Synthesis and biological evaluation of a series of fatty acid amides from Echinacea

Article abstract of DOI:10.1016/j.bmcl.2015.06.024

Abstract Alkylamides are lipophilic constituents of Echinacea and possess numerous biological activities. Although significant effort has been focused on the study of crude Echinacea extracts, very little is known regarding the activities of the individual constituents that make up these herbal treatments. Herein we explore the SAR of simple alkylamides found in Echinacea extracts with respect to their ability to decrease the production of the pro-inflammatory mediator TNF-α. Our results have revealed the key structural requirements for activity and provide lead compounds for further investigation of these poorly understood molecules.

Full text of DOI:10.1016/j.bmcl.2015.06.024

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3091–3094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a series of fatty acid amides
from Echinacea
a,
Yasamin Moazami a, , Travis V. Gulledge b, , Scott M. Laster b, , Joshua G. Pierce
⇑
⇑
a Department of Chemistry, North Carolina State University, Raleigh, NC 27695, United States
b Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Alkylamides are lipophilic constituents of Echinacea and possess numerous biological activities. Although
significant effort has been focused on the study of crude Echinacea extracts, very little is known regarding
the activities of the individual constituents that make up these herbal treatments. Herein we explore the
SAR of simple alkylamides found in Echinacea extracts with respect to their ability to decrease the
Received 13 May 2015
Revised 4 June 2015
Accepted 8 June 2015
Available online 12 June 2015
production of the pro-inflammatory mediator TNF-a. Our results have revealed the key structural
requirements for activity and provide lead compounds for further investigation of these poorly
understood molecules.
Keywords:
Echinacea
Alkylamide
Ó 2015 Elsevier Ltd. All rights reserved.
TNF-a
Anti-inflammatory
Cytokine
Echinacea extracts are widely used as herbal medicines and are
often taken for the treatment of cold-like symptoms due to their
proposed immunomodulating capabilities. Fatty acid amides (alky-
lamides) are the most prevalent lipophilic compounds contained in
Echinacea extracts and the only components known to cross the
intestinal barrier; however, their constituent makeup varies
greatly by species and method of preparation.1 These variances
in crude plant extracts have slowed efforts to utilize such mixtures
as medicinal agents and have complicated the analysis of the var-
ious components’ biological roles.1c Although structurally simple,
fatty acid amides have diverse biological activities and are
involved in signaling pathways relevant to cancer, cardiovascular
disease, pain, inflammation, drug addiction, eating disorders,
sleep deprivation, anxiety and depression.2 Significant efforts have
been focused on the development of fatty acid amides as analgesic
agents due to their inhibition of FAAH, CB-1 and VR-1.3
Surprisingly, relatively little effort has focused on the systematic
study of simple fatty acid amides on pro-inflammatory mediators
cannabinoid receptor (CB)-dependent and CB-independent
mechanisms.4 We have previously demonstrated that diene-con-
taining fatty acid amide 1, a constituent of Echinacea purpurea,
decreases the production of TNF-a from RAW 264.7 macrophage-
like cells in a dose-dependent manner (Fig. 1).5 Herein we report
the chemical synthesis of 1 and a series of analogs to probe the
structural requirements for activity and evaluate the general cyto-
toxicity of these compounds.
We began our synthetic efforts by preparing the lead natural
product (1), as this compound was obtained in extremely small
quantities from natural sources for the previously reported biolog-
ical studies. To this end, two-step oxidation of the commercially
available diene-containing alcohol 2 to carboxylic acid 3 followed
by coupling with isobutyl amine (T3PÒ) provided fatty acid amide
1 in good yield (Scheme 1).6 Compound 1 proved identical to the
natural product by 1H and 13C NMR analysis.
With the natural product in hand, we sought to prepare a series
of analogs to explore the role of the double bonds, alkyl chain
length in the fatty acid unit and the structure of the amide head
group in the observed biological activity of 1. We prepared com-
pound 4 (Fig. 2) which only bears one unsaturation and a series
of chain shortened derivatives (5–9, Fig. 2). In all cases the analogs
were prepared in one step from the coupling of the corresponding
carboxylic acid with isobutyl amine (see SI for details).
(such as TNF-a) even though many members of this family have
demonstrated anti-inflammatory effects.2b This area has been
further complicated by the fact that fatty acid amides have
poorly understood and/or multiple mechanisms of action and are
known to elicit their anti-inflammatory effects via both
To evaluate the impact of these structural changes on the bio-
logical activity of 1, we compared each analog’s ability to inhibit
⇑
Corresponding authors.
E-mail addresses: smlaster@ncsu.edu (S.M. Laster), jgpierce@ncsu.edu
(J.G. Pierce).
TNF-a production by lipopolysaccharide (LPS)-stimulated RAW
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2015.06.024
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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Y. Moazami et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3091–3094
Figure 1. Structure of fatty acid amide 1 from Echinacea.
264.7 cells, a murine, macrophage-like cell line. Each compound
was tested at 100 M in combination with 10 ng/mL of LPS and
incubated for 18 h. TNF- concentrations in the culture
l
a
supernatants were measured after the incubation period using a
commercially available ELISA (eBioscience). The effects of each
compound on TNF-a production were compared to cells
stimulated only with LPS (R595, List Labs). Additionally, RAW
264.7 cells were treated with each compound in the absence of
LPS to determine if these molecules themselves could induce the
production of TNF-
TNF- significantly above levels from unstimulated cells (data not
shown).
As shown in Figure 3, compounds 4 and 5 both significantly
inhibited the production of TNF- , with levels of suppression sim-
ilar to that of 1 indicating that the double bonds along the alkyl
chain are not critical for inhibition of TNF- production.
Significant inhibition was lost with compounds 6–9 (Fig. 2) indicat-
ing that the length of the alkyl chain is critical for determining
inhibitory activity. An alkyl chain with at least 11 carbons is appar-
ently required for this activity, in the absence of any unsaturation.
We next sought to explore the impact of the amide head group
a. None of the compounds were found to induce
a
a
a
Figure 2. Analogs prepared to explore the impact of unsaturation and alkyl chain
length.
110
100
90
on the observed activity of
1 and simplified derivatives.
Accordingly, 10–17 were prepared (see SI for details) to evaluate
the impact of replacing the isobutyl amide with 2-methylbutyl
amide (10–11), benzyl amide (12–13), hexyl amide (14), thiazole
amide isosteres (15–16) and isobutyl amine (17) derivatives
(Fig. 4). This series of alkyl amide derivatives served to not only
evaluate the steric requirements of the amide head group, but also
to answer key questions regarding the amide functionality itself.
The addition of a single carbon to the isobutyl amide head
group in 10 and 11 did not impair their ability to significantly inhi-
80
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70
60
50
40
30
20
10
0
***
bit TNF-a production (Fig. 5). Replacement of the isobutyl amide
group with a benzyl ring in 12 and 13 reduced the overall level
of inhibition from 50.2 5.1% to 29.8 5.5% and 32.4 0.8%,
respectively, although both still produced significant levels of sup-
pression. Similarly, the addition of a 6-carbon alkyl chain on the
amide nitrogen in compound 14 also produced significant suppres-
sion. In contrast, the two thiazole-containing compounds 15 and
16 did not display significant TNF-
a
suppressive activity, highlight-
LPS
1
4
5
6
7
8
9
ing the importance of the amide functionality for the observed
activity. Compound 17, which lacks the carbonyl group present
in most of these compounds, completely inhibited the production
of TNF-a (99.8 0.2%); however, this activity likely arises from cell
death, since 17 displayed significant cytotoxic activity (see below).
Compound Number
Figure 3. TNF-
a production in the presence of alkyl chain analogs. Error bars
indicate means SEM from three independent experiments. One way ANOVA with
Tukey’s post hoc test was used to determine statistical significance. ***p <0.001.
GraphPad Prism software was used for statistical analyses.
Overall, we found that the isobutyl amide head group is not unique
for inhibiting TNF-
a production, although there do appear to be
The cytotoxic effects of each compound were tested to ensure
structural constraints in this region that limit inhibitory activity.
that inhibition of TNF-
a production was not due to cell death.
RAW 264.7 cells were treated with each compound at 100
l
M
and incubated for 18 h before supernatants were collected and
analyzed for lactate dehydrogenase (LDH) activity (Pierce LDH
Cytotoxicity Assay Kit, Thermo Scientific). LDH is a cytosolic
enzyme that is released from dead or dying cells. A lysis buffer
was used to determine the maximal LDH release while sponta-
neous background release was determined by treatment with
media and ethanol. While most compounds did not display signif-
icant cytotoxic effects, 10 and 17 did induce statistically significant
increases in cytotoxicity (Fig. 6). As mentioned above for amine 17,
the suppression of TNF-
a shown in Figure 5 was likely due to its
Scheme 1. Synthesis of fatty acid amide 1.

Y. Moazami et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3091–3094
3093
110
100
90
80
70
60
50
40
30
20
10
0
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**
V
1
4
5
6
7
8
9 10 11 12 13 14 15 16 17 L
Compound Number
Figure 6. Cytotoxicity of the alkylamide structural analogs. Error bars indicate
means SD, n = 2. **p <0.01, ***p <0.001. V, vehicle; L, lysis buffer. GraphPad Prism
software was used for statistical analyses.
In conclusion, the structural requirements of alkylamides to
inhibit production of pro-inflammatory cytokines appear to be
dependent on the alkyl chain length, and while there is structural
flexibility within the amide head group, certain chemical proper-
ties within this region appear to be required to avoid reducing inhi-
bitory effects. The double bonds in the alkyl chain of the natural
Figure 4. Analogs prepared to explore the impact of the amide head group.
product do not appear to be critical for suppression of TNF-a.
This observation is consistent with previous publications in which
additional alkylamides produced by Echinacea, as well as other
plant genera, which vary in the number and placement of unsatu-
ration along the alkyl chain, are also capable of suppressing cyto-
kine production.5,7 It is unlikely that these fatty acid amides are
exerting their activity selectively on LPS or the LPS-signaling
110
100
90
machinery since influenza A-induced production of TNF-
a is also
80
*
blocked. Interestingly, removal of the carbonyl group from the fully
saturated version of the natural alkylamide produced a molecule
with cytotoxic activity. Further experiments to develop an under-
standing of this effect and to further probe the SAR of this family
of alkylamides are underway and will be reported in due course.
**
**
70
60
50
40
30
20
10
0
**
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Acknowledgments
This work was partially funded by NC State’s Office of Research,
Innovation, and Economic Development, in partnership with the
Kenan Institute for Engineering, Technology and Science and the
Center for Comparative Medicine and Translational Research. JGP
gratefully acknowledges North Carolina State University for gener-
ous start-up support. Mass spectra were obtained at the Mass
Spectrometry Laboratory at NC State University.
***
LPS
1
10 11 12 13 14 15 16 17
Compound Number
Figure 5. TNF-
a production in the presence of head group analogs. Error bars
Supplementary data
indicate means SEM from three independent experiments. One way ANOVA with
Tukey’s post hoc test was used to determine statistical significance. ***p <0.001.
*p <0.05, **p <0.01, ***p <0.001. GraphPad Prism software was used for statistical
analyses.
Supplementary data (experimental details) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2015.06.024.
References and notes
significant cytotoxic effects. Other factors, including cell perme-
ability, could play a role in the varying activities of the studied
compounds and these aspects are under additional study.
1. (a) Matthias, A.; Penman, K. G.; Matovic, N. J.; Bone, K. M.; De Voss, J. J.;
Lehmann, R. P. Molecules 2005, 10, 1242; (b) Woelkart, K.; Linde, K.; Bauer, R.

3094
Y. Moazami et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3091–3094
Planta Med. 2008, 74, 633; (c) Todd, D. A.; Gulledge, T. V.; Britton, E. R.;
4. Raduner, S.; Majewska, A.; Chen, J. Z.; Xie, X. Q.; Hamon, J.; Faller, B.; Altmann, K.
H.; Gertsch, J. J. Biol. Chem. 2006, 281, 14192.
5. Cech, N. B.; Kandhi, V.; Davis, J. M.; Hamilton, A.; Eads, D.; Laster, S. M. Int.
Immunopharmacol. 2010, 10, 1268.
Oberhofer, M.; Leyte-Lugo, M.; Moody, A. N.; Shymanovich, T.; Grubbs, L. F.;
Juzumaite, M.; Graf, T. N.; Oberlies, N. H.; Faeth, S. H.; Laster, S. M.; Cech, N. B.
PLoS One 2015, 10, e0124276.
2. (a) Ruiu, S.; Anzani, N.; Orru, A.; Floris, C.; Caboni, P.; Maccioni, E.; Distinto, S.;
Alcaro, S.; Cottiglia, F. Bioorg. Med. Chem. 2013, 21, 7074; (b) Dang, H. T.; Kang, G.
J.; Yoo, E. S.; Hong, J.; Choi, J. S.; Kim, H. S.; Chung, H. Y.; Jung, J. H. Bioorg. Med.
Chem. 2011, 19, 1520; (c) Ezzili, C.; Otrubova, K.; Boger, D. L. Bioorg. Med. Chem.
Lett. 2010, 20, 5959; (d) Matthias, A.; Banbury, L.; Stevenson, L. M.; Bone, K. M.;
Leach, D. N.; Lehmann, R. P. Immunol. Invest. 2007, 36, 117.
3. (a) Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674; (b)
Matovic, N.; Matthias, A.; Gertsch, J.; Raduner, S.; Bone, K. M.; Lehmann, R. P.;
Devoss, J. J. Org. Biomol. Chem. 2007, 5, 169.
6. Matovic, N. J.; Hayes, P. Y.; Penman, K.; Lehmann, R. P.; De Voss, J. J. J. Org. Chem.
2011, 76, 4467.
7. (a) Sasagawa, M.; Cech, N. B.; Gray, D. E.; Elmer, G. W.; Wenner, C. A. Int.
Immunopharmacol. 2006, 6, 1214; (b) Spelman, K.; Iiams-Hauser, K.; Cech, N. B.;
Taylor, E. W.; Smirnoff, N.; Wenner, C. A. Int. Immunopharmacol. 2009, 9, 1260;
(c) Wu, L. C.; Fan, N. C.; Lin, M. H.; Chu, I. R.; Huang, S. J.; Hu, C. Y.; Han, S. Y. J.
Agric. Food Chem. 2008, 56, 2341.