One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

Article abstract of DOI:10.1016/j.bmcl.2014.10.084

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.

Full text of DOI:10.1016/j.bmcl.2014.10.084

Accepted Manuscript
One-pot Synthesis of Polyunsaturated Fatty Acid Amides with Anti-prolifera-
tive Properties
Hugo Tremblay, Catherine St-Georges, Marc-André Legault, Caroline Morin,
Samuel Fortin, Eric Marsault
PII:
S0960-894X(14)01155-X
http://dx.doi.org/10.1016/j.bmcl.2014.10.084
BMCL 22139
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 August 2014
24 October 2014
27 October 2014
Please cite this article as: Tremblay, H., St-Georges, C., Legault, M-A., Morin, C., Fortin, S., Marsault, E., One-pot
Synthesis of Polyunsaturated Fatty Acid Amides with Anti-proliferative Properties, Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.10.084
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
One-pot Synthesis of Polyunsaturated Fatty Acid Amides with Anti-proliferative Properties
Hugo Tremblaya, Catherine St-Georgesa, Marc-André Legaulta, Caroline Morinb, Samuel Fortinb and Eric Marsaulta
     
  
ARTICLE INFO
ABSTRACT
A one-
.
Received
-3 fatty acid ethyl esters to
amides and mono- or diacylglycerols was investigated via the use of a polymer-
supported lipase. The method was used to synthesize a library of fatty acid
monoglyceryl esters and amides. These new derivatives were found to have potent
growth inhibition effects against A549 lung cancer cells.
Revised
Accepted
2009 Elsevier Ltd. All rights reserved.
Available online
. polyunsaturated fatty acid amides, transamidation, environmentally
..
Keyword_1
friendly synthesis, growth inhibition
Keyword_2Keu
Keyword_3
Keyword_4
Keyword_5
11
-3 polyunsaturated fatty acids
class 3 organic solvents10,
and minimal waste generation.
(PUFA) have recently been associated with a number of
beneficial health effects, such as cardiovascular and cerebral
health,1 and shown some potential against asthma and2 lipid
malabsorption.3 They also possess anti-inflammatory,4 anti-
proliferative and antioxidant5 properties which may be beneficial
Moreover, it should be noted that polyunsaturated fatty acids are
prone to double bond migration or degradation when subjected to
harsh conditions.12 Reported PUFA amide derivatives have been
prepared by standard peptide coupling reactions from the
corresponding acids and amines.9 In order to avoid additional
reagents such as base and peptide coupling reagents, we resorted
to direct transamidation from monoethyl esters, which are
directly available in large quantities and cheaper.
7
against cancer.6, Their metabolism is well documented with
minimal side effects,8 and their presence in fish oil extracts has
been shown to be beneficial as dietary intake. We have recently
demonstrated the anti-proliferative properties of MAG-DHA and
MAG-EPA against colon and lung cancer cell lines.6, 7 In order to
better understand the structure-activity relationship (SAR) of this
compound class and improve its chemical and metabolic stability
compared to native monoacylglycerol esters, a reliable synthetic
method allowing rapid analog generation was required. Our first
goal was to generate fatty acid amide analogs, with the
expectation that they may be more stable compared to their ester
counterparts (Fig. 1). Fatty acid amides may additionally possess
beneficial anti-inflammatory properties.9
After careful review of existing methods corresponding to the
above requirements13-19
,
we retained two approaches:
organocatalysis using triazabicyclodecene (TBD), and enzyme-
catalyzed transamidation with lipases.
<Insert Figure 1>
Most reported examples of transesterification explored the
transformation of triacylglycerol esters to simpler esters such as
methyl esters as the final product, using the simpler alcohol as
the reaction solvent.1 This approach turned out to be impractical
for the transesterification from ethyl to glyceryl esters or amides,
-3 fatty acid ethyl ester are usually
non miscible, (2) the high viscosity of glycerol or its derivatives
precluded efficient stirring of the reaction mixture, and (3)
glyceryl amines being more expensive than glycerol, their use as
a solvent would be prohibitive. Furthermore, the method had to
We thus embarked on the synthesis of a small library of fatty
acid amides of three important polyunsaturated fatty acids: DHA,
EPA and DPA. From the onset, we opted for mild,
environmentally friendly conditions to effect
these
transformations. This includes mild reaction conditions, the use
of enzymatic or organic catalysis, as well as the preference for

be mild enough to preserve the unconjugated 1,4--diene units
occur to yield the terminal ester as an intermediate, however in
those instances no trace of terminally substituted monoester was
observed by proton NMR and no other isobaric product was
present as measured by LC-MS. Reaction with 1,3-
diaminopropan-2-ol provided a mixture of mono amide 2 (48%)
and diamide 3 (38%), which were easily separated by flash
chromatography. Most derivatives reacted in the same way with
similar yields and ease of purification. Exceptions to this
observation were the amino acids producing amides 11, 14 and
20, which gave lower yields and were more difficult to isolate. In
the case of N,N-dimethylaminopropanol, only the alcohol reacted
to give ester 27 in low yield. Finally, in 28 the methylamide was
obtained preferentially but in low yield, owing to difficult
purification.
-3-PUFA.
Synthesis of glyceryl esters and amides
First attempts of transesterification with triazabicyclodecene,
which may have been later transferred onto solid support, were
not satisfactory.20 Reaction optimization along various
parameters, including solvent (neat, THF, DMSO, pyridine,
water, acetonitrile or mixtures), temperature (0 100oC), reagent
and catalyst (5-20 mol%) did not produce clean mixtures. The
reaction was generally very slow even under the best conditions
(4 days for 50% conversion in DMSO at 100oC). Traces of water
-3-
fatty acid as a hydrolysis product. Decomposition and untractable
mixtures were frequently observed.
We then turned our attention to lipase-mediated
transesterification of DHA ethyl ester to monoacylglycerol as a
test reaction. After initial screen we selected two lipases:
Lipozyme® and Novozyme 435®, both immobilized on
polystyrene beads. After 24 h reaction in glycerol, the reaction
with Lipozyme® did not produce any desired MAG ester, while
the reaction with Novozyme 435® showed almost complete
consumption of the starting ethyl ester by TLC, albeit with
Growth inhibition results of new derivatives
New compounds were tested for their growth inhibitory
effects on lung A549 lung cancer cells (Table 2). An initial
screen was performed at a single concentration of 3 M. In this
assay, MAG-DPA gave superior results to MAG-EPA and MAG-
DHA, with cell growth inhibition of 74.5%, 51.4% and 59.1%,
respectively. In the DHA series, replacement of the glyceryl
ester (59.1% growth inhibition) for either a primary (4, 88.7%) or
secondary amide (1, 77.4%) improved growth inhibition. The
same held true for diamide 3 (83.8%), whereas monoamide 2
(38.3%) was less efficient than the parent MAG-DHA. Similarly
to secondary amide 1, the analogue with a carboxylate substituent
(20) displayed reasonable growth inhibition (68.7%). The best
compounds in this series were analogs 7 and 15 (98.4 and 98.3%,
respectively), whereas truncated mono- and diamide derivatives
16 and 17 (29.3 and 13.0% growth inhibition, respectively) were
less efficacious compared to MAG-DHA (59.1% growth
inhibition).
1
several products (Scheme 1). Further analysis by LC-MS and H-
NMR showed the presence of appreciable amounts of
diacylglycerol, some acid, and a small quantity of the desired
compound (see below).
<Insert Scheme 1>
For example, reaction of DHA ethyl ester in glycerol at 35oC
for 24 h yielded a 16:40:30:14 mixture of monoacylglycerol:
diacylglycerol:fatty acid:starting ethyl ester. The predominance
of the diester can be explained by the poor solubility of the
starting ethyl ester in glycerol compared to the MAG ester, which
would favour subsequent reaction of the MAG ester over the
ethyl ester. No triacylglycerol was detected in any of these
experiments. Formation of the acid was readily explained by the
reaction of the ethyl ester with a water molecule; however the
occurrence of free fatty acid was much lower in the enzyme-
catalyzed compared to the TBD-mediated reaction.
<Insert Table 2>
Interestingly, the DHA and DPA series gave remarkably
different SARs. Indeed, whereas DHA ethanolamide 16 had
modest inhibitory effect on growth of A549 cells, its DPA analog
21 demonstrated a robust effect (29.3 vs 96.9%). On the other
hand, both DHA and DPA diamides 3 and 22 displayed robust
growth inhibition, the latter being again stronger (83.8 vs 97.7%).
Likewise, the same trend was observed for 2-substituted amides 1
and 25 (77.4 vs 97.4%), in favour of DPA.
Reaction conditions were subsequently optimized to promote
the formation of the MAG ester. Toward this end, several co-
solvents able to better mix the starting materials and decrease
reaction time were screened. THF, 1,4-dioxane, DMF, DMSO,
acetonitrile and acetone were tested at 35oC to optimize enzyme
activity,21 increase fluidity and minimize decomposition. From
those experiments, acetone gave the best results (over 50% of the
-3 fatty acid MAG ester after 5 h of reaction). Acetone is an
environmentally benign, low toxicity class 3 solvent10 so it was
chosen as the solvent for further optimization with the amides.
Typically, a minimal amount of acetone was required (2 mL per
gram of ester).
<Insert Figure 2>
The IC50 of DHA analogs 1 and 15 was further determined as
indicated in Figure 2, with values of 0.48 and 0.25 µM,
respectively, superior to the parent ester MAG-DHA (IC50 = 1.25
M).
Conclusion
A one-pot environmentally friendly method for the
-3 fatty amides derived from various amines
The first reaction between DHA ethyl ester and 2-Amino-1,3-
propanediol gave 87% of the desired amide 1 in 4 h, the main by-
product being the acid as a result of hydrolysis. Similar yields
were obtained using EPA, DHA and DPA with various amines
(Table 1).
and ethyl esters of three important -3 polyunsaturated fatty
acids has been optimized. The method uses minimal
quantities of acetone and mild temperature, combined with
enzyme-catalyzed transamidation to afford the desired
molecule in high yields, without decomposition or
conjugation of the unsaturations. It allowed the synthesis of
several amides and supported the rapid determination of the
SAR of this new class, particularly with respect to the
growth inhibitory properties of the compounds on the A549
lung cancer cell line. These are very promising results, and
subsequent biological results will be further reported shortly.
<Insert Table 1>
As can be seen in Table 1, the reaction is broadly applicable,
both in terms of PUFA ethyl ester and amine. In general, amides
23
were formed preferentially to esters.22,
In the case of more
hindered amides (i.e., position 2), an initial reaction of the
terminal alcohol followed by intramolecular transamidation may

Funding from the NSERC (Natural Sciences and Engineering
Research Council of Canada) Engage program is gratefully
acknowledged. The Faculty of Medicine and Health Sciences of
Université de Sherbrooke is acknowledged for summer internship
to C. St-Georges and M.-A. Legault.
Experimental
Representative procedure: synthesis of amide 25
To 500 mg (1.5 mmol) of DPA ethyl ester as an oil were
added 100 mg of Novozym 435© lipase on resin and 5 eq (7.5
mmol) of serinol. A minimum amount of acetone was then added
to decrease viscosity (1 mL). The mixture was magnetically
stirred for 4-18 h at 35°C until TLC indicated complete
disappearance of the ethyl ester (to be noted, LC-MS methods
with UV detection are not useful to follow these reactions owing
to the very low absorbance of the chromophores). The reaction
was then filtered, acetone was evaporated then water added. The
mixture was extracted with a minimal amount of diethyl ether (1
mL) thrice. The organic phase was dried with magnesium
sulphate, filtered then evaporated under reduced pressure. The
crude product was purified by flash chromatography with
hexane/ethyl acetate to give the desired amide 25 as a colorless
oil in 60% yield (> 90% purity by1H NMR).
References and notes
1.
2.
3.
Feltes, M. M. C.; Oliveira, D; Block, J. M.; Ninow, J. L.;
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Morin, C.; Fortin, S.; Cantin, A. M.; Rousseau, E.;  
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Cho, K. L.; Lee, J. H.; Kim, J. M.; Park, S. H.; Choi, M. S.;
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1H NMR (300MHz, CDCl3) (ppm): 6.52 (d, 1H, 7.2 Hz), 5.43-
5.29 (m, 10H), 4.98 (s, 2H), 3.94-3.91 (m, 1H), 3.81 (dd, 2H, 3.9
Hz, 4.2 Hz), 3.70 (dd, 2H, 4.8 Hz, and 5.1 Hz), 2.89-2.79 (m,
10H), 2.32 (t, 2H, 8.3 Hz), 2.22 (t, 2H, 7.5 Hz), 2.07 (quint, 2H,
7.1 Hz), 1.63 (quint, 4H, 7.2Hz), 1.42-1.30 (m, 6H), 1.26-1.24
(m, 2H), 0.97 (t, 3H, 7.7 Hz).
6.
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 2013, , 319.
Chon, S. H.; Zhou, Y. X.; Dixon, J. L.; Storch, J.; 
 2007, , 33346.
Dang, H. T.; Kang, G. J.; Yoo, E. S.; Hong, J.; Choi, J. S.;
Kim, H. S.; Chung, H. Y.; Jung, J. H.; 
2011, , 1520.
http://www.ich.org/products/guidelines/quality/quality-
single/article/impurities-guideline-for-residual-
solvents.html.
8.
9.
13C NMR (75.5 MHz, CDCl3) (ppm): 178.57, 174.71, 132.02,
130.01, 128.56, 128.41, 128.23, 128.13, 128.01, 127.90, 127.03,
61.93, 52.45, 36.55, 34.19, 29.31, 28.91, 28.80, 27.07, 25.64,
25.53, 24.78, 20.56, 14.29.
10.
IR (CHCl3) (cm-1): 3330, 3011, 2931, 2858, 1709, 1650, 1547,
1461, 1266, 1051, 975, 720.
HRMS (M-H+) C25H42NO3; calc: 404.3159; measured: 404.3164
11.
Prat, D. H.; Hayler, J.; Wells, A.; 2014, DOI:
10.1039/c4gc01149j.
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Ast, H.,  1963, , 1539.
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2011, , 15.
Anitescu, G.; Bruno, T.J.;    2012, ,
133.
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, 471.
Veras, I. C.; Silva, F. A.; Ferrao-Gonzales, A. D.; Moreau,
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Cervero, J. A.; Alvarez, JR; Luque, S,  
.2014, , 131.
Poppe, J. K.; Garcia-Galan, J.; Matte, C. R.; Fernandez-
Lafuente, R.; Rodrigues, R. C.; Ayub, M. A. Z.,  
 . 2013, , 51.
Weiberth, F. Y.; Yu, Y.; Subotkowski, W.; Pemberton, C;
2012, , 1967.
Islam, M. K.; Curie, Y. M. ; Gossage, J.L.; Benson, T.J.;
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Chang, H. M.; Liao, H. F.; Lee, C. C. ; Shieh, C. J. 
  2005, , 307.
Mergen, F. L.; Lambert, D. M.; Poupaert, J. H. ; Bidaine,
A.; Dumont, P.; .1991, , 267.
Zhang, J. C.; Cai, D.; Tang, W.; Zhang, Z.; Liu, Y.; Gao,
X.;    2014, , 871.
Cell culture
Human A549 lung adenocarcinoma cells were obtained from
the American Type Culture Collection (ATCC). A549 cells were
maintained in RPMI 1640 (Wisent, St-Bruno, QC, Canada)
containing 10 % FBS and 10 units/mL penicillin, 100 µg/mL
streptomycin. Cells were grown in a 5% CO2 incubator at 37°C
and used between passage 3 to 6 for all conditions and assays
tested in this study.
14.
15.
16.
17.
18.
Growth inhibition assay
A predefined number of A549 cells were allowed to grow in
24 wells plates (1 × 104 cells/well) for 3 days until cells reached
60% confluence. They were then starved in RPMI medium
without FBS for 8 h, then the culture medium was replaced with
RPMI + 0.2 % FBS and 3 µM of test compound were added to
each well. Culture media were changed every 24 h and cells were
treated for 48 h. After 48 h, the medium was removed from the
culture plates and 0.05% trypsin EDTA was used to detach the
cells from the surface of the culture plates. The harvested cells
were counted, both the viable and dead ones, using a Countess
Automated Cell Counter (Invitrogen Inc.). Briefly, for each
condition an appropriated cell dilution was prepared from the
harvested cells and an aliquot was mixed with an equal volume
of 0.4% trypan blue, and 10 µL was transferred into each side of
concentrations tested were
19.
20.
21.
22.
23.
performed in triplicata and were representative of 5 independent
experiments.
Acknowledgments

Figures
Figure 1. Analogs of DHA, DPA and EPA explored in this
study
Scheme 1. First results of transesterification between DHA
ethyl ester and glycerol
Figure 2. Concentration-response curve to MAG-DHA,
analogs 1 and 15 on A549 cell growth

(48%)
a:
DHA=
(4Z,7Z,10Z,13Z,16Z,19Z)--4,7,10,13,16,19-
docosahexaenoic acid EPA= (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-
icosapentaenoic acid DPA= (7Z,10Z,13Z,16Z,19Z)-
7,10,13,16,19-docosapentaenoic acid b: Isolated yield. c: Excess
EPA ethyl ester was used to force the formation of the ester.
Tables
Table 1 Results of transamidation
Table 2 Inhibition of cell growth
Product
(Nr)
Product
(yield)b
Time
(h)
Estera
Amine
growth inhibition
growth inhibition
Product
(Nr)
Product
(Nr)
(%) at 3
M
(%) at 3
M
1
2
DHA
DHA
Amide (87%)
Amide (48%)
4
8
mean
SEM
Mean
SEM
MAG-
DHA
59.1
3.1
15
98.3
1.3
3
4
5
6
DHA
DHA
EPA
EPA
Diamide 38%)
Amide (80%)
Amide (40%)
Amide (75%)
8
8
8
8
MAG-EPA 51.4
MAG-DPA 74.5
2.5
1.7
1.5
5.0
1.4
2.1
0
16
17
18
19
20
21
22
23
24
25
26
27
28
29.3
13.0
48.1
42.3
68.7
96.9
97.7
47.4
31.6
97.4
nd
3.5
2.0
2.1
2.6
2.5
0.5
0.5
3.4
3.2
0.7
77.4
38.3
83.8
88.7
98.4
70.9
1
2
3
4
7
EPA
Amide (78%)
8
7
8
9
EPA
EPA
EPA
Amide (80%)
Amide (70%)
Amide (70%)
8
8
8
8
1.9
nd
nd
nd
9
10
11
12
13
14
10
84.2
1.3
95.0
67.8
1.2
2.3
11
EPA
Amide (60%)
8
nd
nd
nd: not determined
12
EPA
EPA
Amide (80%)
Ester (25%)
8
13c
18
EPA
Amide (55%)
Amide (70%)
8
8
14
15
DHA
16
17
18
19
DHA
DHA
DHA
DHA
Amide (75%)
Diamide (60%)
Amide (70%)
Amide (65%)
8
8
8
8
20
21
DHA
DPA
Amide (55%)
Amide (60%)
8
8
DPA
DPA
DPA
DPA
Diamide (45%)
Amide (80%)
Amide (70%)
Amide (60%)
8
8
8
8
22
23
24
25
26
27
28
EPA
EPA
EPA
Diamide (40%)
Ester (28%)
8
18
16
NMe-Amide