Chemical synthesis and biological evaluation of ω-hydroxy polyunsaturated fatty acids

Article abstract of DOI:10.1016/j.bmcl.2016.12.002

ω-Hydroxy polyunsaturated fatty acids (PUFAs), natural metabolites from arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared via convergent synthesis approach using two key steps: Cu-mediated C[sbnd]C bond formation to construct methylene skipped poly-ynes and a partial alkyne hydrogenation where the presence of excess 2-methyl-2-butene as an additive that is proven to be critical for the success of partial reduction of the poly-ynes to the corresponding cis-alkenes without over-hydrogenation. The potential biological function of ω-hydroxy PUFAs in pain was evaluated in naive rats. Following intraplantar injection, 20-hydroxyeicosatetraenoic acid (20-HETE, ω-hydroxy ARA) generated an acute decrease in paw withdrawal thresholds in a mechanical nociceptive assay indicating pain, but no change was observed from rats which received either 20-hydroxyeicosapentaenoic acid (20-HEPE, ω-hydroxy EPA) or 22-hydroxydocosahexaenoic acid (22-HDoHE, ω-hydroxy DHA). We also found that both 20-HEPE and 22-HDoHE are more potent than 20-HETE to activate murine transient receptor potential vanilloid receptor1 (mTRPV1).

Full text of DOI:10.1016/j.bmcl.2016.12.002

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical synthesis and biological evaluation of
polyunsaturated fatty acids
x-hydroxy
Sung Hee Hwang a, Karen Wagner a, Jian Xu b, Jun Yang a, Xichun Li b, Zhengyu Cao b
Christophe Morisseau a, Kin Sing Stephen Lee a, Bruce D. Hammock a,
⇑
a Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
b Jiangsu Provincial Key Laboratory of TCM Evaluation and Development, China Pharmaceutical University, 639, Longmian Ave, Jiangning District, Nanjing, Jiangsu 211198, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
x-Hydroxy polyunsaturated fatty acids (PUFAs), natural metabolites from arachidonic acid (ARA), eicos-
Received 11 October 2016
Revised 30 November 2016
Accepted 1 December 2016
Available online xxxx
apentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared via convergent synthesis
approach using two key steps: Cu-mediated CAC bond formation to construct methylene skipped
poly-ynes and a partial alkyne hydrogenation where the presence of excess 2-methyl-2-butene as an
additive that is proven to be critical for the success of partial reduction of the poly-ynes to the corre-
sponding cis-alkenes without over-hydrogenation. The potential biological function of
PUFAs in pain was evaluated in naive rats. Following intraplantar injection, 20-hydroxyeicosatetraenoic
acid (20-HETE, -hydroxy ARA) generated an acute decrease in paw withdrawal thresholds in a mechan-
ical nociceptive assay indicating pain, but no change was observed from rats which received either 20-
hydroxyeicosapentaenoic acid (20-HEPE, -hydroxy EPA) or 22-hydroxydocosahexaenoic acid (22-
HDoHE, -hydroxy DHA). We also found that both 20-HEPE and 22-HDoHE are more potent than 20-
HETE to activate murine transient receptor potential vanilloid receptor1 (mTRPV1).
Ó 2016 Elsevier Ltd. All rights reserved.
x-hydroxy
Keywords:
x
-Hydroxy PUFA
x
20-HETE
20-HEPE
22-HDoHE
TRPV1
x
x
Pain
Polyunsaturated fatty acids (PUFAs) are mainly converted to
20-HETE. The same CYP isoforms also metabolize both x-3 PUFAs,
oxylipin metabolites by cytochrome P450 (CYP) enzymes that
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to
catalyze hydroxylation or epoxidation.1 Arachidonic acid (ARA),
the corresponding epoxy- and hydroxy-PUFAs.3 Recently, benefi-
an
x
-6 PUFA, is metabolized by the CYP enzymes to hydroxye-
cial effects from dietary supplements of fish oil including x-3 fish
icosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs).
While EET regioisomers can be found in roughly similar amounts,
oil prescriptions have triggered interests in the biological functions
of their metabolites.4 For example, epoxides from DHA can reduce
pain perception,5 blood pressure,6 and angiogenesis.7 Herein, we
20-HETE is a product of
x-hydroxylation that is a major regioiso-
mer of HETEs derived from ARA. All of these metabolites are impor-
tant lipid mediators that play critical roles in various diseases.1,2
Interestingly, EETs and HETEs generally have opposing biological
functions, e.g., EETs are known to be anti-inflammatory and
anti-hypertensive, but 20-HETE shows the opposite effect.2 Among
CYP enzymes, isoforms of the CYP2 family, such as CYP2C and
CYP2J, are frequently implicated in production of EETs, but
isoforms of the CYP4 family, such as CYP4A and CYP4F, produce
are exploring biological roles of x-hydroxy PUFAs (20-HEPE and
22-HDoHE) derived from EPA and DHA, respectively, compared
to the relatively well-studied 20-HETE derived from ARA.
20-HETE has been shown to have detrimental effects in several
diseases such as hypertension,8 cancer,9 and cardiovascular and
kidney diseases,10 despite its low in vivo concentrations due to
reincorporation into membrane phospholipid pools, and plasma
protein binding similar to other fatty acids.1 In addition, signifi-
cantly increased 20-HETE resulting from chronic administration
of rofecoxib to mice suggests that it may contribute to the cardio-
vascular risks associated with coxibs and nonselective nonsteroidal
anti-inflammatory drugs (NSAIDs).11 There is also growing
evidence that 20-HETE is a potent agonist of a transient receptor
potential vanilloid receptor 1 (TRPV1)12 whose activation by
endogenous lipid mediators is closely associated with pain.13
Abbreviations: ARA, arachidonic acid; COX, cyclooxygenase; CYP 450, cyto-
chrome P450; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FLIPR,
fluorescent imaging plate reader; 22-HDoHE, 22-hydroxydocosahexaenoic acid; 20-
HEPE, 20-hydroxyeicosapentaenoic acid; 20-HETE, 20-hydroxyeicosatetranoic acid;
PUFA, polyunsaturated fatty acids; TRPV1, transient receptor potential vanilloid 1.
⇑
Corresponding author.
E-mail address: bdhammock@ucdavis.edu (B.D. Hammock).
http://dx.doi.org/10.1016/j.bmcl.2016.12.002
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hwang S.H., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.002

2
S.H. Hwang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
However, little is known about the biological roles of
x
-hydroxy
heavy atom standards.16 Thus, we report here the practical chem-
ical syntheses of all three -hydroxy PUFAs and their initial biolog-
metabolites derived from -3 PUFAs due in part to their difficult
x
x
synthesis and therefore limited availability. These are 20-hydrox-
yeicosapentaenoic acid (20-HEPE) and 22-hydroxydocosahex-
aenoic acid (22-HDoHE) derived from EPA and DHA, respectively.
While several methods for the chemical and biosynthesis of 20-
HETE have been reported,14 there is only one example for 20-HEPE
chemical synthesis.15 To our knowledge, even though bioconver-
sion of 22-HDoHE using fungi or enzymes has been recently repor-
ted,14f–h its chemical synthesis, which renders the ease of scale-up
compared to its bioconversion, has not yet been reported. These
compounds can be also used as standards for expanding
ical evaluation in vitro and in vivo.
Our convergent synthetic approach of the
x-hydroxy PUFAs,
20-HETE, 20-HEPE, and 22-HDoHE, is summarized in Scheme 1.
During their total syntheses, serial Cu-mediated CAC bond
formation to construct required methylene skipped poly-ynes
and their partial hydrogenation to obtain the desired cis-double
bonds of each
x-hydroxy PUFA have been used as two key reaction
steps. All of the compounds were prepared by partially sharing
common fragments 3, 6, 11, and 16, which were also obtained
via serial Cu-mediated CAC bond formation reactions from the
corresponding terminal alkynes 1, 4, 7, and 12, respectively. While
the preparation of the fragments 3, 6, and 16 was straightforward,
the initial attempt of bromination of 8 to prepare 11 in a similar
metabolite analysis of oxylipins in the ARA cascade to the
x-3
PUFAs such as EPA and DHA. In particular, the chemical synthetic
approach to form these molecules will provide a facile route to
O
OH
MeO
1
4
c
a
OH
O
Si
R 1
5
d
MeO
1
2
3
, R = OH
b
1
. R = Br
OH
6
Me3Si
7
MeO
a
O
12
Me3Si
R 2
a
2
8
, R = OH
MeO
R 3
e
2
9
, R = OTs
3
O
13
14
, R = OH
f
b
b
3
. R = Br
Me3Si
g
OH
10
R 3
d
MeO
3
15
16
, R = OH
O
OH
3
. R = Br
11
3
6
3
11
h
16 11
+
+
+
h
h
O
O
OMe
OMe
OMe
O
OH
OH
OH
17
i
19
i
21
i
O
OMe
O
OMe
O
OMe
OH
HO
22
OH
18
20
j
j
j
20-HETE
20-HEPE
22-HDoHE
Scheme 1. Synthetic routes of compounds 20-HETE, 20-HEPE, and 22-HDoHE. Reagents and conditions: (a) CuI, NaI, Cs2CO3, 4-chloro-2-butyn-1-ol, DMF, rt, 12 h; (b) PPh3,
CBr4, DCM, 0 °C, 2 h; (c) CuI, NaI, Cs2CO3, 3-bromo-1-(trimethylsilyl)-1-propyne, DMF, rt, 12 h; (d) 1 M TBAF in THF, AcOH, THF, rt, 12 h; (e) Et3N, DMAP, p-TsCl, DCM, 0 °C to
rt, 12 h; (f) CuI, NaI, Cs2CO3, 3-butyn-1-OH, DMF, rt, 12 h; (g) CuI, NaI, Cs2CO3, propargyl alcohol, DMF, rt, 12 h; (h) CuI, NaI, Cs2CO3, DMF, rt, 12 h; (i) Lindlar catalyst, 2-
methyl-2-butene:MeOH:pyridine (4:4:1), H2, rt, 18 h–2 d (note: 1:1 MeOH/EtOAc was used instead of MeOH for both 20 and 22); (j) 1 N NaOH, MeOH, rt, 5 h.
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S.H. Hwang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
manner to the preparation of 15 from 13 was problematic due to
the instability and volatility of the brominated product. Therefore,
a tosylate 9, which is solid and is easily separated by column chro-
matography, was prepared instead. In addition, desilylation of ter-
minal alkynes, as in the compounds 5 and 10, often suffer from
isomerization of the desilylated product in a basic condition, but
the compounds 6 and 11 were exclusively prepared using Balas’
method where the reaction was performed in neutral condition
by adding the same ratio of TBAF:AcOH.17
hydrolysis of the esters 18, 20, and 22 gave the desired free acids,
20-HETE, 20-HEPE, and 22-HDoHE, respectively (synthetic proce-
dure, characterization and purity data of 20-HETE, 20-HEPE, and
22-HDoHE are available in Supporting information).
Several studies suggest that 20-HETE has effects in multiple car-
diovascular disease states, including ischemic disease, hyperten-
sion, and stroke and that this activity is dependent on its ability
to activate TRPV1.12 TRPV1 channels are also expressed in periph-
eral sensory neurons which mediate pain sensation activated by
inflammation.21 Therefore TRPV1 has been a therapeutic target
for pain management.22 Repeated sensitization of TRPV1, however,
induces desensitization to the stimuli, and thus both TRPV1 ago-
nists such as capsaicin and TRPV1 antagonists such as capsazepine
have been used to treat pain.23 Several TRPV1 antagonists have
With the required fragments 3, 6, 11, and 16 for the total syn-
theses of all three
x-hydroxy PUFAs in hand, their convergent syn-
theses by cross-mixing the fragments were performed to prepare
20-HETE (a combination of 3 and 6), 20-HEPE (a combination of
3 and 11), and 22-HDoHE (a combination of 11 and 16) as in
Scheme 1. The most challenging step during the synthesis was
the difficulty in controlling the partial hydrogenation of multiple
triple bonds in 17, 19, and 21 to convert them into the correspond-
ing desired cis-double bonds without over-hydrogenation in com-
pounds 18, 20, and 22. Partial hydrogenation of the triple bonds
using the Lindlar catalyst is a well-known approach to stop the
reaction of alkynes at the stage of desired alkenes without over-
hydrogenation to alkanes.18 Even though palladium metal itself
inside the Lindlar catalyst is intentionally deactivated with various
forms of lead, further deactivation by adding an additive such as
quinoline or pyridine is often required to prevent over-hydrogena-
tion to alkanes. Other methods such as Ni/B reduction have also
been used for the partial hydrogenation of alkynes. Therefore,
these conventional methods were applied to synthesize 20-HETE
and other PUFAs such as DHA. Interestingly, none of the reported
procedures, however, described the exact same conditions such
as amounts of reagents, temperature, reaction times, or addi-
tives.14b,19 This is likely because the reaction is hard to control
and variable depending on nature of starting materials. Due to
this reason, we were unable to stop the partial hydrogenation
reaction of the alkyne 17 to obtain the desired alkene 18 using
such conventional partial hydrogenation conditions, which
yielded significant amounts of over-hydrogenated alkanes. To
overcome this, we tested whether an additional additive, 2-
methyl-2-buene (bp 39 °C), which is easily removable to due to
its lower boiling point, could prevent undesired over-
hydrogenation of 17 based on Ho’s finding where an extra
additive, cyclohexene (bp 83 °C), which possesses a double-bond,
prevents
over-hydrogenation
of
the
desired
partially
hydrogenated product.20 Therefore 17 was subjected to partial
hydrogenation with the Lindlar catalyst in MeOH in the presence
of both additives – pyridine and 2-methyl-2-butene – to obtain
the desired 20-HETE methyl ester 18 with none or minimum
amounts of the corresponding over-hydrogenated alkanes.
Fig. 1. Comparison of pain induction by
22-HDoHE, in naïve rats. (A) Intraplantar injection of the corresponding methyl
esters (1 g in 1:9 EtOH/saline) revealed -6 hydroxy PUFA (20-HETE), but not -3
hydroxy PUFAs (20-HEPE and 22-HDoHE), changed mechanical withdrawal sensi-
tivity in the von Frey assay (Two Way Analysis of Variance, Holm-Sidak post hoc
p < 0.001, n = 6, male Sprague Dawley rats). (B) Intraplantar injection of 20-HETE
x-hydroxy PUFAs, 20-HETE, 20-HEPE, and
l
x
x
To test if this method is repeatable and scalable, the reaction
condition was confirmed by performing the reaction in duplicate
(entry I, Table 1) and in a 5-fold scale (entry II, Table 1). With these
results in hand, the partial hydrogenation of both 19 and 21 was
successfully applied to reduce their multiple triple bonds to the
desired cis-double bonds of 20, and 22, respectively. Finally,
(1 lg of 20-HETE methyl ester in 1:9 EtOH/saline) decreased mechanical with-
drawal thresholds in a dose-dependent manner indicating that pain behavior was
localized to the injected (ipsilateral) hind paw (Two Way Analysis of Variance,
Holm-Sidak post hoc p = 0.005, n = 6, male Sprague Dawley rats).
Table 1
Partial hydrogenation of poly-yne compound 17 to the corresponding alkene methyl ester 18.
17
18
Time (h)a
18
Yield (%)
43b
I
100 mg
40 mg
(0.3 mmol)
II
500 mg
250 mg
28
48
(1.6 mmol)
a
Reaction condition: Lindlar catalyst, 2-methyl-2-butene:MeOH:pyridine (4:4:1), room temperature, H2 gas (1 atm).
Average yield in duplicates.
b
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S.H. Hwang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Fig. 2. Calcium influx assay. (A) Capsaicin, 20-HETE, 20-HEPE and 22-HDoHE-induced Ca2+ influx in mTRPV1-transfected HEK293 cells as a function of time. (B) Quantification
of the Ca2+ responses (area under curve, AUC) induced by capsaicin, 20-HETE, 20-HEPE and 22-HDoHE. The experiments were repeated twice each in triplicates with similar
results.
been developed for pain treatment and have reached clinical tri-
als.23b However, several of these compounds failed due to
adverse effects or poor efficacy.23b,24 Given that TRPV1 activation
by capsaicin induces pain25 and the 20-HETE is known to be an
agonist of TRPV1 which modulates nociception, we tested the three
in vivo. The methyl esters of the metabolites were used to enhance
absorption. For the assay each ester (1 g) was administered by
l
intraplantar injection in naïve rats. Pain response was assessed
using an electronic von Frey aesthesiometer over a time course
(Figs. 1A and S1). While 20-HETE acutely decreased nociceptive
thresholds suggesting that 20-HETE induced pain in the rats, 20-
HEPE and 22-HDoHE did not elicit any change. 20-HETE also
decreased the thresholds in a dose-dependent manner (ipsilateral,
Fig. 1B) and the effects were local in that there was no change in
the contralateral paw even at a higher dose.
x
-hydroxy PUFAs for their pain induction in vivo and activation of
TRPV1 in vitro.
We conducted an in vivo investigation in naïve rats to better
observe the individual action of the directly applied metabolites
and avoid complications of inflammation induced activation and
upregulation of the TRP channels or their sensitization. The methyl
esters 18, 20, and 22 of 20-HETE, 20-HEPE and 22-HDoHE, respec-
tively, were tested for their ability to induce mechanical allodynia
To study whether the in vivo pain data are associated with
TRPV1 activation by these
x-hydroxy PUFAs, the influence of 20-
HETE, 20-HEPE, and 22-HDoHE on mTRPV1 were measured using
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S.H. Hwang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
5
a
calcium influx assay in HEK293 cells that heterologously
addition, we found that both 20-HEPE and 22-HDoHE are potent
TRPV1 agonist but do not induce pain like 20-HETE. These com-
pounds may have therapeutic property where TRPV1 activation is
expressed mTRPV1. Capsaicin was applied to stimulate the Ca2+
influx in mTRPV1 expressed HEK293 cells as a positive control
(Fig. 2). We found that 20-HEPE and 22-HDoHE produced signifi-
beneficial.23b Overall, this current practical synthesis of these
x-
cant Ca2+ influx at 10
l
M concentration, even though their activity
hydroxy PUFAs can be a useful tool to further investigate their
was less than that of the classical agonist capsaicin. However, 20-
roles in biological systems.
HETE produced marginal Ca2+ response at the concentrations
examined. (Fig. 2). It is worth noting that 20-HETE at 10 lM con-
Acknowledgments
centration activated hTRPV1 in a previous study.26 The discrepancy
between two studies could be derived from the difference in spe-
cies (human (hTRPV1) versus murine (mTRPV1) receptors). Never-
theless, the data presented here demonstrate that both 20-HEPE
and 22-HDoHE are more potent (or more efficacious) in stimulat-
ing mTRPV1 than 20-HETE.
This work was supported in part by NIEHS grant R01 ES02710,
NIEHS Superfund Basic Research Program grant P42 ES04699, and
NIHLB grant HL059699, National Institute of Neurological Disor-
ders and Stroke (NINDS) U54 NS079202. NIH 5T32DC008072-05
and 5T32HL086350-08 (to K.W.). K.S.S.L. has been partially sup-
ported by the NIH Pathway to Independence Award from NIH/
NIEHS (1K99ES024806-01).
While the 20-HETE was able to induce pain in naïve rats, the
effect was minimal compared to a potent algogen (pain-producing
agent), prostaglandin E2 (PGE2), which is an arachidonic acid
metabolite formed by cyclooxygenases (COXs). In a previous
study,27 PGE2 (100 ng) induced a 60% decrease in withdrawal
thresholds indicating pain. However, only 30% decrease in with-
drawal thresholds (indicating less pain) was observed with 20-
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.
002.
HETE methyl ester (1 lg) (Fig. 1A). TRPV1 channels are subject to
both desensitization, where agonists can induce a conformational
change and acutely block the opening of the channel, and tachy-
phylaxis, where the channel can be stimulated by an agonist into
a reoccurring intermediate state and become refractory to further
agonists and other types of nociceptive stimuli.13,23 Therefore it
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there was a distinct difference between the effects of the
x-6 ver-
sus -3 derived metabolites in the nociceptive assay with 20-HETE
x
alone demonstrating painful effects. In contrast to the in vivo
results, our in vitro data showed that 20-HEPE and 22-HDoHE acti-
vate mTRPV1 more than 20-HETE does. We cannot rule out that 20-
HEPE and 22-HDoHE may bind a distinct TRPV1 agonist-binding
site from 20-HETE, which may contribute to the different out-
come.31 We also tested their COX inhibition, but no COX activity
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A practical, stereospecific route to 18-, 19-, and 20-hydroxyeicosa-5(Z),8(Z),11
changes were found up to 100
porting information). Taken together,
derived from EPA and DHA may be beneficial, unlike 20-HETE. In
l
M concentrations (see the Sup-
x
-3 hydroxy metabolites
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