Development of Robust 17(R),18(S)-Epoxyeicosatetraenoic Acid (17,18-EEQ) Analogues as Potential Clinical Antiarrhythmic Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b00952

17(R),18(S)-Epoxyeicosatetraenoic acid (EEQ) is a cytochrome P450 metabolite of eicosapentaenoic acid (EPA) and a powerful negative chronotrope with low nanomolar activity in a neonatal rat cardiomyocyte (NRCM) arrhythmia model. Prior studies identified oxamide 2b as a soluble epoxide hydrolase (sEH) stable replacement but unsuitable for in vivo applications due to limited oral bioavailability and metabolic stability. These ADME limitations have been addressed in an improved generation of negative chronotropes, e.g., 4 and 16, which were evaluated as potential clinical candidates.

Full text of DOI:10.1021/acs.jmedchem.9b00952

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Article
Development of Robust 17(R),18(S)-Epoxyeicosatetraenoic Acid
(17,18-EEQ) Analogs as Potential Clinical Antiarrhythmic Agents
Adeniyi Michael Adebesin, Tim Wesser, Jonnalagadda Vijaykumar, Anna
Konkel, Mahesh Paudyal, Janine Lossie, Chen Zhu, Christina Westphal,
Narender Puli, Robert Fischer, Wolf-Hagen Schunck, and John R. Falck
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00952 • Publication Date (Web): 06 Nov 2019
Downloaded from pubs.acs.org on November 7, 2019
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Development of Robust 17(R),18(S)-Epoxyeicosatetraenoic Acid (17,18-EEQ)
Analogs as Potential Clinical Antiarrhythmic Agents
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†
#
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Adeniyi Michael Adebesin, Tim Wesser, Jonnalagadda Vijaykumar, Anne Konkel, Mahesh P.
†
#
†
§
†
#
Paudyal, Janine Lossie, Chen Zhu, Christina Westphal, Narender Puli, Robert Fischer, Wolf-
,
§
,†
Hagen Schunck,* and John R. Falck*
†Division of Chemistry, Department of Biochemistry, University of Texas Southwestern, Dallas,
TX 75390 USA
#OMEICOS Therapeutics GmbH, Robert-Rössle-Straße 10, 13125 Berlin, Germany
§Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin,
Germany
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ABSTRACT: 17(R),18(S)-Epoxyeicosatetraenoic acid (EEQ) is a cytochrome P450 metabolite of
eicosapentaenoic acid (EPA) and a powerful negative chronotrope with low nanomolar activity in
a neonatal rat cardiomyocyte (NRCM) arrhythmia model. Prior studies identified oxamide 2b as a
soluble epoxide hydrolase (sEH) stable replacement, but unsuitable for in vivo applications due to
limited oral bioavailability and metabolic stability. These ADME limitations have been addressed
in an improved generation of negative chronotropes, e.g., 4 and 16, which were evaluated as
potential clinical candidates.
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INTRODUCTION
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Cardiac arrhythmias often progress into serious cardiovascular complications such as cardiac
arrest or stroke and, thus, are important contributors to morbidity and mortality worldwide.2
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Current treatment options include potentially risky and invasive procedures, e.g., electric shock
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therapy (cardioversion) and catheter ablation, and usually long-term medication. Unfortunately,
extant antiarrhythmic drugs, when used prophylactically or therapeutically, are often poorly
5
tolerated because of their side effects and have a disconcerting propensity toward inducing future
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arrhythmias. Due to the various etiologies of arrhythmias, the development of safer and more
effective antiarrhythmic drugs is very challenging, and requires the discovery of pleiotropic agents
effective at various stages of an arrhythmic episode.
In recent decades, a large body of evidence has been garnered from epidemiological, animal, and
clinical studies that purport polyunsaturated -3 fatty acids (-3 PUFAs), primarily the
eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids commonly found in fish oils, have
rhythm stabilizing effects and are beneficial for the prevention and/or amelioration of arrhythmias
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-9
arising from diverse origins. Specifically, EPA prevents and/or terminates arrhythmias caused
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by β-adrenergic agonists and calcium overload, which suggest EPA offers multi-modal benefits
in prophylactic and therapeutic applications for the management of arrhythmias.11 It is also
reassuring that the above referenced clinical studies concluded -3 PUFA supplementation is well
tolerated, safe, and synergistic with existing therapies. On the other hand, some clinical studies of
dietary fish oil or EPA ester dietary supplementation have proven inconclusive12,13 or proposed
narrowly defined utility.14
1
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Pioneering studies by Schunck and colleagues into the mechanisms of action and molecular
species responsible for the antiarrhythmic and cardioprotective effects of EPA and its congeners
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implicated the cytochrome P450 (CYP) branch of the eicosanoid cascade as an important
participant.16 Noteworthy observations include: (i) dietary EPA/DHA ester supplementation
reduced arrhythmia as well as sudden cardiac death in an angiotensin (Ang) II-induced
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hypertension study using rats; (ii) EPA is equal or preferred over arachidonic acid (AA) as a
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substrate by most CYP epoxygenases; (iii) CYP-epoxyeicosanoid reservoirs are present at the
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sn-2 position of phospholipids and readily released by phospholipase (PL) A2; (iv) the principal
CYP epoxygenase isoforms preferentially metabolize EPA to the 17,18-epoxide (17,18-EEQ),
including most relevantly the major cardiac isoform, CYP2J;18,20-22 (v) transgenic overexpression
of human CYP2J2 protects against arrhythmia susceptibility in mouse models of cardiac
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hypertrophy; (vi) 17,18-EEQ is the main fatty acid epoxide in the heart and other organs of rats
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fed an -3 PUFA rich diet, but not when ingesting an -6 PUFA diet; (vi) EPA epoxides and/or
their hydrolysis products have been detected in humans in vivo and their levels were found to
increase in the plasma and urine of human volunteers ingesting daily multigram amounts of -3
PUFA esters;24-26 and (vii) EPA and 17,18-EEQ are non-angiogenic unlike some AA metabolites.27
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The effects of EPA (3-10 M) in a neonatal rat cardiomyocyte (NRCM) arrhythmia model ,
2+
e.g., restoration to basal beating rate and termination of Ca -induced fibrillation, could be
reproduced by 17,18-EEQ, but at a 1000-fold lower concentration and with almost immediate
effect as compared with the 10-30 min delay needed for EPA (Figure 1).18,28 Reflective of the
dominant stereoselectivity in the biosynthesis of 17,18-EEQ (vide supra), the R,S-enantiomer
dose-dependently reduces the spontaneous beating rate of neonatal cardiomyocytes with an EC50
of 1-2 nM, whereas its antipode has the opposite, albeit far less potent, effect.28
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esterification
beta-oxidation
O
O
O
O
O
OH
OH
OH
OH
X
CYP
1st Gen
2nd Gen
Y
N
O
O
H
EPA
highly labile
low water sol
17(R),18(S)-EEQ
1
2a: X = NH, Y = CH2
b: X = C(O), Y = NH
sEH stable



highly labile
autooxidation stable
COX/LOX stable
easier synthesis
1000x more potent
immediate onset
 beat rate: -22.5±0.8)
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10-30 min onset
partial water sol
no stereocenters
a:  beat rate: -27.0±1.2)
b:  beat rate: -33.7±1.3)
 beat rate: -20.3±1.2)
(
(
Figure 1. Structure and properties of eicosapentaenoic acid (EPA), 17,18-EEQ and analogs.
Negative chronotropic effects on NRCM beating rate at 30 nM.18,28
In common with other CYP PUFA autacoids,29,30 17,18-EEQ is chemically and metabolically
labile which constrains its study in vivo as well as its therapeutic utility. Prominent
degradative/catabolic processes for epoxyeicosanoids include (i) autooxidation, (ii) -oxidation,
(
iii) esterification, and (iv) metabolism by other pathways of the eicosanoid cascade, viz.,
cytochromes P450 (CYPs), cyclooxygenases (COXs), lipoxygenases (LOXs), and soluble epoxide
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hydrolase (sEH). The latter converts 17,18-EEQ to the corresponding vic-17,18-diol and nullifies
the ability to regulate NRCM beating rates.18
A systematic program to elucidate the pharmacophore responsible for the chronotropic activity
of 17,18-EEQ identified the carboxylic acid, cis-11,12-olefin, and 17(R),18(S)-epoxide as key
structural elements.28 Consequently, 17,18-EEQ could be simplified to 1 which was more
accessible synthetically and precluded autooxidation as well as metabolism by COXs and LOXs
(Figure 1). The second generation of analogs, illustrated by 2, imparted more drug-like properties
and witnessed the introduction of epoxide bioisosteres including ureas and oxamides. These
epoxide mimetics were conveniently often solids, dispensed with the two stereocenters without
loss of efficacy, modestly improved hydrophilicity, and, most importantly, obviated sEH
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inactivation. In the present study, we address the remaining -oxidation and esterification
liabilities in preparation for pharmacokinetic (PK) studies, in vivo evaluation in animal arrhythmia
models, and to expedite progress toward the clinical introduction of a first-in-class therapeutic.
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RESULTS AND DISCUSSION
The third generation analogs shown in Table 1 were screened initially for their chronotropic effects
in our NRCM assay and for inhibition of recombinant sEH at 1 and 10 M (Table 1). Since
replacement of the -carbon of PUFAs with oxygen abolishes32,33 -oxidation and profoundly
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limits esterification into phospholipids and glycerides, the 3-oxa versions of 2a and b, i.e., 3 and
4
, respectively, were prepared. In addition to improving water solubility by 2-3 orders of
magnitude (vide infra), this simple atomic substitution boosted the negative chronotropic impact
of urea 3 by ca. 40% whereas that of oxamide 4 remained essentially unchanged. As expected
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based upon earlier studies, 4 was also the weaker sEH inhibitor of the two analogs. The aqueous
solubilities of triethylene glycol (TEG) esters 5 and 6 as well as glycine amide 7 improved
compared with their parent carboxylates, but later studies revealed they were quickly hydrolyzed
in biologic milieu. The more hindered N-methylglycine amide 8 and alanine amide 9 proved less
proficient than 7 at suppressing the beating rate of the NRCMs. This can be attributed to the
reduced rate of amidase hydrolysis of 8 and 9 to the free carboxylic acid which is necessary for
biological activity. With its ionizable proton that mimics the carboxylate in 1 and 2 at physiologic
pH, N-phenylsulphonimide 10 restored the activity to an acceptable level as did sulphonate 11 and
phosphonate 13. Since the latter is dibasic, it was of interest to probe the influence of charge on
3
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biologic activity. We anticipated phosphodiesterases would rapidly cleave 12 into dianion 13,
whereas the mixed methyl/pivaloyloxymethyldiester analog 14 would in the short term furnish a
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monoanion, and the dipivaloyloxymethyldiester 15 might survive intact long enough to penetrate
the cellular membrane and undergo intracellular cleavage. In the event, there was no clear
advantage from any of the three compared with disodium salt 13. Of several traditional carboxylate
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bioisosteres, tetrazole 16 was the most potent in the NRCM assay followed by oxathiadiazole 17
while the structurally related oxadiazol-5-one 18 proved disappointing, likely due to the much
reduced acidity of oxadiazol-5-ones compared with tetrazoles and oxathiadiazoles. Despite the
utilization of an oxamide epoxide bioisostere, 19 was second only to urea 6 for inhibition of sEH;
this would appear to be an unrecognized property of the benzothiazolylacetamide moiety. The poor
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performance of phenol 20 is consistent with the earlier SAR study that identified a carboxylic
acid or comparably acidic functional group as essential for full biological activity of CYP
epoxyeicosanoids. Departing from the linear aliphatic template utilized thus far, analogs 21 and
2
2 demonstrate the 11,12-olefin can be replaced with an arene without loss of efficacy, thus
suggesting wider options for future development.
Oxamides 4 and 16 were selected for the next tier of evaluations based upon potency and their
minimal sEH inhibition which in toto more closely represent the pharmacological profile of 17,18-
EEQ. The potential advantages and disadvantages of sEH inhibitors versus epoxyeicosanoid
16
mimetics as therapeutics have been thoroughly discussed recently. Chief amongst the many
strategic considerations of sEH inhibition by the analogs was the unpredictable consequences of
the accumulation of endogenous epoxy-PUFAs [e.g., leukotoxins (EpOMEs) and
epoxyeicosatrienoic acids (EETs)] which can vary depending amongst other things on tissue,
disease state, and nutritional intake of PUFAs.
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Table 1. Negative Chronotropic Effects of 17,18-EEQ Analogs on NRCMs and Inhibition of
Soluble Epoxide Hydrolase (sEH)a,b
sEHi (%)b
1 M 10 M
sEHi (%)b
1 M 10 M
Change
Change
Compd
3
Analog
O
Compd
13
Analog
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(beats/min)
(beats/min)
O
O
ONa
ONa
P
OH
O
c,
c,
O
-38.0±3.3
-33.6±1.1
3.6±2.3 *10.4±4.1 *
H
-21.3±1.0
-16.2±0.9
6.8±3.2* 37.7±2.9*
5.4±2.8* 18.6±3.6*
N
N
H
N
CH3
CH3
N
CH3
OCH3
H
H
O
P
O
O
O
O
H
OPiv
OH
O
4
5
O
0.8±1.5d 0.8±2.5d
14
15
H
N
N
CH3
N
H
N
H
O
O
P
O
O
O
OPiv
OPiv
O
O
OH
O
O
3
1.3±1.2d
1.9±0.9d
O
H
-23.5±1.0
-30.9±5.5
5.1±2.1* 38.4±6.7*
O
-25.2±2.3
H
N
N
CH3
N
H
CH3
N
H
O
N
N
O
N
OH
N
O
O H H
N
3
26.5±2.5* 76.6±3.0*
0.9±6.0 19.2±5.0*
-0.6±3.8 17.8±4.0*
1.9±5.7 18.7±3.4*
16
-1.3±4.6
1.9±4.6
6
7
8
O
-19.8±1.0
-21.1±1.4
CH3
CH3
N
H
N
H
N
O
O
H
O
N
S
O
N
CO Na
N
2
O
H
O H
H
-24.3±1.3
-11.0±1.2
-21.1±1.1
-0.2±4.0 10.0±3.1*
-5.0±3.5 -9.2±4.1*
17.0±3.1* 61.8±2.8*
1
7
N
N
H
N
CH3
N
H
CH3
H
O
O
O
N
CH3
O
N
N
O
H CO2Na -15.6±4.3
O H H
18
19
N
N
N
H
CH3
N
H
CH3
O
O
O
NHAc
CH3
N
O
S
N
CO Na
2
O
H
O
9
H
-1.1±1.5
-23.1±1.2
-20.7±0.9
H
N
N
N
H
CH3
N
H
CH3
O
O
O
O
O
O
HO
S
S
N
10
H H
-3.2±5.0
1.1±2.7
N
20
O
H
-7.5±4.5
0.4±4.1
1.5±3.8
N
N
H
CH3
CH3
O
O
N
H
O
O
O
ONa
S
P
ONa
ONa
1
1
2
O
H
5.2±1.8* 0.0±3.8
5.0±1.72* 6.4±2.7*
O
-24.5±0.8
-21.8±1.0
-1.0±5.5 -2.2±2.2
2
1
H
N
N
CH3
CH3
N
H
N
O
P
O
O
H
O
OCH3
ONa
P
OCH3
ONa
4
O
-18.0±1.0
O
H
1.3±5.8
1.8±2.6
1
H
22
N
N
CH3
N
CH3
N
H
H
O
O
aAnalogs tested at 30 nM final concentration using n = 18-24 clusters originating from at least three independent
b
NRCM preparations. Data presented as mean ± SE. sEH = soluble epoxide hydrolase. Assay used recombinant human
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sEH with 14,15-EET as substrate. Data represent mean ± SD (n = 4). AUDA, a potent sEH inhibitor, produced 98.4
c
±
0.1% inhibition at 1 M. sEHi = soluble epoxide hydrolase inhibition. sEH inhibition by 2a is 4.7 ± 1.7%* and
d
2
3.4 ± 2.9%* at 1 and 10 M, respectively. sEH inhibition by 2b is -4.1 ± 2.9% and -8.3 ± 6.3% at 1 and 10 M,
respectively. *Statistically significant differences (t-test versus vehicle control) were observed (p<0.05). Piv =
pivaloyl.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inspection of the dose response curves for 17,18-EEQ, 2b, 4, and 16 (Figure 2) reveals a
progressive generational improvement in potency in the order of 17,18-EEQ < 2b < 4 ≈ 16. The
Emax for negative chronotropic activity, on the other hand, was about the same for all of the
synthetic analogs (Figure 2).
Figure 2. Concentration-response curves for (±)-17,18-EEQ, 2b, 4 and 16 using NRCMs.
Data points are mean±SEM (n = 24, each concentration). Curves were fitted using the standard
slope model in GraphPad Prism. The basal beating rates of the spontaneously contracting
cardiomyocyte clusters ranged between 120-150 beats/min.
Table 2. in vitro ADME Profile of 2b, 4 and 16
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Compound
4
ADME Parameter
2b
16
phosphate
buffer, pH7.4
Solubility
LogD
mg/mL
0.08 ± 0.01 1.96 ± 0.04 0.04± 0.01
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
n-octanol/
aqueous phase
ratio
TRIS buffer,
pH7.4
2.01
-0.03
2.35
pKa
calculated
4.95
no
4.23
no
5.08
no
human
plasma, half-
life
Plasma
stability
min
degradation degradation degradation
PPB
human plasma
Clint
% binding
99.91
16.53
52.43
99.37
2.48
99.97
4.77
µL/min/million
cells
Metabolic
stability
in human
hepatocytes
half-life
1A2
min
349.72
181.50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
46.20
>50
>50
>50
>50
2
2
B6
C8
>50
CYP Inhibition
2C9
C19
IC (µM)
5.40
0.21
43.20
4.70
5
0
2
2
D6
3A4
Papp
a-b and b-a
-6
×
10 cm/sec
5.3/2.3
28.2/15.8
10.0/8.0
Caco-2
Papp (b-a)/
Papp (a-b)
Efflux ratio
0.40
0.60
0.80
PPB = plasma protein binding; Clint = intrinsic clearance; Papp = apparent permeation coefficient, a = apical, b =
basolateral.
2
b, 4 and 16 displayed similarly high human plasma stability and percent plasma protein binding
(
PPB), but the in vitro clearance rate of 2b using human hepatocytes was ~6.6 and ~3.5 times faster
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
4
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4
4
4
4
4
4
4
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5
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5
5
5
5
6
than 4 and 16, respectively (Table 2). However, it was the latter's significant inhibition of CYP2C9,
CYP2C19, and CYP3A4 that relegated this analog to a lower priority.
0
1
2
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4
5
6
7
8
9
0
1
2
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9
0
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9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Effects of 2b, 4, 16, amiodarone, dronedarone, and terfenadine on CYP2J2-catalyzed
EPA epoxidation. Epoxygenase activities were calculated from the sum of EEQs and vic-
dihydoxyeicosatetraenoic acids (DiHETEs) formed by the recombinant enzyme in incubations
containing 10 µM EPA as substrate and 1 mM NADPH as electron donor. All reactions were
performed in triplicate as described in the Experimental Section. The inhibitory effects of
amiodarone, dronedarone, and terfenadine on CYP2J2-catalyzed EPA epoxidation were analyzed
in parallel incubations. Data are mean±SD.
In contrast to 2b and 4, analog 16 also inhibited the biosynthesis of EEQs from EPA by CYP2J2,
the major CYP epoxygenase in human heart (Figure 3). Analog 16 shared this property with the
currently used antiarrhythmic drugs amiodarone and dronedarone as well as with the antihistamine
terfenadanine (Figure 3). As discussed recently, drug-mediated inhibition of CYP2J2-catalyzed
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formation of endogenous cardioprotective epoxyeicosanoids might induce adverse cardiac
effects.37,38
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
4
4
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4
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4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
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8
9
0
1
2
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8
9
0
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2
3
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8
9
0
1
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0
1
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1
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8
9
0
Figure 4. NADPH-dependent metabolism of oleic acid (reference compound) and analogs 2b, 4,
and 16 by human liver microsomes. The incubations contained 0.5 mg/mL of human liver
microsomes and the test compound at a final concentration of 1 µM as described in the
Experimental Section. LC-MS in single ion monitoring mode was used to follow the time-
dependent loss of the respective parental compound and the formation of primary metabolites
resulting from monooxygenation (hydroxylation or epoxidation; [+O] metabolites) as well as
secondary metabolites originating from epoxidation and addition of the elements of water,
presumably from epoxide hydrolysis; ([+O + H O] metabolites). Data are mean values from
2
reactions run in duplicate for each time point and represent the peak areas of the molecular ions as
detected by LC-MS. No product formation occurred when NADPH was omitted from the
incubations.
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Studies with human liver microsomes indicate that analog 16 is not only a potent inhibitor, but
also an effective substrate for CYP enzymes. As shown in Figure 4, 16 was rapidly metabolized
to [+O] and [+O+H O] metabolites. The reaction sequence presumably consisted of CYP-
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
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5
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0
1
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9
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8
9
0
1
2
3
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8
9
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1
2
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0
1
2
3
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9
0
catalyzed epoxidation of the double bond, followed by hydration to a diol through the action of
3
9
microsomal and/or soluble epoxide hydrolases, which are present in human liver microsomes. In
contrast to 16, analogs 2b and 4 remained comparatively stable (Figure 4). After 60 min of
incubation, about 40 % of 16, 3.9 % of 2b, and 1.5 % of 4 were converted to the corresponding
oxygenated metabolites (sum of [+O] and [+O + H O] metabolites), as calculated from the relative
2
peak areas in LC-MS analysis.
The influences of the 3-oxa and tetrazole bioisostere on PK are evident in the 24 h plasma
concentration-time curves of 4 and 16 vs. 2b in rat (Figure 5). Maximum plasma levels were
reached about 1.6 (2b and 4) and 2 h (16) after oral administration, indicating rapid absorption of
all three analogs. However, the maximum plasma levels for 4 and 16 were approximately two
orders of magnitude higher and remained elevated far longer than for 2b (Figure 5, Panel A). Oral
bioavailabilities were almost 100 % with 4 and 35 % with 16, as calculated from the dose-corrected
areas under the curve after oral (Figure 5, Panel A) versus intravenous (Figure 5, Panel B)
administration. Factors contributing to the higher oral bioavailability of 4 compared to 16 are so
far unknown, but may include differences in their physicochemical properties (for pKa and logD
values see Table 2) as well as gastrointestinal absorption sites. In the elimination phase, 4 showed
a considerably higher half-life than 16 (9.6 vs. 3.6 h after oral administration), in line with the
different in vitro stabilities of these analogs in liver microsomes (Figure 4) and hepatocytes
(Table 2).
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1
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2
3
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0
1
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0
1
2
3
4
5
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0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Plasma concentration-time curves of 2b, 4, and 16 in rat. (A) Time course of uptake,
distribution, and elimination after a single oral dose of 3 mg/Kg. (B) Time course of distribution
and elimination after a single intravenous dose of 1 mg/Kg. Data are mean±SEM; n=4 per group;
n.d.=not detectable (below LOQ).
Based upon the foregoing results, 4 was selected for allometric scaling in three mammalian
species using a linear regression model with the log of animal mass as the X variable and log of
half-life as the Y variable (Figure 6). Based on this allometric scaling, a t of 37.9 h is predicted
1
/2
in humans with a standard weight of 75 kg (Y=0.2742*X+1.064).
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9
0
1
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6
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8
9
0
Figure 6. Plasma half-life of 4 in different animal species and predicted value for human. Data are
based on PK studies following single dose oral administration of 4 to mouse (n=3, 8 mg/Kg bw),
rat (n=4, 3 mg/Kg bw), dog (n=4, 3 mg/Kg bw).
To obtain a first in vivo proof of concept regarding the antiarrhythmic properties of synthetic
1
7,18-EEQ analogs, compound 5 was tested in a rat model of acute myocardial infarction. In this
model, coronary artery ligation is used to induce the development of spontaneous ventricular
tachycardia. Continuous ECG recording showed that pretreating the animals with compound 5
significantly reduced the total time in arrhythmia as well as the severity of the arrhythmic episodes
compared with the vehicle control (Figure 7).
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Figure 7: Effect of 5, the triethylene glycol (TEG)-prodrug of 4, on the development of ventricular
tachycardia in a rat model of acute myocardial infarction. The animals received single i.v. bolus
injections of 5 (n=10) or vehicle (n=9) 10 min before inducing ventricular arrhythmia by coronary
artery ligation. Continuous ECG monitoring was performed over the whole observational period
of 45 min. The severity score was calculated as described in the Experimental Section. Data are
mean±SD; *(p<0.05).
CHEMISTRY
Syntheses of analogs 11, 19, and 20 are summarized in Scheme 1. Alkylation of silyl protected
4
0
41
5
-hexyn-1-ol 23 with 2-(9-bromononaloxy)tetrahydro-2H-pyran in THF/HMPA gave 24 in
good yield. Removal of the silyl protecting group, followed by semi-hydrogenation of the alkyne
over catalytic P2-nickel, and substitution of the hydroxyl group for an azide under Mitsunobu
conditions provided 25. Staudinger reduction of the azide and subsequent substitutive amidation
4
2
of the crude amine with ethyl 2-(methylamino)-2-oxoacetate gave 26 (71% over two steps).
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The tetrahydropyranyl group was uneventfully removed under acidic condition, and the resulting
alcohol was converted to either iodide 27 or bromide 28 under Appel-type conditions.43
4
4
Substitution of the iodide in 27 with sodium sulfite, phenol 29, or o-thiophenol gave 11, 19,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and 20, respectively.
Scheme 1. Syntheses of 11, 19, and 20a
OTHP
OTBDPS
a
OTBDPS b-d
THPO
8
3
H
23
24
25
N3
OTHP
X
e,f
g,h or i
O
O
H
N
H
N
N
CH3
N
CH3
26
H
O
27: X = I
8: X = Br
H
O
2
O
O
S
ONa
j
O
H
N
N
H
CH3
11
19
O
O
I
S
k
O
O
N
H
H
N
N
NHAc
N
H
CH3
N
H
CH3
27
O
O
S
HO
S
N
NHAc
l
O
H
N
OH
29
N
H
CH3
20
O
aReagents and conditions: (a) n-BuLi (1.1 equiv), THF/HMPA (5:1), -78 to -20 °C, 1 h; Br(CH
4 h, 88%. (b) n-Bu NF, THF, rt, 12 h, 80%. (c) P-2 Ni/(H NCH , H (1 atm), EtOH, rt, 98%. (d) DIAD, Ph
20 °C, 30 min; (PhO) P(O)N , 0 °C to rt, 10 h, 86%. (e) Ph P, THF/H O, rt, 14 h. (f) EtO(CO)(CO)NHCH
5 °C, 10 h, 71% (over two steps). (g) 10 mol% p-TSA, MeOH, rt, 10 h, 98%. (h) Ph P, imidazole, I , CH Cl
P, CBr , CH Cl , 0 °C to rt, 14 h, 67%. (j) Na SO , cyclohexene, rt, 27%. (k) 29, K CO , DMF,
rt, 12 h, 29%. (l) KHCO , o-thiophenol, DMF, rt, 79%.
OTHP, -78 °C to rt,
P, THF,
, EtOH,
, 0 °C
2 9
)
1
4
2
2
)
2
2
3
-
2
3
3
2
3
8
3
2
2
2
to rt, 14 h, 70%. (i) Ph
3
4
2
2
2
3
2
3
3
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2 outlines the syntheses of analogs 12-15 and is representative of the approach utilized
for 21 and 22. Briefly, bromide 28 was transformed into dimethylphosphonate 12 via a Michaelis-
Arbuzov reaction. Demethylation of the phosphonate esters using trimethylsilyl bromide afforded
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
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9
0
1
2
3
4
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9
0
3 after basification with aqueous sodium carbonate and SM-2 Bio-BeadTM (Bio-Rad) extraction.
The mixed methyl/pivaloyloxymethyl (POM) ester 14 and di-POM ester 15 were obtained from
2 by substitution with POM iodide.
1
1
Scheme 2. Syntheses of 12, 13, 14, and 15a
O
P
ONa
ONa
b
O
O
H
N
OCH3
P
N
CH3
OCH3
13
O
P
H
O
a
O
28
H
O
P
N
OR
OR
OR
N
CH3
H
OCH3
12
O
c
O
+
O
H
N
H
N
N
CH3
N
CH3
O
H
H
14
15
O
O
O
I
O
tBu
POM-I
R =
O
tBu
aReagents and conditions: (a) (MeO)
P, reflux, 48 h, 54%. (b) TMSBr, CH
Cl (5:1), rt, 48 h: 14 (3.8%) and 15 (3.3%).
3
2 2 2 3
Cl , rt, 4 h; Na CO , 1 h, SM-2 Biobeads,
3
0%. (c) POM-I (5 equiv), MeCN/CH
2
2
Heterocyclic analogs 16, 17 and 18 were obtained from the common precursor, 30, that in turn
4
5
was secured from bromide 28 via S 2 cyanation (Scheme 3). Following literature precedent, 1,3-
N
dipolar cycloaddition of 30 with hydrazoic acid, generated in situ from sodium azide and
triethylamine hydrochloride, in dioxane provided 16 in moderate yield. Addition of hydroxylamine
to 30 provided amidoxime 31, which was reacted with either thionyl chloride or 1,1'-
carbonyldiimidazole to furnish 17 and 18, respectively.
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Scheme 3. Syntheses of 16, 17, and 18a
N
N
N
N
H
b
O
H
N
N
H
CH3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
1
6
O
O
S
N
CN
N
a
28
O
H
H
d
e
N
O
OH
H
N
CH3
N
O
N
O
30
H
O
N
H
CH3
17
NH2
O
O
H
N
O
c
N
N
H
CH3
N
H
3
1
O
H
N
N
H
CH3
18
O
aReagents and conditions: (a) KCN, DMSO, rt, 20 h, 99%. (b) NaN
NH OH·HCl, Na CO , MeOH, rt, 10 h, 69%. (d) SOCl , pyridine, CH
CH Cl , rt, 14 h, 18%.
, Et
N·HCl, dioxane, 120 °C, 24 h, 37%. (c)
CO,
3
3
2
2
3
2
2
Cl , 0 °C to rt, 14 h, 63%. (e) (imidazole)
2
2
2
2
The syntheses of 3 and 4 were accomplished using a more convergent synthetic strategy
involving Suzuki-Miyaura coupling between vinyl iodide 35 or 36 and borane 39 (Scheme 4). The
4
6
latter was generated in two steps via chemoselective In(OTf) mediated diazo O-H insertion
3
using commercial 6-hepten-1-ol (37) and ethyl diazoacetate followed by hydroboration of the
resultant ether 38 with 9-BBN. Urea 35 and oxamide 36 were synthesized from the known alkyne
4
7
3
2 in four routine steps involving silver-mediated alkyne iodination to give 33, disiamylborane
reduction, phthalimide deprotection affording vinyl iodide 34, and subsequent addition to ethyl
isocyanate or acylation with ethyl (methylamino)(oxo)acetate, respectively. Pd-catalyzed cross-
coupling led to 40 and 41. Final ester hydrolysis and acidification delivered 3 and 4, respectively.
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2
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2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4. Syntheses of 3 and 4a
O
I
a
b,c
d
or e
Y
NH2
3
N
X
CH3
NPth
NPth
3
3
3
H
I
I
32
33
34
35: X = NH, Y = CH2
36: X = CO, Y = NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
f
g
OH
O
3
OEt
OEt
3
3
37
38
9-BBN
39
O
CO2Et
O
CO2H
h
i
O
O
Y
Y
N
H
X
CH3
N
X
CH3
H
4
0: X = NH, Y = CH2
3: X = NH, Y = CH2
4: X = CO, Y = NH
41: X = CO, Y = NH
aReagents and conditions: (a) N-iodosuccinimide, AgNO
h. (c) 40% aq. MeNH , EtOH, rt, 35% (two steps). (d) EtNCO, THF, rt, 20 h, 94%. (e) EtO(CO)(CO)NHCH
5 °C, 10 h, 60%. (f) 20 mol% In(OTf) , ethyl diazoacetate, toluene, rt, 3 h, 70%. (g) 9-BBN, THF, 0 °C to rt, 2 h. (h)
5, 10 mol% Pd(Ph P) Cl , 2 M Na CO , THF, 50 °C, 14-16 h, 40 (70%); 36, same conditions, 41 (90%). (i) LiOH,
O, 0 °C to rt, 18-20 h; 1 M oxalic acid, 3 (82%) and 4 (67%).
, THF, rt, 14 h, 97%. (b) disiamylborane, THF, 0 °C to rt, 4
3
2
3
, EtOH,
8
3
3
3
2
2
2
3
THF, H
2
Finally, esters 5 and 6 as well as amides 7-10 were obtained from 2a, 2b, and 4 as described in
Scheme 5. Routine EDCI/DCC couplings and ester hydrolysis under basic condition provided the
desired compounds.
Scheme 5. Syntheses of 5-10a
2
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8
9
O
O
O
OH
N
CO Na
2
O
H
N
e,f
Me
a
3
4
O
O
O
O
O
O
H
N
N
H
CH3
N
H
CH3
Me
CO2Na
8
9
5
O
O
O
O
OH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
b
g,h
3
H
2b
H
N
N
H
N
H
O
CH3
N
H
CH3
6
7
O
O O
2
a
O
S
N
H
CO Na
2
N
H
H
N
i
c,d
N
H
N
H
CH3
N
H
CH3
10
O
aReagents and conditions: (a) triethylene glycol (TEG), EDCI, CH
85%. (c) methyl glycinate, EDCI, MeCN/CH Cl , 40 °C, 18 h, 71%. (d) NaOH, THF/MeOH/H
SM-2 Bio-Beads, 79%. (e) methyl sarcosinate, EDCI, MeCN/CH Cl , rt, 48 h, 74%. (f) NaOH, THF/H
8 h; SM-2 Bio-Beads, 84%. (g) NHS, DCC, cat. DMAP, EtOAc, 40 °C, 24 h. (h) L-alanine, DMAP,
Cl
2
, rt, 48 h, 53%. (b) TEG, EDCI, DMF, rt, 14 h,
O, 0 °C to rt, 48 h;
O, 0 °C to rt,
2
2
2
2
2
2
2
4
MeCN/DMF/H
h, 84%.
2 3
O, 48 h; 1 N HCl; NaHCO , 37% (2 steps). (i) benzenesulfonamide, EDCI, cat. DMAP, DMF, rt, 24
CONCLUSION
The identification of 17(R),18(S)-epoxyeicosatetraenoic acid (EEQ) as the primary cytochrome
P450 metabolite of EPA and the recognition of EEQ’s powerful biological activities provided a
cogent mechanistic foundation to help rationalize many of the cardiovascular benefits attributed
to diets rich in fish oils. SAR studies in these laboratories elucidated the pharmacophore
responsible for EEQ’s antiarrhythmic effects and supported significant progress towards more
drug-like analogs; however, these were still unsuitable for in vivo applications due to limited oral
bioavailability and metabolic stability. Subsequent investigations as described herein of prodrug
modifications, carboxylate bioisosteres, and carbon backbone substitutions identified the 3-oxa
2
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
analog 4 whose in vivo ADME/PK properties make it a potential candidate for further pre-clinical
and clinical development.
EXPERIMENTAL
Chemical Syntheses
0
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9
0
General Information. Nuclear magnetic resonance (NMR) spectra were recorded in CDCl with
3
1
TMS as internal standard, unless otherwise stated. H NMR data are reported as follows: chemical
shift (ppm), multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, app
q = apparent quartet, qn = quintet, app qn = apparent quintet, m = multiplet) and coupling constant
(Hz). Melting points were measured using an automated melting point apparatus and are
uncorrected. Analytical thin layer chromatography (TLC) used EMD Chemicals TLC silica gel 60
F254 plates (0.040-0.063 mm) with visualization by UV light and/or KMnO or phosphomolybdic
4
acid (PMA) solution followed by heating. All oxygen and/or moisture sensitive reactions were
performed under an argon atmosphere using oven-dried glassware and anhydrous solvents.
Extracts were dried over anhydrous Na SO and filtered prior to removal of all volatiles under
2
4
reduced pressure. Chromatographic purifications utilized preparative TLC or flash

chromatography using commercial pre-packed SiO columns on a CombiFlash R 200
2
f
chromatograph (Teledyne Isco). Unless otherwise noted, yields refer to isolated, purified material
with spectral data consistent with assigned structures or, if known, were concordant with published
data. Reagents were the highest commercial quality available and used without further purification,
unless otherwise noted. POM-I was purchased from Enamine LLC, Monmouth Jct., NJ 08852
USA. Reaction solvents, except anhydrous CH CN, were dried via passage under argon through
3
activated, neutral alumina columns immediately prior to use. The purity of all test compounds was
1
3
1
determined to be ≥95% by a combination of high-field C/ H NMR and LC-MS using a Luna
2
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C18(2) (3 µm), 100 × 2 mm and/or an Agilent ZORBAX Eclipse XDB-C18 (3.5 µm), 50 × 2.1
mm, using a gradient of aq. ammonium acetate (50 mM) and CH CN or MeOH.
3
Synthesis of 2-((13-(3-ethylureido)tridec-8(Z)-en-1-yl)oxy)acetic acid (3). To a rt solution of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0 (60 mg, 0.161 mmol) in THF (0.5 mL) and MeOH (0.5 mL) was added LiOH·H O (20.38 mg,
2
0
.485 mmol, 3.0 equiv) in H O (0.25 mL). After 20 h, the reaction was cooled to 4 °C and acidified
2
to pH 4 using aq. 2 N HCl. The mixture was diluted with H O (10 mL) and extracted with EtOAc
2
(
20 mL × 3). The combined organic extracts were dried over Na SO , filtered through a fritted
2 4
funnel, and concentrated in vacuo. Flash chromatography of the crude material (4 g SiO column
2
eluted with CH Cl , 3 min; 0-10% MeOH/CH Cl , 12 min; 10%, 10 min; 10-20%, 5 min; 20%, 5
2
2
2
2
min) gave 3 (46 mg, 82.0%) as a white solid, mp 56.5-57.5 °C. TLC: 10% MeOH/CH Cl , R ~
2
2
f
1
0
.20. H NMR (400 MHz, CDCl ) δ 5.50–5.35 (m, 1H), 5.35–5.21 (m, 1H), 4.05 (s, 2H), 3.54 (t,
3
J = 6.2 Hz, 2H), 3.24 (q, J = 7.2 Hz, 2H), 3.03 (t, J = 7.3 Hz, 2H), 2.17–1.96 (m, 4H), 1.68–1.50
1
3
(m, 4H), 1.50–1.28 (m, 10H), 1.15 (t, J = 7.2 Hz, 3H); C NMR (101 MHz, CDCl ) δ 174.18,
3
1
2
3
59.66, 130.79, 129.23, 71.66, 68.10, 41.20, 35.55, 29.53, 29.31, 29.15, 29.01, 28.67, 27.19, 26.99,
+
6.87, 25.84, 15.41. HRMS (ESI-TOF) m/z: [M+H] Calcd for C H N O 343.2591; Found
1
8
34
2
4
43.2597.
Synthesis of 2-[(13-(2-(methylamino)-2-oxoacetamido)tridec-8(Z)-en-1-yl)oxy]acetic acid (4).
To a rt solution of 41 (1.08 g, 2.80 mmol) in THF (60 mL) was added LiOH (90 mg, 3.76 mmol,
1
.3 equiv) in H O (20 mL). After 48 h, the reaction was cooled to 4 °C and acidified to pH 4 using
2
aq. 2 N HCl. The mixture was diluted with H O (10 mL) and extracted with EtOAc (20 mL × 3).
2
The combined organic extracts were dried over Na SO , filtered through a fritted funnel, and
2
4
concentrated in vacuo. Flash chromatography of the crude material (24 g SiO column eluted with
2
0-30% EtOAc/hexanes, 5 min; 30%, 5 min; CH Cl , 3 min; 0-10% MeOH/CH Cl , 10 min; 10%,
2
2
2
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
0 min; 10-20%, 5 min; 20%, 5 min) gave 4 (676 mg, 67%) as a white solid, mp 94.6-94.7 °C. H
NMR (500 MHz, CDCl ) δ 7.90 (s, 1H), 7.66 (s, 1H), 5.48–5.22 (m, 2H), 4.10 (s, 2H), 3.58 (t, J =
3
6.5 Hz, 2H), 3.32 (app q, J = 6.9 Hz, 2H), 2.91 (d, J = 5.2 Hz, 3H), 2.16–1.90 (m, 4H), 1.71–1.48
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
(
m, 4H), 1.45–1.18 (m, 10H); C NMR (101 MHz, CDCl ) δ 172.98, 160.86, 159.82, 130.65,
3
1
29.22, 72.12, 67.91, 39.85, 29.50, 29.46, 29.05, 28.96, 28.81, 27.14, 26.98, 26.80, 26.40, 25.91.
+
HRMS (ESI-TOF) m/z: [M+H] Calcd for C H N O 357.2384; Found 357.2376.
18 32
2
5
Synthesis
of
2-(2-(2-hydroxyethoxy)ethoxy)ethyl
(5).
2-((13-(2-(methylamino)-2-
mixture of 1-ethyl-3-(3-
oxoacetamido)tridec-8(Z)-en-1-yl)oxy)acetate
A
dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl, 275 mg, 1.43 mmol, 1.30 equiv)
and triethyleneglycol (1.5 mL, 11 mmol, 10 equiv) was dried under high vacuum for 90 min. The
reaction flask was flushed with argon and 4-dimethylaminopyridine (DMAP, 175 mg, 1.43 mmol,
1
.30 equiv), acid 4 (395 mg, 1.10 mmol), and anhydrous CH Cl (55 mL) were added. After 2
2 2
days, the reaction mixture was diluted with brine (20 mL) and 1N aq. HCl (10 mL). The organic
phase was separated and the aqueous phase was re-extracted with CH Cl (20 mL × 2). The
2
2
combined organic extracts were dried over Na SO , filtered, and concentrated in vacuo. Flash
2
4
chromatographic purification of the residue (12 g SiO column eluted with 0-100%
2
EtOAc/hexanes, 15 min; EtOAc, 15 min; 5% MeOH/CH Cl , 5 min) gave 5 (286 mg, 53%) as a
2
2
1
white solid, mp 65.3-65.8 °C. H NMR (400 MHz, CDCl ) δ 7.55 (br s, 2H), 5.40–5.21 (m, 2H),
3
4
.32–4.27 (m, 2H), 4.08 (s, 2H), 3.73–3.67 (m, 4H), 3.66–3.60 (m, 4H), 3.60–3.55 (m, 2H), 3.49
(t, J = 6.7 Hz, 1H), 3.28 (app q, J = 7.2 Hz, 2H), 2.87 (d, J = 5.2 Hz, 3H), 2.55 (br s, 1H), 2.08–
13
1
1
.90 (m, 4H), 1.65–1.48 (m, 4H), 1.42–1.17 (m, 10H); C NMR (101 MHz, CDCl ) δ 170.68,
3
60.68, 159.80, 130.63, 128.97, 72.59, 72.05, 70.63, 70.39, 69.04, 68.20, 63.67, 61.79, 39.63,
2
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8
9
2
9.68, 29.57, 29.37, 29.26, 28.87, 27.27, 26.93, 26.76, 26.23, 25.99. HRMS (ESI-TOF) m/z:
+
[M+Na] Calcd for C H N O Na 511.2990; Found 511.3010.
2
4
44
2
8
Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 16-(3-ethylureido)hexadec-11(Z)-enoate
6). A mixture of EDCI·HCl (6.40 mg, 0.034 mmol, 1.10 equiv) and triethyleneglycol (42.0 mg,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
0
.277 mmol, 10 equiv) was dried under high vacuum for 90 min. After flushing the flask with
argon, DMAP (4.20 mg, 0.034 mmol, 1.2 equiv), 16-(3-ethylureido)hexadec-11(Z)-enoic acid28
(2a, 10.0 mg, 0.028 mmol), and anhydrous DMF (3 mL) were added. After 14 h, the reaction
mixture was diluted with EtOAc (10 mL) and washed with water (5 mL). The organic phase was
concentrated in vacuo, and the residue was purified by column chromatography (SiO column,
2
eluted with 0-100% EtOAc/hexanes) to give 6 (11.1 mg, 85%) as a low melting white solid. TLC:
1
1
00% EtOAc, R ~ 0.2. H NMR (300 MHz, CDCl ) δ 5.42–5.27 (m, 2H), 4.34 (br s, 1H), 4.23 (t,
f
3
J = 5.8 Hz, 2H), 3.74–3.59 (m, 10H), 3.24–3.12 (m, 4H), 2.46 (br s, 1H), 2.33 (t, J = 7.3 Hz, 2H),
1
3
2.07–1.96 (m, 4H), 1.62–1.25 (m, 18H), 1.13 (t, J = 7.3 Hz, 3H); C NMR (75 MHz, CDCl ) δ
3
1
3
74.21, 158.42, 130.66, 129.44, 72.70, 70.79, 70.57, 69.44, 63.46, 61.98, 40.74, 35.59, 34.40,
0.11, 29.88, 29.63, 29.60, 29.44, 29.42, 29.31, 27.41, 27.22, 27.08, 25.10, 15.73. HRMS (ESI-
+
TOF) m/z: [M+H] Calcd for C H N O 473.3585; Found 473.3585.
25 48
2
6
Synthesis of sodium (16-(3-ethylureido)hexadec-11(Z)-enoyl)glycinate (7). To a mixture of 2a
80 mg, 0.234 mmol), glycine methyl ester hydrochloride (38.3 mg, 0.305 mmol, 1.30 equiv), and
EDCI·HCl (62.8 mg, 0.327 mmol, 1.40 equiv) in MeCN/CH Cl (1.5/1.5 mL) was added DMAP
(
2
2
(80.0 mg, 0.655 mmol, 2.80 equiv) at 0 °C. After stirring for 18 h at 40 °C, the reaction mixture
was diluted with water (5 mL) and extracted with EtOAc (3  10 mL). The combined organic
extracts were washed with 1N aq. HCl (2 mL), brine (10 mL), dried over Na SO , concentrated
2
4
under reduced pressure, and the residue was purified via flash chromatography (4 g SiO column
2
2
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
eluted with 0-80% EtOAc/hexanes) to give methyl (16-(3-ethylureido)hexadec-11(Z)-
1
enoyl)glycinate (75 mg, 71%) as a white solid, mp 92.4-93.6 °C. TLC: EtOAc, R ~ 0.2. H NMR
f
(400 MHz, CDCl ) δ 6.13 (br s, 1H), 5.43–5.24 (m, 2H), 4.32 (br s, 2H), 4.05 (d, J = 5.2 Hz, 2H),
3
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
1
2
.76 (s, 3H), 3.26–3.07 (m, 4H), 2.24 (t, J = 7.6 Hz, 2H), 2.11–1.93 (m, 4H), 1.70–1.59 (m, 2H),
1
3
.55–1.44 (m, 2H), 1.44–1.21 (m, 14H), 1.13 (t, J = 7.2 Hz, 3H); C NMR (101 MHz, CDCl ) δ
3
73.77, 170.72, 158.66, 130.47, 129.40, 52.46, 41.28, 40.50, 36.46, 35.31, 30.11, 29.63, 29.43,
9.37, 29.35, 29.31, 29.12, 27.18, 27.16, 27.01, 25.69, 15.66. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C H N O 412.3170; Found 412.3201.
22
41
3
4
To a 0 °C solution of the above amide (70 mg, 0.156 mmol) in THF/MeOH/H O (0.500 mL
2
each) was added 2M NaOH solution (0.200 mmol, 1.30 equiv, 100 µL). The mixture was stirred
at rt for 48 h, and then concentrated in vacuo. The residue was dissolved in H O (2 mL) and pre-
2
washed (alternate washes with EtOH and H O, 3 times) Bio-Beads™ SM-2 absorbent (Bio-Rad,
2
20-50 mesh, 3 g) was added to the solution. The mixture was allowed to stand at rt for 4 h and
then the beads were collected on a Buchner funnel. The beads were washed with cold H O (2 mL),
2
and the product was stripped from the beads with EtOH. Concentration of the EtOH filtrate gave
1
7
(52 mg, 79%) as a white solid, mp 163.8-169.8 °C. H NMR (400 MHz, CD OD) δ 5.44–5.27
3
(m, 2H), 3.74 (s, 2H), 3.19–3.03 (m, 4H), 2.24 (t, J = 7.8 Hz, 2H), 2.11–1.98 (m, 4H), 1.69–1.55
1
3
(
m, 2H), 1.55–1.42 (m, 2H), 1.43–1.23 (m, 14H), 1.09 (t, J = 7.2 Hz, 3H); C NMR (101 MHz,
CD OD) δ 176.59, 175.80, 161.24, 131.14, 130.48, 44.54, 40.88, 37.17, 35.75, 31.01, 30.83, 30.59,
3
30.48, 30.46, 30.39, 30.32, 28.14, 28.10, 27.91, 26.87, 15.85. HRMS (ESI-TOF) m/z: [M+2H-
+
Na] Calcd for C H N O Na 398.3013; Found 398.3013.
2
1
38
3
4
1
2
Synthesis of sodium N -methyl-N -(16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-
enoyl)glycinate (8). To a mixture of 16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoic
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acid acid (2b, 50 mg, 0.141 mmol), sarcosine methyl ester hydrochloride (23.6 mg, 0.169 mmol,
1
.20 equiv), and EDCI·HCl (40.5 mg, 0.212 mmol, 1.50 equiv) in MeCN/CH Cl (3:1, 4 mL) was
2
2
added DMAP (48.2 mg, 0.395 mmol, 2.80 equiv). After stirring for 48 h at rt, the reaction mixture
was diluted with water (5 mL) and extracted with EtOAc (3 × 10 mL). The combined organic
extracts were washed with brine (10 mL), dried over Na SO , concentrated under reduced pressure,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
1
2
and the residue was purified by TLC (55% EtOAc/hexanes, 4 times) to give methyl N -methyl-N -
(
16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoyl)glycinate (46 mg, 74%) as a white
1
solid, mp 79.4 °C. TLC: 65% EtOAc, R ~ 0.2. H NMR (500 MHz, CDCl 4.5:1 mixture of amide
f
3;
rotamers) δ 7.59 (br s, 1H), 7.54 (br s, 1H), 5.43–5.20 (m, 2H), 4.10 (s, 1.59H), 4.03 (s, 0.34H),
3.75 (s, 0.6H), 3.70 (s, 2.2H), 3.34–3.24 (m, 2H), 3.05 (s, 2.3H), 2.95 (s, 0.5H), 2.88 (d, J = 5.2
Hz, 3H), 2.35 (t, J = 7.7 Hz, 1.66H), 2.19 (t, J = 7.6 Hz, 0.31H), 2.08–1.89 (m, 4H), 1.67–1.48 (m,
1
3
4
H), 1.41–1.14 (m, 14H); C NMR (101 MHz, CDCl ) δ 173.98, 173.53, 170.06, 169.70, 160.66,
3
159.79, 130.70, 128.91, 52.49, 52.12, 51.57, 49.33, 39.63, 36.62, 36.41, 34.89, 33.21, 33.01, 29.76,
2
9.55, 29.53, 29.50, 29.44, 29.35, 28.87, 27.30, 26.93, 26.76, 26.24, 25.12, 24.94. HRMS (ESI-
+
TOF) m/z: [M+H] Calcd for C H N O 440.3119; Found 440.3123.
2
3
41
3
5
To a 0 °C solution of the above amide (34 mg, 0.077 mmol) in THF/H O (0.900/0.250 mL) was
2
added 2M aq. NaOH solution (0.100 mmol, 1.30 equiv, 50 µL). The solution was stirred at rt for
4
8 h, and then concentrated in vacuo. The residue was dissolved in H O (2 mL) and pre-washed
2
(
alternate washes with EtOH and H O, 3 times) Bio-Beads™ SM-2 absorbent (Bio-Rad, 20-50
2
mesh, 3 g) was added. The mixture was allowed to stand at 2-4 °C for 14 h and then the beads
were collecetd on a Buchner funnel. The beads were washed with cold H O (2 × 2 mL), and the
2
product was stripped from the beads with EtOH. Concentration of the EtOH filtrate gave 8 (29 mg,
1
84%) as a white solid, mp 145.6-163.4 °C. H NMR (400 MHz, CD OD; 1:1 mixture of amide
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2
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2
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rotamers) δ 5.43–5.26 (m, 2H), 3.93 (s, 1H), 3.87 (s, 1H), 3.30–3.18 (m, 2H), 3.05 (s, 1.4H), 2.94
(s, 1.6H), 2.82 (s, 3H), 2.46–2.36 (m, 1H), 2.35–2.25 (m, 1H), 2.12–1.97 (m, 4H), 1.67–1.50 (m,
1
3
4H), 1.44–1.23 (m, 14H); C NMR (101 MHz, CD OD) δ 176.63, 176.29, 175.93, 175.71, 166.55,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
66.16, 162.39, 161.53, 131.29, 131.21, 130.34, 130.27, 55.19, 52.63, 40.37, 37.02, 35.47, 34.25,
3
4.10, 30.82, 30.64, 30.61, 30.59, 30.57, 30.53, 30.33, 30.06, 29.87, 28.14, 28.05, 27.84, 27.78,
+
2
6.38, 26.30, 26.16. HRMS (ESI-TOF) m/z: [M+2H-Na] Calcd for C H N O Na 426.2962;
22 38
3
5
Found 426.2961.
Synthesis of sodium (16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoyl)-L-
alaninate (9). To a mixture of 2b (55 mg, 0.155 mmol), N-hydroxysuccinimide (NHS, 21.4 mg,
0.186 mmol, 1.20 equiv), and dicyclohexylcarbodiimide (DCC, 35.2 mg, 0.171 mmol, 1.10 equiv)
in dry EtOAc (2.55 mL) was added DMAP (1.89 mg, 0.016 mmol, 0.1 equiv). After stirring for
®
2
4 h at 40 °C, the reaction mixture was cooled to rt, filtered through a pad of Celite with EtOAc,
and the filtrate was concentrated to give the crude NHS ester.
To a mixture of the above crude NHS ester and L-alanine (18 mg, 0.202 mmol, 1.30 equiv) in
MeCN/H O/DMF (3/0.5/0.5 mL) was added DMAP (28 mg, 0.233 mmol, 1.50 equiv). After 2
2
days, the reaction mixture was diluted with EtOAc (12 mL) and H O (6 mL), cooled to 0 °C and
2
acidified to pH 2.0 with 2N aq. HCl. The organic phase was separated and the aqueous phase was
re-extracted with EtOAc (12 mL × 2). The combined organic extracts were washed with brine,
dried over Na SO , filtered, and concentrated in vacuo. Flash chromatography of the residue (4 g
2
4
SiO column eluted with CH Cl , 5 min; 0-5% MeOH/CH Cl , 40 min; 5% MeOH/CH Cl , 50
2
2
2
2
2
2
2
min) gave the adduct (25 mg, 37%; TLC: 10% MeOH/CHCl , R ~ 0.13) as a white solid. The
3
f
crude product (15 mg, 0.035 mmol) was dissolved in MeOH (4 mL) and NaHCO (3.85 mg, 0.046
3
mmol, 1.30 equiv) was added. After stirring for 6 h, the product was filtered through a syringe
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filter (Millex-FH, 0.45 µM, PTFE) and concentrated to give 9 (15 mg) as a white solid, mp 129.3-
1
1
29.4 °C. H NMR (500 MHz, CD OD) δ 5.42–5.29 (m, 2H), 4.28 (q, J = 7.1 Hz, 1H), 3.25 (t, J
3
= 7.1 Hz, 2H), 2.82 (s, 3H), 2.22 (t, J = 7.6 Hz, 2H), 2.11–1.98 (m, 4H), 1.66–1.52 (m, 4H), 1.43–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
1
5
1
.24 (m, 17H); C NMR (101 MHz, CD OD) δ 178.56, 175.44, 162.42, 161.56, 131.31, 130.28,
3
0.98, 40.38, 37.12, 30.82, 30.60, 30.58, 30.47, 30.31, 29.87, 28.13, 28.05, 27.77, 26.94, 26.29,
+
8.85. HRMS (ESI-TOF) m/z: [M+2H-Na] Calcd for C H N O Na 426.2963; Found 426.2962.
22 39
3
5
1
2
Synthesis of N -methyl-N -(16-oxo-16-(phenylsulfonamido)hexadec-5(Z)-en-1-yl)oxalamide
10). To a mixture of 2b (30 mg, 0.091 mmol) and benzenesulfonamide (13 mg, 0.091 mmol) in
(
anhydrous DMF (5 mL) were added DMAP (13 mg, 0.120 mmol, 1.30 equiv) and EDCI·HCl (16
mg, 0.120 mmol, 1.30 equiv). After stirring for 24 h at rt, the reaction mixture was diluted with
water (5 mL) and extracted with EtOAc (3 × 10 mL), The combined organic extracts were washed
with brine (10 mL), dried over Na SO , concentrated under reduced pressure, and the residue was
2
4
purified by TLC using 70% EtOAc as eluent to give 10 (35 mg, 84%) as a white solid, mp 71.6
1
°
C. TLC: 50% EtOAc, R ~ 0.28. H NMR (500 MHz, CDCl ) δ 9.75 (s, 1H), 8.08–8.01 (m, 2H),
f
3
7
.89 (br s, 1H), 7.72 (br s, 1H), 7.67–7.60 (m, 1H), 7.57–7.50 (m, 2H), 5.39–5.27 (m, 2H), 3.31
(app q, J = 7.0, 2H), 2.92 (d, J = 5.2 Hz, 3H), 2.27 (t, J = 8.0, 2H), 2.09–1.94 (m, 4H), 1.63–1.51
1
3
(
m, 4H), 1.44–1.35 (m, 2H), 1.35–1.27 (m, 2H), 1.20 (s, 10H); C NMR (101 MHz, CDCl ) δ
3
1
2
71.38, 160.63, 160.00, 138.91, 133.93, 130.76, 129.08, 129.04, 128.38, 39.83, 36.43, 29.27,
8.97, 28.94, 28.88, 28.67, 27.06, 26.86, 26.82, 26.44, 24.41. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C H N O S 494.2683; Found 494.2690.
25
39
3
5
Synthesis of sodium 15-(2-(methylamino)-2-oxoacetamido)pentadec-10(Z)-en-1-sulfonate
11). A mixture of 27 (0.2 g, 0.460 mmol), Na SO (0.231 g, 1.83 mmol, 4 equiv), EtOH (3 mL),
(
2
3
cyclohexene (0.932 mL, 9.20 mmol, 20 equiv) and water (1.5 mL) was heated at 85 °C in a sealed
2
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