synthesis of novel long chain unsaturated fatty acid
TRANSCRIPT
University of Mississippi University of Mississippi
eGrove eGrove
Honors Theses Honors College (Sally McDonnell Barksdale Honors College)
Spring 4-30-2021
Synthesis of Novel Long Chain Unsaturated Fatty Acid Analogs of Synthesis of Novel Long Chain Unsaturated Fatty Acid Analogs of
Capsaicin Capsaicin
Eli Bettiga University of Mississippi
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Recommended Citation Recommended Citation Bettiga, Eli, "Synthesis of Novel Long Chain Unsaturated Fatty Acid Analogs of Capsaicin" (2021). Honors Theses. 1785. https://egrove.olemiss.edu/hon_thesis/1785
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SYNTHESIS OF NOVEL LONG CHAIN UNSATURATED FATTY ACID ANALOGS OF
CAPSAICIN
By
Eli Thomas Bettiga
A thesis submitted to the faculty of The University of Mississippi in partial fulfillment of the
requirements of the Sally McDonnell Barksdale Honors College.
Oxford, MS
May 2021
Approved By
______________________________
Advisor: Professor John M. Rimoldi
______________________________
Reader: Dr. Rama Gadepalli
______________________________
Reader: Professor Sudeshna Roy
ii
© 2021
Eli Thomas Bettiga
ALL RIGHTS RESERVED
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DEDICATION
This thesis is dedicated to my family. I couldn’t have done any of this without you.
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ACKNOWLEDGEMENTS
I would like to thank Dr. John M. Rimoldi for guiding me throughout my time at the
University of Mississippi. Dr. Rimoldi took me on as an undergraduate assistant in his lab
despite my lack of experience, and he challenged me to achieve goals I thought were
unattainable. He has gone out of his way to ensure my growth and success, and I am so thankful
for all he has done for me and my academic career. Moving forward in science and medicine, I
hope to be as gracious and kind as he has been to me.
I would also like to thank Dr. Rama S. Gadepalli for always working alongside me and
patiently advising me throughout my research. Dr. Gadepalli has been a source of endless
patience and knowledge for me as I found my way in the lab. He is a passionate scientist who has
inspired me to take pride in my work and academics. He has been instrumental to the completion
of my research and I am very grateful for the opportunity to learn and grow under his leadership.
I would also like to thank all of the past instructors in my life who have encouraged me to
never settle. You gave me all of the tools I needed to succeed, and I’m so grateful for your
efforts.
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ABSTRACT
ELI THOMAS BETTIGA: Synthesis of Novel Long Chain Unsaturated Fatty Acid Analogues of
Capsaicin
(Under the direction of John M. Rimoldi)
A number of key studies have shown that the natural product capsaicin displays in vitro
and in vivo antitumor effects against a variety of cancers, however, both the analgesic and
cancer-related therapeutic potential of capsaicin have been limited by its unpleasant pungent side
effects and modest potency. N-acyl vanillylamides (N-AVAMs) are a unique class of
compounds that are simple side chain modified capsaicin derivatives. Limited structure-activity
relationship (SAR) studies of N-AVAMs demonstrated that substitution of the capsaicin side-
chain with longer unsaturated fatty acid groups results in non-pungent analogs with improved
anti-invasive activity relative to capsaicin. In an effort to ultimately create and test new analogs
for in vitro and in vivo biological evaluation studies this project is centered on the synthesis of
rare and unique N-AVAMs composed of -linoleic acid (GLA, 10) and stearidonic acid (SDA,
11), rare -3 fatty acids that are reported to be found in high concentrations in the dietary
supplements Echium seed oil and Ahiflower oil. Each of these oils were evaluated indirectly for
GLA and SDA content by invoking a chemical capture reaction using the plant oils as stand-
alone reagents in a condensation reaction with vanillamine. The products obtained were purified
and analyzed using a combination of analytical techniques, including mass spectrometry and
NMR spectroscopy. Although the research was disrupted by COVID-19, the preliminary results
obtained from these studies suggest that natural product oils may be a valuable source of rare
polyunsaturated fatty acids to be used as building blocks in the synthesis of novel N-AVAMs.
vi
TABLE OF CONTENTS
COPYRIGHT ii
ACKNOWLEDGEMENTS iv
ABSTRACT v
TABLE OF CONTENTS vi
LIST OF FIGURES vii
LIST OF TABLES viii
LIST OF ABBREVIATIONS ix
INTRODUCTION
Capsaicin and its anticancer activity
Caveats of the analgesic and cancer related therapeutic potential of capsaicin
N-AVAM analogues of capsaicin
Specific Aims
The unsaturated fatty acids γ -linoleic acid (GLA) and stearidonic acid (SDA)
Purification of Fatty acids from natural product oils
1
2
3
4
5
7
8
RESULTS AND DISCUSSION
Discussion
Future Work
9
14
15
EXPERIMENTAL METHODS
Preparation of free fatty acids from Echium seed oil
Condensation reaction of Echium seed oil free fatty acids with vanillamine
Preparation of free fatty acids from Ahiflower seed oil
Condensation of Ahiflower oil free fatty acids with vanillylamine: Small scale
Condensation of Ahiflower oil fatty acids with vanillylamine: Scaled reaction
16
16
17
18
18
19
BIBLIOGRAPHY 21
APPENDIX 25
vii
LIST OF FIGURES
FIGURE 1 Capsaicin 1
FIGURE 2 Saturated and Unsaturated N-AVAMs 5
FIGURE 3 Target N-AVAMs derived from γ -linoleic acid and stearidonic acid 6
FIGURE 4 Chemical capture reaction of fatty acids from plant oils 6
FIGURE 5 Commercial sources of Echium seed oil and Ahiflower oil used in this
study
8
FIGURE 6 13C NMR resonances in C=C region of spectrum 11
FIGURE 7 Purification flowchart of N-AVAMs from Ahiflower Oil 12
FIGURE 8 Experimental and predicted ESI+ MS spectrum for the N-AVAM
derived from Ahiflower SDA (Fraction D8-2)
13
FIGURE 9 Partial 1H NMR spectrum of Fraction D8-2 in CDCl3 13
FIGURE 10 Partial 13C-NMR spectrum of Fraction D8-2 in CDCl3-Expaned C=C
olefinic region
14
FIGURE 11 Stearidonic acid amide 14
viii
LIST OF TABLES
TABLE 1 Percent fatty acid composition of plant oils reported in two
supplements
8
ix
LIST OF ABBREVIATIONS
PHN Postherpetic neuralgia
DPN Diabetic peripheral neuropathy
TRPV Transient receptor potential vanilloid
SAR Structure activity relationship
EMT Epithelial-mesenchymal transition
ROS Reactive oxygen species
N-AVAM N-acyl vanillylamide
SCLC Small cell lung cancer
GLA γ -linoleic acid
SDA Stearidonic acid
LCFA Long chain fatty acid
NMR Nuclear magnetic resonance spectroscopy
HPLC High-performance liquid chromatography
TBTU 2-(1H-Benzotriazole-1-yl)-1,2,3,3,-tetramethyl-uronium
tetrafluoroborate
TLC Thin layer chromatography
LA Linoleic acid
ALA α-Linoleic acid
TEA Triethylamine
EtOAc Ethyl Acetate
1
INTRODUCTION
The natural product capsaicin (1) the active pungent component from hot chili peppers is
used in many over-the-counter topical formulations to treat pain and inflammation (Figure 1).
Qutenza® (capsaicin) 8% topical system was approved by the FDA in 2009 and indicated for the
management of neuropathic pain associated with postherpetic neuralgia. In 2020, an expanded
indication was approved use in adults for the treatment of neuropathic pain associated with
postherpetic neuralgia (PHN) and for neuropathic pain associated with diabetic peripheral
neuropathy (DPN) of the feet. Capsaicin analgesic activity is mediated by the transient receptor
potential vanilloid (TRPV) receptor superfamily of ion-channel receptors on target cells,
specifically the TRPV1 receptor, of which capsaicin is a potent agonist (Benítez-Angeles et al.,
2020). When capsaicin binds to this receptor (Elokely et al., 2016) it results in a cascade of
cellular signaling events that leads to the downregulation of the neuropeptide Substance P,
involved in the inflammatory response through interaction with nociceptive nerve endings and
cytokines. This process essentially turns off the affected nociceptive fibers, leading to a decrease
in the sensation of pain.
Figure 1: Capsaicin (1)
2
Structure Activity Relationship (SAR) studies of capsaicin have been conducted in an
attempt to identify key structural motifs responsible for its TRPV receptor activity and to create
novel analogs that display improved potency and efficacy (Thomas et al., 2011). Capsaicin
comprises two structural regions; an aromatic vanillin amine core and a long chain alkyl
hydrophobic side chain linked by an amide group. Synthetic and natural product derivatives
have been evaluated to select for greater analgesic activities while decreasing off-target effects
(Wrigglesworth et. al, 1996; Suh et al., 2005; Huang et al., 2013).
Capsaicin and its anticancer activity
Numerous studies have shown that capsaicin and its structurally similar analogs display
potent in vitro and in vivo antitumor effects against human cancers; these findings have been
extensively reviewed (Freidman et al., 2018; Richbart et al., 2021). The anticancer activity of
capsaicin has been confirmed in a number of human cancers including breast, lung, prostate,
gastric, renal, and hepatocellular carcinoma (Friedman et al, 2019) and is mediated by TRPV-
dependent and TRPV-independent mechanisms. Capsaicin also modulates tumor angiogenesis
and metastasis; two key processes in cancer progression (Min et al., 2004). To much surprise,
capsaicin’s mechanism to downregulate tumor angiogenesis does not involve the TRPV1
receptor that is responsible for its analgesic effects. The antiproliferative properties of capsaicin
is derived from its ability to block multiple survival pathways that are vital to cancer progression
including epithelial-mesenchymal transition (EMT), invasion and metastasis (Caprodossi et al,
2011)
Capsaicin recruits numerous growth inhibitory signaling pathways, and include but are
not limited to activation of calpain family of apoptotic proteases, disruption of mitochondrial
3
respiration, regulation of intracellular calcium, induction of apoptosis and autophagy, generation
of reactive oxygen species (ROS), and the inhibition of transcription factors like p53, STAT3
and NF-κB (Zhang et al., 2020). Additionally, capsaicin can inhibit cancer progression through
its ability to downregulate tumor angiogenesis, its primary antitumor mechanism (Chakraborty et
al., 2014) and through its interference of pathways like epithelial-mesenchymal transition
(EMT), invasion and metastasis (Amantini et al, 2016). A number of studies have shown
capsaicin to be an effective chemo-and radio-sensitizer in cell lines and animal models,
reinforcing established chemotherapy drugs and radiotherapy respectively (Zhang et al., 2020).
Caveats of the analgesic and cancer-related therapeutic potential of capsaicin
The analgesic and anticancer therapeutic potential of capsaicin have been limited by its
physicochemical properties including its poor aqueous solubility and oral bioavailability, and
also because of its unpleasant pungency. For example, clinical trials exploring the pain-relieving
activity of orally administered capsaicin resulted in patients discontinuing its use due to its strong
pungency and other adverse effects including hyperalgesia, nausea, vomiting, abdominal pain,
and stomach cramps (Basith et al., 2016; Drewes et al., 2003). Clinical trials investigating the
analgesic activity of orally administered capsaicin for visceral pain have shown that such
disagreeable side effects have led to some patients discontinuing its use (Hammer 2006). The
therapeutic potential of capsaicin as an anticancer drug also has liabilities. It has been shown to
promote the growth of skin cancer, stomach cancer, colon cancer and gastric cancers (Bode &
Dong 2011). Indeed, the presence of adverse side effects has led to increased interest in research
focused on the discovery and design of novel synthetic capsaicin analogs devoid of these effects.
4
N-AVAM analogues of capsaicin
The compounds that belong to the N-acyl vanillylamide (N-AVAM) family of capsaicin
mimetics represent some of the most intriguing drug leads reported. Specifically, the N-AVAM
analogues have been studied for their functional activity, specificity and selectivity,
pharmacokinetics, and bioavailability and represent compounds with diverse fatty acid side
chains. Several analogs display greater analgesic effects than capsaicin itself, although the
antineoplastic activity of N-AVAM capsaicin analogs has only been reported in a few studies.
The anticancer activity of N-AVAM capsaicin analogs is known to be mediated via multiple
signaling networks. For example, in human breast epithelial cells and in small cell lung cancer
cells (SCLC), capsaicin’s anticancer activity is directly associated with its ability to change pro-
apoptotic calpain proteases’ functional activity. Specifically, in human SCLCs, it has been
shown that the growth suppressive activity of N-AVAM capsaicin analogs is associated with the
activation states of calpain 1 and calpain 2 (Friedman et al., 2018).
Limited structure-activity relationship (SAR) studies of N-AVAM demonstrated that
substitution of the capsaicin side-chain with longer unsaturated fatty acid groups results in non-
pungent analogs with improved anti-invasive activity relative to capsaicin in a panel of human
small cell lung cancer cell lines. Conversely, substitution of the acyl side chain of capsaicin
with saturated long-chain lipophilic groups resulted in compounds devoid of activity (see Figure
2 for structures). For example, potent activation of the TRPV1 receptor was achieved with a
series of C20 and C18 unsaturated N-AVAMs, while the saturated C18 analog palvanil (4) was
inactive (Melck et al., 1999). This prevailing trend has been documented with N-AVAM analog
evaluation in cancer cell lines. Olvanil (6) and arvanil (9), which contain unsaturated oleic
(C18:1) and arachidonic acid (C20:4) acyl groups respectively, retain a capsaicin-like anti-
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invasive activity (Hurley et al., 2017). Other longer chain capsaicin analogs synthesized by Dr.
Rama Gadepalli and evaluated by our collaborator Dr. Piyali Dasgupta include those containing
a saturated fatty acid group [stevanil (5)] and unsaturated fatty acid groups [livanil (7) and
linvanil (8)] (Dasgupta, unpublished data). Although incomplete, studies to date suggest that
growth-inhibition of these N-AVAM capsaicin analogs are directly proportional to both the chain
length of the fatty acyl group and the number of double bonds present within this fatty acid side
chain. (Richbart et al., 2021)
Figure 2: Saturated (left panel) and Unsaturated (right panel) N-acyl vanillamides (N-AVAMs)
Specific Aims
In an effort to ultimately create and test new analogs for in vitro and in vivo biological
evaluation studies (with collaboration at Marshall University, Dr. Piyali Dasgupta), this thesis
project is centered on the synthesis of rare and unique N-AVAMs containing long chain
unsaturated fatty acid groups. The target compounds of interest are composed of -linoleic acid
(GLA, 10) and stearidonic acid (SDA, 11), rare -3 fatty acids that are reported to be found in
high concentrations in the dietary supplements Echium seed oil and Ahiflower oil (Figure 3).
6
Figure 3: Target N-AVAMs derived from -linoleic acid and stearidonic acid.
These plant oils represent a cost-effective source to obtain unique long chain fatty acids
(LCFAs) to be used as starting materials in the synthesis of novel capsaicin derivatives (Kobata
et al, 2014). Each of these oils will be evaluated indirectly for GLA and SDA content by
invoking a chemical capture reaction using the plant oils as stand-alone reagents in a
condensation reaction (Figure 4). Each of the fatty acids in the source oils will be reacted with
vanillamine producing five distinct fatty acid amides. The amides will be purified and analyzed
using a combination of analytical techniques, including mass spectrometry and NMR
spectroscopy.
Figure 4. Chemical capture reaction of fatty acids from plant oils.
7
The Unsaturated fatty acids γ -linoleic acid (GLA) and stearidonic acid (SDA)
Stearidonic acid [SDA;(C18:4)] is a dietary long-chain omega-3 polyunsaturated fatty
acid that is found in small quantities in seafood, fish oil, and in larger concentrations from a few
select natural product seed oils (Whelan 2009). SDA dietary intake has been linked to reductions
in cardiovascular disease, inflammation, and cancer. γ -Linoleic acid (GLA) is an endogenous
and dietary fatty acid found in vegetable oils with anti-inflammatory profiles (Sergeant et al.,
2016). While GLA is commercially available and relatively inexpensive ($1/1 milligram), SDA
is limited by availability and price. Only three commercial US suppliers offer SDA with an
average cost of $100/1 milligram. Echium seed oil (Echium plantagineum L.) represents a
practical source of SDA, and is available to the public as a beauty product containing five
polyunsaturated fatty acids (Miquel 2008). Ahiflower oil is also a good source oil for SDA and is
available to the public as a dietary supplement. It contains five polyunsaturated fatty acids and is
reported to contain greater quantities of SDA when compared to Echium seed oil (Table 1). Not
only is Ahiflower oil’s SDA percentage greater than Echium seed oil, it also is reported to
contain the highest SDA quantities derived from natural product origin. Based on the percent
fatty acid composition and price of Ahiflower oil capsules ($30/66 g oil), this oil represents an
economical source of large quantities of SDA (11 g) and GLA (2.6 g) from a 66 g bottle of
softgel capsules.
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Fatty Acid Echium Seed Oil
(H&B Oils Center)
Ahiflower Oil
Softgels
(Clean Machine)
Capsaicin
derivative
MWT
oleic acid 16% 6% Olvanil (6) known 417.3
linoleic acid 19% 9% Livanil (7) known 415.3
α-linoleic acid 30% 42% Linvanil (8) known 413.3
γ-linoleic acid 10% 4% New 413.3
stearidonic acid 13% 17% New 411.3
Table 1: Percent fatty acid composition of plant oils reported in two supplements.
Figure 5: Commercial sources of Echium seed oil (Health and Beauty Oils Center) and
Ahiflower oil (Clean Machine) used in this study.
Purification of fatty acids from natural product oils
In order to access fatty acids like SDA and GLA from plant oils, we have considered
several methods based on published accounts of long chain unsaturated fatty acid analysis,
purification and isolation. Fatty acid mixtures can be purified in advance using preparative
reverse-phase HPLC, low-temperature crystallization, fractional distillation, or esterification
followed by chromatography and subsequently used in chemical reactions. Each of these
methods suffer from drawbacks related to the inherent structural similarities of long chain
unsaturated fatty acids, and the time- and labor-intensive protocols involved. An alternative
9
approach is to conduct a reaction with the plant oil directly invoking a chemical capture reaction
using vanillamine (4-hydroxy-3-methoxybenzylamine), which is the starting material required to
synthesize capsaicin analogs (Richbart et al., 2021). In this fashion, the fatty acids in the plant oil
will be converted to five distinct fatty acid amides.
The benefits of the chemical capture approach with plant oils are that the resulting
products represent stable, UV-active, and chromatographically friendly compounds that can be
easily separated, purified, and analyzed in a preparative fashion using standard methods of
normal-phase silica gel flash column chromatography (see Figure 4).
The reaction between vanillamine and plant oil will be conducted using a standard
carboxylic acid-amine condensation reaction to afford a mixture of fatty acid amides (and
represent the final capsaicin analogs containing long chain unsaturated fatty acids) in a single
step. The five fatty acid amide products derived from the reaction will be purified (silica gel,
ethyl acetate/hexanes mobile phase) and analyzed using mass spectrometry for confirmation of
molecular weight and 1H- and 13C-NMR for confirmation of structure.
Results and Discussion
The first plant oil investigated was Echium seed oil (Health and Beauty Oils Center,
Westchester, Illinois USA), which is reported to contain a mixture of five unsaturated fatty acids
(Table 1). Prior to exploring this reaction, saponification of the commercial oil was necessary to
convert triacyl- and diacyglycerides to free fatty acids (Baik et al., 2015). Echium oil was
treated with an aqueous solution of sodium hydroxide in ethanol and subsequently acidified and
extracted into hexanes to yield the free fatty acid mixture.
10
Initial condensation reactions with 2-(1H-Benzotriazole-1-yl)-1,2,3,3,-tetramethyl-
uronium tetrafluoroborate (TBTU) and vanillamine afforded the crude product; the reaction
course was monitored using thin layer chromatography (TLC) and after the reaction was
quenched and product extracted, the crude material was initially purified on silica gel using flash
chromatographic methods and a mobile phase consisting of ethyl acetate/hexanes to afford
purified fractions and those molecular weights that corresponded to the anticipated products
(MWT range 412-420 Daltons for M+H+ ions or 434-442 for M+Na+ ions) were pursued.
Further purification using silica gel flash chromatography yielded sub fractions that were
analyzed by MS and NMR. The m/z product ion observed using electrospray ionization
corresponded to a M+Na+ ion peak of m/z = 436. The 1H NMR spectrum was indicative of new
olefinic protons which resonate at 5.3 and 6.0 ppm. 13C NMR (1H- decoupled) analysis
confirmed the presence of 4 additional unsaturated carbon atoms in addition to the vanillamine
aromatic carbons and the amide carbonyl group (Figure 6). Although these carbon signals
overlap, 13C –DEPT NMR analysis was performed to identify the C=C resonances ( 127.89,
128.05, 130.00, 130.22). The product isolated was suspected to correspond to Livanil (7),
derived from the condensation of vanillamine with linoleic acid (a major fatty acid in Echium
oil). This was further confirmed by comparing the proton and carbon spectra with a synthetic
sample of Linvanil (7) (prepared in our lab previously by Dr. Rama Gadepalli by condensation of
vanillamine with linoleic acid)
11
Figure 6: 13C NMR resonances in C=C region of spectrum obtained from Echium seed
oil (Left panel) and from sample prepared from vanillamine and linoleic acid (Right panel)
Numerous attempts were made to identify additonal N-AVAM products from Echium oil but
purification of the constituents in the mixture proved to be difficult. We next focused our
attention on Ahiflower oil, and created a purification protocol that was more amenable to
fractionation of structurally related compounds. Twenty softgel capsules of Ahiflower oil was
carefully cut open and the oil (about 10 g) was collected into a clean round bottom flask. A
modified saponification method was used, that included aqueous sodium hydroxide and ethanol
as used in the Echium oil hydrolysis, but was heated to reflux to ensure complete hydrolysis of
the acyl-glycerides (Delmonte et al., 2018). The purification protocol was changed to include an
automated flash chromatography step using a Teledyne-Isco Combiflash system to purify
constituents based on mass-directed analysis. Figure 7 depicts the separation protocols
employed.
12
Figure 7: Purification Flowchart of N-AVAMs from Ahiflower oil.
The product mixture resulting from the vanillamine chemistry post-workup was fractionated
using the Combiflash system and from this Fractions D1-12 were collected and mass was
directed to identify peaks corresponding to m/z 411 [MH+ 412], the molecular mass
corresponding to the N-AVAM comprising stearidonic acid (compound 11). Of these fractions,
D8 contained material that was enriched in compounds corresponding to 11. Fraction D8 was
further purified using standard silica gel flash chromatography and a mobile phase consisting of
ethyl acetate and hexanes to afford sub-fractions D8-(1-14), of which D8-2 constituted an
enriched fraction that contained a constituent with a m/z =434 [M+Na+]-matching the expected
molecular mass of the N-AVAM derived from stearidonic acid, in addition to higher mass peaks
of m/z = 438 and 440 [M+Na+] (Figure 8).
13
Figure 8. Experimental (top panel) and Predicted (bottom panel) ESI+ MS spectrum (sodium
adducts) for the N-AVAM derived from Ahiflower stearidonic acid (Fraction D8-2).
1H NMR analysis of the enriched product fraction revealed a complex spectrum (Figure 9,
partial NMR spectrum). The protons on the structure dervied from the vanillamine group are
diagnostic for quantifying peak areas and include aromatic protons [ 6.6-6.8 ppm (3H)], the
methylene CH2 resonance [ 4.25 ppm (2H)] and the methoxy protons [ 3.75 ppm (3H)]. The
olefinic signals appear as muliplets centered at approximately 5.3 ppm (< 2H) and 5.7 ppm (<
6H). 13C NMR experiments were performed to analyze the number of carbon olefinic signals,
which was equally complex, due to the presence of at least one additional N-AVAM in the
sample.
Figure 9. Partial 1H NMR spectrum of Fraction D8-2 in CDCl3
14
Figure 10. Partial 13C-NMR spectrum of Fraction D8-2 in CDCl3 (Left panet) and Expaned C=C
olefinic region (Right panel)
Analysis of the carbon spectrum in the C=C olefinic region clearly shows one major compound
and one minor compound. Although carbon NMR is not quantitative as per area under the peak
integration, the combination of mass analysis, proton NMR, and carbon NMR analysis is
suggestive of the presence of one of the target molecules, stearidonic acid amide (11).
Discussion
The results from the Echium oil experiments were disappointing, and did not result in
identification or separation/purification of N-AVAMs resulting from SDA or GLA condensation.
Potential pitfalls that led to the failure with the Echium oil experiments point to front-end
problems, namely, the incomplete hydrolysis of the acyl-glycerides to free fatty acids. This is the
most logical issue that explains the lack of identifying other N-AVAMs in the sample. Another
difficulty that was anticipated and observed by experiment involved purification. The fatty acid
derivatives of vanillamine that were synthesized from the condensation reaction are structurally
similar, and differ only in the unsaturation number and/or double bond location. Nonetheless,
15
this thesis project represents a first step in using inexpensive and commercially available oils as
reagents to produce high-value compounds, of particular interest in the quest to create novel
capsaicin analogs as potentially useful therapeutic drug leads.
Future Work
Anticipating this research will be continued by others, the obvious goal that remains to be
met is to improve the methods for purification of minor constituents from Echium or Ahiflower
oils, including GLA and SDA. Once this has been accomplished, the vanillamine chemical
capture of fatty acid mixtures and subsequent purification/analysis could be extended to other
commercially available and inexpensive dietary seed oils containing unique long unsaturated
fatty acids, like the seeds of the Chinese violet cress (Orychophragmus violaceus) which is
known to contain unusual C24 hydroxylated fatty acids. (Li et al., 2018). The intended outcome
is to create new capsaicin analogs which display improved anticancer bioactivity, a greater
therapeutic index, and diminished adverse and off-target effects.
16
Experimental Methods
NMR spectral data 1H and 13C, were recorded in deuterated chloroform on either a Bruker 400 or 500 MHz
spectrometer. Chemical shifts (δ) were reported relative to tetramethylsilane or solvent as an internal
standard. Spin multiplicities were given as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet),
dd (double doublet), or m (multiplet). Coupling constants (J) are reported in Hertz (Hz). Experiments
requiring anhydrous conditions were performed using glassware that was flame-dried under vacuum and
purged with argon. Transfer of reagents was performed by syringes equipped with stainless steel needles
that were flushed with argon and kept under positive argon pressure until use. Mass spectral analysis was
performed using a Waters ZQ single quadrupole instrument and electrospray ionization in either positive
or negative mode. Samples were analyzed by direct injection. The automated purification and mass-directed
analysis was performed using a Teledyne-Isco Combiflash system. Thin layer chromatography (silica gel
plates, UV detection) were used to monitor reaction progress. Chemicals, reagents, and solvents were
purchased from Fisher Scientific or Sigma-Aldrich.
Preparation of free fatty acids from Echium seed oil
Echium seed oil (10 g), which is claimed to be a mixture of 16% Oleic acid (18:1), 19%
Linoleic acid (LA, 18:2), 10% γ-linolenic acid (GLA, 18:3), 30% α-linolenic acid (ALA, 18:3)
and 13% stearidonic acid (SDA, 18:4) in the form of diacyl- and triacyl-glycerol esters was
treated with a solution of sodium hydroxide (48 g) in distilled water (10 mL) and ethanol (30
mL). The mixture stirred for 1 hr. Water (20 mL) was added to the saponified mixture and the
aqueous layer containing the organic acids was acidified by adding an aqueous solution of 6 N
HCl and adjusted to pH 1. The upper layer containing the fatty acid mixture was extracted into n-
hexane and washed twice with distilled water. The n-hexane layer containing the fatty acid
mixture was then dried over anhydrous sodium sulfate. Then n-hexane was removed from the
17
fatty acid by evaporation in a rotary evaporator. The residual n-hexane in the remaining fatty
acid crude product, (A) was removed by placing the sample under high vacuum and the resulted
colorless viscous oily residue (Yield: 8.65 g) converted to white viscous wax on cooling.
Condensation reaction of Echium seed oil free fatty acids with vanillamine
2-(1H-Benzotriazole-1-yl)-1,2,3,3,-tetramethyluronium tetrafluoroborate (TBTU, 1 equiv; 1.41
mmol; 452 mg) was added to Echium seed oil free fatty acids (400 mg) with triethylamine (TEA,
2 equiv; 2.82 mmol; 285.4 mg; 400 mL) in EtOAc (30 mL). After stirring for 1 h at room
temperature, 4-hydroxy3-methoxy-benzylamine hydrochloride (vanillamine hydrochloride) (2
equiv; 2.82 mmol; 535 mg) was added and the reaction mixture was stirred overnight. The mixture
was washed with water then with brine, dried over anhydrous sodium sulfate, filtered and
concentrated to give crude product (B), a pale yellow oily residue. This crude reaction product
which was considered to be a mixture of more than one N-AVAM was subjected to purification
using a preparative column packed with silica gel to isolate enriched fractions using a mobile phase
consisting of 50% ethyl acetate/50% hexanes for elution. Purification afforded one enriched
fraction that was obtained as a colorless oil. 1H NMR (500 MHz, Chloroform-d) δ 6.90 – 6.68 (m,
3H), 6.15 – 5.89 (m, 1H), 5.55 – 5.20 (m, 3H), 4.33 (d, J = 5.6 Hz, 2H), 3.85 (s, 3H), 2.77 (d, J =
6.6 Hz, 1H), 2.20 (t, J = 7.6 Hz, 2H), 2.04 (d, J = 4.9 Hz, 3H), 1.64 (t, J = 7.4 Hz, 2H), 1.45 – 1.20
(m, 15H), 0.94 – 0.81 (m, 3H). 13C NMR (126 MHz, CDCl3) δ 173.09, 146.80, 145.18, 130.27,
130.21, 130.00, 128.05, 127.89, 120.69, 114.47, 110.75, 77.37, 77.11, 76.86, 55.87, 43.50, 36.77,
31.52, 29.71, 29.61, 29.34, 29.32, 29.30, 29.28, 29.16, 27.20, 25.81, 25.63, 22.58, 14.08.
18
Preparation of free fatty acids from Ahiflower oil
Twenty softgel capsules of Ahiflower oil was carefully cut open and the oil (about 10 g) was
collected into a clean round bottom flask comprising a solution of sodium hydroxide (48 g) in
distilled water (10 mL) and ethanol (30 mL). The mixture was refluxed with stirring for 1 hr.
Water (20 mL) was added to the saponified mixture and the aqueous layer containing the free
fatty acids was acidified by adding an aqueous solution of 6 N HCl and adjusted to pH 1 to
release the free fatty acids. Litmus paper was used to measure the pH of the crude product
intermittently throughout the titration. After the pH was brought to 1, the contents were taken
into a separation funnel and extracted with ethyl acetate (100 ml x 2) and the organic portions
were combined washed twice with water and then with brine, dried the organic portion with
anhydrous sodium sulfate, filtered and evaporated under reduced pressure to yield the fatty acid
mixture. Mass analysis of this fatty acid mixture concentrate after saponification revealed the
following data: MS in MeOH (ESI+): m/z [MH+] 276.45; [MNa+] 299.36 [MNa+] 302.39
[MNa+]; 304.48 [MNa+].
Condensation of Ahiflower oil free fatty acids with vanillylamine: Small scale reaction
2-(1H-Benzotriazole-1-yl)-1,2,3,3,-tetramethyluronium tetrafluoroborate (TBTU, 1 equiv; 110
mg) was added to a (100 mg) mixture of Ahiflower oil fatty acids and triethylamine (TEA, 2
equiv; 135 mg) in EtOAc (11 mL). After stirring for 1 h at room temperature, 4-hydroxy3-
methoxy-benzylamine hydrochloride (vanillamine hydrochloride) (2 equiv; 0.712 mmol; 135
mg) was added and the reaction mixture was stirred overnight. The mixture was washed with
water (2x 12 mL), then with brine and dried over anhydrous sodium sulfate, filtered and
concentrated to give crude product as a pale yellow oily residue.
19
Condensation of Ahiflower oil fatty acids with vanillylamine: Scaled reaction
The reaction was repeated by reacting 2-(1H-benzotriazole-1-yl)-1,2,3,3,-
tetramethyluronium tetrafluoroborate (TBTU, 3 equiv; 348 mg) with mixture of Ahiflower oil
fatty acids (320 mg) and triethylamine (TEA, 3 equiv; 400 mg) in EtOAc (33 mL). After stirring
for 1 h at room temperature, 4-hydroxy3-methoxy-benzylamine hydrochloride (vanillamine
hydrochloride) (3 equiv; 405.5 mg) was added and the reaction mixture was stirred overnight.
The mixture was washed with water (2x 12 mL), then with brine and dried over anhydrous
sodium sulfate, filtered and concentrated to give crude product amides as a pale yellow oily
residue. The crude material was subjected to automated column purification using the
Combiflash/ Teledyne-Isco flash chromatography system resulting in isolated fractions depicted
as D1 to D12. The D8 fraction was considered to be the major fraction (310 mg) containing the
N-AVAMs. This D8 fraction was further subjected to meticulous gradient flash chromatography
purification (ethyl acetate/hexanes mobile phase gradient). to yield fourteen fractions, D8-1 to
D8-14 that contained closely overlapping constituents when monitored by TLC. The fraction
D8-2 was analyzed.
Mass Analysis : ESI+MS: m/z 434.30 [M+Na] (calculated m/z: 434.27)
NMR analysis: 1H NMR (500 MHz, CDCl3) δ 6.86 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 1.9 Hz,
1H), 6.76 (d, J = 1.9 Hz, 1H), 5.93 (d, J = 28.9 Hz, 2H), 5.37 (ddd, J = 6.7, 3.3, 2.0 Hz, 5H), 4.34
(d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 2.82 (td, J = 5.6, 1.8 Hz, 3H), 2.20 (dd, J = 8.5, 6.8 Hz, 2H),
2.07 (dt, J = 11.7, 4.1 Hz, 4H), 1.75 – 1.59 (m, 2H), 1.40 – 1.21 (m, 9H), 1.03 – 0.81 (m, 3H).
13C NMR (126 MHz, CDCl3) δ 173.05, 146.77, 145.16, 131.96, 130.28, 130.23, 128.29, 128.23,
20
127.74, 127.10, 120.73, 114.44, 110.73, 77.35, 77.10, 76.84, 55.90, 43.52, 36.80, 29.59, 29.30,
29.28, 29.15, 27.21, 25.80, 25.63, 25.54, 20.56, 14.30.
21
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25
APPENDIX
26
1H and 13C of major N-AVAM isolated from reaction of Echium seed oil and vanillamine:
corresponds to Livanil (7)
27
13C DEPT NMR of major N-AVAM isolated from reaction of Echium seed oil and
vanillamine: corresponds to Livanil (7)
28
1H and 13C NMR spectra of Livanil (7)-synthetic standard produced from reaction of
vanillamine and linoleic acid (by Rama Gadepalli)
29
1H and 13C NMR spectra of Fraction D8-2: produced from reaction of Ahiflower oil fatty
acids and vanillamine.