH 3 CN/H 2 O at a flow rate of 1.0 mL/min following a linear gradient of 40→80% CH 3 CN in H 2 O over 45 min.Online UV spectra were collected between 200-400 nm.Compound quantification was carried out by calculating the UV response relative to the internal standard 7-hydroxy-(E)-N-isobutylundeca-2-ene-8,10-diynamide (C 15 H 21 O 2 ), which has been found suitable for use as an internal standard for these two amides because it was not found in Echinacea plants and does not overlap with any other metabolites found in Echinacea.Amides 1 and 5 were quantified at UV 260 nm with respect to the internal standard, using relative response factors to correct for absorbance differences between these two amides and the standard.These relative response factors for amides 1 and 5 were calculated at UV 260 nm.Various amount of authentic amide 1 or amide 5 (0.625 -3.125 µg) with internal standard (2.5 µg) were injected to give average relative response factors of 0.0677 (R 2 = 0.99) for amide 1, and 0.0669 (R 2 = 0.99) for amide 5, respectively.The internal quantification method used here can account for variations in extraction efficiencies in different extracts.The HPLC detection limit for both of the amides was approximately 0.02 µg mL −1 .

Statistical analysis
Statistical analyses were performed using SAS software version 8.02 (SAS Institute Inc., Cary, NC).One-way analysis of variance followed by the Tukey test was used to compare means.Significance of difference was defined at p < 0.05.
Dimethylsulfoxide (0.766 mL, 10.8 mmol) was added dropwise at -78 o C to a solution of oxalyl chloride (0.471 mL, 5.4 mmol) in CH 2 Cl 2 (10 mL).The mixture was stirred at same temp.for 20 min and triethylamine (2.25 mL, 16.2 mmol) was added dropwise and stirred at same temp.for 20 min.To the mixture was added the alcohol synthesized above (0.487 mg, 2.7 mmol) at -78 o C and stirred for 80 min while slowly warmed to room temperature.The reaction was quenched with sat NH 4 Cl (aq) and the aqueous layer was extracted with CH 2 Cl 2 (2 x 20 mL).The combined organic layers were washed with water (2 x 10 mL), dried (MgSO 4 ), filtered and concentrated in vacuo.The crude residue was purified via flash column chromatography to give the aldehyde (0.409 g, 85 % yield).

2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (1).
To a solution of compound 7b (0.437 g, 2.3 mmol) in 20 mL of THF was added DIBAL (4.6 mL,1.0Msoln in THF) at -78 o C in Ar.After stirring for 2 h at -78 o C, the reaction was quenched with ethyl acetate (30 mL) at -78 o C and warmed to rt.The mixture was washed with 10% HCl (aq) (10 mL), brine, dried (MgSO 4 ), filtered and concentrated in vacuo.The crude residue was purified via flash column chromatography to give the allylic alcohol (0.28 g, 81 % yield).Dimethylsulfoxide (0.530 mL, 7.48 mmol) was added dropwise at -78 o C to a solution of oxalyl chloride (0.326 mL, 3.74 mmol) in 20 mL of CH 2 Cl 2 .The mixture was stirred at the same temp for 20 min and triethylamine (1.56 mL, 11.2 mmol) was added dropwise and stirred at same temperature for 20 min.The above alcohol (0.277 g, 1.87 mmol) was added to the mixture at -78 o C and stirred for 80 min while slowly warming to rt.The reaction was quenched with sat NH 4 Cl (aq) and aqueous layer was extracted with CH 2 Cl 2 (2x 10 mL).Combined organic layer was washed with water (2x 10 mL), dried (MgSO 4 ), filtered and concentrated in vacuo.The crude residue was purified via flash column chromatography to give aldehyde (0.229 g, 84 % yield).To a solution of diphenylphosphonoacetamide (0.370 g, 1.06 mmol) in THF (10 mL) was added NaHMDS (1.06 mL, 1 M soln in THF) at -78 o C and stirred at same temp for 20 min.To the mixture was added the above aldehyde (0.140 g, 0.97 mmol) in THF (2 mL) via cannula and the resulting mixture was warmed to 10 o C over 2h.The reaction was quenched with NH 4 Cl (aq), washed with water, brine, dried (MgSO 4 ), filtered and concentrated in vacuo.The crude residue was purified via

Figure 2 .
Figure 2. Concentration of amides 1 and 5 in roots (A), flowers (B), and leaves (C) from 6-monthold plants of nine species of Echinacea.Error bars indicate standard deviations of means of triplicate experiments.For each amide, different letters (a -g) indicate a significant difference (p < 0.05).