Synthesis of Novel Long Chain Unsaturated Fatty Acid ...

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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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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.

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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.

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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.

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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)

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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.

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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).

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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

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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.

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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.