14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates: Carboxylate modifications

Article abstract of DOI:10.1021/jm500262m

The cytochrome P450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a powerful endogenous autacoid that has been ascribed an impressive array of physiologic functions including regulation of blood pressure. Because 14,15-EET is chemically and metabolically labile, structurally related surrogates containing epoxide bioisosteres were introduced and have become useful in vitro pharmacologic tools but are not suitable for in vivo applications. A new generation of EET mimics incorporating modifications to the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase (sEH). Tetrazole 19 (ED₅₀ 0.18 μM) and oxadiazole-5-thione 25 (ED₅₀ 0.36 μM) were 12- and 6-fold more potent, respectively, than 14,15-EET as vasorelaxants; on the other hand, their ability to block sEH differed substantially, i.e., 11 vs >500 nM. These data will expedite the development of potent and specific in vivo drug candidates.

Full text of DOI:10.1021/jm500262m

Article
pubs.acs.org/jmc
Open Access on 08/13/2015
14,15-Epoxyeicosa-5,8,11-trienoic Acid (14,15-EET) Surrogates:
Carboxylate Modifications
John R. Falck,*,† Sreenivasulu Reddy Koduru,† Seetaram Mohapatra,† Rajkumar Manne,† Raju Atcha,†
Vijaya L. Manthati,† Jorge H. Capdevila,‡ Sarah Christian,§ John D. Imig,§ and William B. Campbell§
†Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, Texas 75390, United States
‡Departments of Medicine and Biochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232, United States
§Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States
S
* Supporting Information
ABSTRACT: The cytochrome P450 eicosanoid 14,15-epox-
yeicosa-5,8,11-trienoic acid (14,15-EET) is a powerful
endogenous autacoid that has been ascribed an impressive
array of physiologic functions including regulation of blood
pressure. Because 14,15-EET is chemically and metabolically
labile, structurally related surrogates containing epoxide
bioisosteres were introduced and have become useful in vitro
pharmacologic tools but are not suitable for in vivo applications. A new generation of EET mimics incorporating modifications to
the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase (sEH). Tetrazole 19
(ED50 0.18 μM) and oxadiazole-5-thione 25 (ED50 0.36 μM) were 12- and 6-fold more potent, respectively, than 14,15-EET as
vasorelaxants; on the other hand, their ability to block sEH differed substantially, i.e., 11 vs >500 nM. These data will expedite the
development of potent and specific in vivo drug candidates.
substructures characteristic of polyunsaturated fatty acids,
necessitates handling and/or storage under strict conditions
that minimize exposure to oxygen and trace transition metals.16
A precis of inactivating enzymatic processes includes
esterification,17 metabolism by other pathways of the
arachidonate cascade,18 conjugation,19 β-oxidation,20 chain
elongation,21 and hydrolysis of the epoxide.22 In biological
milieu, hydration of the epoxide by soluble epoxide hydrolase
(sEH) is a major determinant in maintaining the steady state
levels of 14,15-EET,23 whose half-life has been estimated at no
more than seconds to minutes.24 The pioneering studies of
Hammock25 and others26 confirmed inhibition of sEH can
elevate EET levels both in vitro and in vivo, thus offering an
indirect means for pharmacologic intervention in EET-
mediated processes. However, this strategy might prove less
efficacious than an agonist replacement therapy whenever
endogenous EET biosynthesis is compromised, for instance, as
a consequence of disease, inflammation, radiotherapy, aging,
and/or exposure to xenobiotics and drugs that inhibit
cytochromes P450.27
INTRODUCTION
■
An imposing body of studies,1 spanning more than three
decades, has cogently elucidated the involvement of epox-
yeicosatrienoic acids (EETs) in a wide array of critical
physiological functions, inter alia, blood pressure regulation,2
nociception,3 adipogenesis,4 anti-inflammatory activity,5 organ
regeneration,6 insulin potentiation,7 podocyte integrity,8 and
cellular responses to bacterial infection.9 The most prominent
regioisomer,10 14,15-epoxyeicosa-5(Z),8(Z),11(Z)-trienoic acid
(14,15-EET), along with other members of this autacoid family,
arise via metabolism of arachidonic acid by cytochromes P450,
especially members of the 2C11 and 2J12 families. The ratio of
EET isomers and their stereocomposition are CYP P450
isoform-dependent13 and, thus, species- and tissue-specific.14
Numerous chemical and metabolic factors conspire to
complicate investigations into the roles of 14,15-EET and
restrain its potential therapeutic utility (Figure 1).15 Its
susceptibility toward aerial oxidation or auto-oxidation, i.e., a
nonenzymatic, free radical process involving the 1,4-dienyl
RESULTS AND DISCUSSION
■
To address some of the stability limitations of natural EETs,
our laboratories previously prepared several iterations of EET
surrogates with improved stability.28,29 Advanced versions were
Received: February 19, 2014
Published: August 13, 2014
Figure 1. Major routes of metabolism/degradation (oxid = oxidation).
© 2014 American Chemical Society
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Table 1. Vasorelaxation of Precontracted Bovine Coronary Artery and in Vitro Inhibition of Recombinant Human Soluble
a,b
Epoxide Hydrolase
a
At 10 μM, 14,15-EET induces 85% of maximum vasorelaxation and its ED50 is 2.2 μM. For recombinant human sEH, the IC50 for 12-(3-adamantan-
b
1-yl-ureido)dodecanoic acid (AUDA) is 3 nM. Bioassay determinations (n) = 3−5.
evaluated for relaxation of precontracted bovine coronary artery
rings and for in vitro inhibition of recombinant human sEH.15
Depending upon the bioisostere and its position along the
carbon chain, varying levels of vascular relaxation and/or sEH
inhibition were observed. Generally, oxamides and N-iPr-
amides displayed useful 14,15-EET agonist activities but were
modest to poor sEH inhibitors. Unsubstituted ureas proved to
be both potent 14,15-EET agonists and sEH inhibitors. The in
vitro success of this generation of analogues prompted us to
consider further structural iterations.30
The surrogates described herein modify the free carboxylic
acid of the previous generation of 14,15-EET mimics while, for
the most part, retaining some key structural features of the
pharmacophore identified earlier (i.e., cis-Δ8,9-olefin and an
epoxide bioisostere). They were evaluated as described before
in precontracted bovine coronary artery rings for (i) %
vasorelaxation at 10 μM relative to a 14,15-EET control, (ii)
EC50 for vasorelaxation, and (iii) IC50 for sEH inhibition (Table
1).15 Interestingly, the simple expedient of conjugating the
carboxylate with a short poly(ethylene oxide) (PEG) unit
improved both % vasorelaxation and the EC50 somewhat
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compared with the parent free acids31 regardless the type of
epoxide bioisostere, viz., N-iPr-amide 1, urea 2, and oxamide 3;
on the other hand, the soluble epoxide hydrolase (sEH)
inhibitory activity of urea 2 was significantly compromised
versus the parent free acid (EC50 7.5 μM, IC50 46 nM). Linkage
of the carboxylate to the nitrogen of glycine (4 and 5) or
aspartic acid (6) resulted in a modest boost to the vasoactivities
while the sEH IC50 of 6 dropped by an order of magnitude
compared to the parent free acid. Conversion to the N-phenyl
and N-methylsulfonimides 7 and 8, respectively, left the
biological activities virtually unchanged in all categories while
the simplified phenylsulfonamide 9 led to an improvement in
the EC50 by a factor of ∼2 and smaller improvements in the
other parameters.
olefin was deleted, leading to N-iPr-amide 30, urea 31, and
oxamide 32, whose total chain length more closely resembled
that of 14,15-EET. As hoped, the EC50 values increased relative
to 29, although the % vasorelaxation was variable. All were
comparatively poor sEH inhibitors, including unexpectedly urea
31.
Contrary to expectations, there was no evident correlation
between the pKa of the EET analogues and the ED50 for
vasorelaxation (Figure 2), suggesting factors other than ionic
interactions are involved in binding the carboxylate at the
putative EET binding site.
Given the generally lackluster behavior of the esters and
amides, our focus changed to replacement of the carboxylate
with a variety of heterocyclic bioisosteres identified from
literature sources.32 For phosphonate 10 and sulfonate 11, the
increase in polarity did not improve potency as seen with the
PEG esters. Interestingly, sulfonate 11 retained its ability to
suppress sEH, in contrast to phosphonate 10 and PEG ester 2.
Vasorelaxation by S-alkylthiocatechol 12 was poor, but its IC50
for sEH was pushed down into the single digits.33 The sulfone
variant 13 regained some vasopotency, probably due to an
increase in the acidity of the phenol but not sufficiently to be
viable. Replacement of the phenol moiety in 12 with a tetrazole
ring34 produced 14 and was encouraging, but this trend was not
continued in triazole 15. Sequential oxidation of the sulfur to
sulfoxamide 16 and sulfonamide 17, as seen for 12 → 13,
incrementally improved the EC50. The ED50 jumped in the
tetrazole bioisostere series 18−21, achieving a submicromolar
value for urea 19 (12-fold better than 14,15-EET), while the %
vasorelaxation versus 14,15-EET peaked. Factoring in a very
respectable low nanomolar IC50, 19 is the best dual activity
analogue in the study and a leading candidate for further
development. Notably, all three parameters began to erode,
albeit minimally, in the one-carbon homologue 20 and further
in oxamide 21. The performance of another five-membered
heterocycle,35 oxadiazol-5-one 22, while acceptable, was not
comparable to urea 19. The closely related oxathiadiazole-2-
oxides35 23 and 24, on the other hand, demonstrated good
vasorelaxant activities; sEH inhibition potency was consistent
with our previous observations that ureas ≫ N-iPr-amides. The
isomeric thioxo-1,2,4-oxadiazol35 25 registered a 6-fold
improvement in ED50 over 14,15-EET yet was a comparatively
poor sEH inhibitor and, thus, is a good option whenever an
EET agonist with minimal impact on sEH function is required.
The corresponding urea thioxo-oxadiazol,36 26, could not
sustain the vasopotency; its ED50 sagged by almost an order of
magnitude versus 25 while at the same time its sEH IC50
improved by more than an order of magnitude. A final variant
on this heterocyclic theme, 5-thioxothiadiazol36 27, fell short of
expectations on all levels and further study of this particular
heterocycle was terminated. Despite its success in the glitazone
series of antidiabetic drugs,37 the 2,4-thiazolidinedione
modification proved disappointing when incorporated into
28. Given the larger size of the N-(4-hydroxy-2-
benzothiazolyl)acetamide bioisostere,38 the carbon chain
utilized in most of the analogues (Figure 1) was trimmed by
four carbons before attachment via the phenolic oxygen to
generate 29. The undistinguished results caused us to re-
evaluate our assumptions. Following inspection of molecular
models, the carbon chain was shortened even further and the
Figure 2. Plot of calculated pKa vs ED50 (μM) of analogues in Table 1.
The protonated form of analogues 6, 11, and 12 was used for the
calculation.
Chemistry. The syntheses of tetrazole 19 and thioxo-1,2,4-
oxadiazol 25 are summarized in Scheme 1 and illustrate the
approach used to prepare the other analogues. Semihydroge-
nation of the known39 acetylene 34 using P-2 nickel and H2 led
to cis-olefin 35 that was subjected to azidation using
diphenylphosphoryl azide (DPPA) under Mitsunobu condi-
tions. Staudinger reduction of the product, azide 36, led to
primary amine 37 that was reacted without purification with n-
pentyl isocyanate to furnish urea 38. An uneventful series of
functional group interchanges proceeding through alcohol 39,
bromide 40, and ending with nitrile 41 set the stage for the zinc
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TLC or flash chromatography using prepacked SiO2 columns on a
CombiFlash Rf200 chromatograph (Teledyne Isco). Unless otherwise
noted, yields refer to isolated, purified material with spectral data
consistent with assigned structures or, if known, were in agreement
with published data. Reagents were purchased at the highest
commercial quality available and used without further purification,
unless otherwise noted. Anhydrous solvents were dried using a Glass
Contours solvent system by passage through columns of activated
packing material under argon immediately prior to use.
Scheme 1. Synthesis of Representative 14,15-EET
Analogues
a
1-(12-(1H-Tetrazol-5-yl)dodec-5(Z)-en-1-yl)-3-pentylurea
(19). A mixture of 1-(12-cyanododec-5(Z)-enyl)-3-n-pentylurea (41).
(500 mg, 1.55 mmol), sodium azide (100 mg, 1.55 mmol), and zinc
bromide (335 mg, 1.48 mmol) was heated at 110 °C in 2-propanol/
H2O (1:3, 8 mL) while stirring vigorously in a sealed tube.40 After 18
h, the mixture was cooled to room temperature and the pH was
adjusted to 1 using aq HCl (3 N, 4 mL). Ethyl acetate (10 mL) was
added, and the stirring was continued until no solid was present. The
organic layer was isolated and the aqueous layer extracted with EtOAc
(2 × 25 mL). The combined organic fractions were washed with water
(3 × 25 mL), dried, and concentrated in vacuo. The residue was
purified by silica gel column chromatography to give the tetrazole 19
(431 mg, 76%) as a colorless solid, mp 205.6−205.8 °C. TLC: 10%
a
Reagents and conditions: (a) P2-Ni/(H2NCH2)2, H2 (1 atm), EtOH,
1
MeOH/CH2Cl2, Rf ∼ 0.30. H NMR (CD3OD, 300 MHz) δ 5.40−
rt, 1 h (96%); (b) DIAD/Ph3P/Ph2P(O)N3, THF, −20 to 23 °C, 4 h
(72%); (c) Ph3P, H2O/THF, rt, 12 h (76%); (d) C5H11NCO, THF,
rt, 3 h (76%); (e) nBu4NF, THF, rt, 12 h (82−89%); (f) CBr4/Ph3P,
CH2Cl2, rt, 2 h (83−84%); (g) KCN, DMSO, rt, 12 h (78−81%); (h)
NaN3/ZnBr2, iPrOH/H2O (1:3), 110 °C, 18 h (76%); (i) HO-
(CO)2NH(CH2)3CH3, EDCI, DMF, rt, 12 h (73%); (j) H2NOH/
Na2CO3, MeOH/H2O (4:1), 60 °C, 18 h (62%); (k) Im2C(S), THF,
rt, 45 min (63%).
5.30 (m, 2H), 3.06−3.11 (m, 4H), 2.93 (t, J = 8.0 Hz, 2H), 1.98−2.10
(m, 4H), 1.70−1.82 (m, 2H), 1.24−1.50 (m, 16H), 0.90 (t, J = 7.6 Hz,
3H). 13C NMR (CD3OD, 75 MHz) δ 160.16, 156.81, 129.77, 129.47,
39.81, 39.68, 29.88, 29.80, 29.35, 28.99, 28.69, 28.55, 27.48, 26.85,
26.81, 26.68, 22.96, 22.31, 13.22. HRMS calcd for C19H37N6O [M +
1]+ 365.3029, found 365.3030.
N1-Butyl-N2-(12-(2-oxido-3H-1,2,3,5-oxathiadiazol-4-yl)-
dodec-5(Z)-en-1-yl)oxalamide (25). A mixture of N1-(13-amino-
13-(hydroxyimino)tridec-5(Z)-enyl)-N2-n-butyloxalamide (46) (100
mg, 0.27 mmol) and 1,1′-(thiocarbonyl)diimidazole (57 mg, 0.32
mmol) in dry THF (5 mL) was stirred at room temperature for 45
min. The mixture was diluted with water (20 mL) and extracted with
ethyl acetate (3 × 10 mL). The combined organic extracts were
washed with water and dried, and the solvent was evaporated in vacuo.
The residue was dissolved in acetonitrile (5 mL) to which was then
added DBU (61 mg, 0.40 mmol). After stirring at room temperature
for 1 h, the mixture was diluted with water (10 mL), adjusted pH ∼ 4
with 1 N HCl, and extracted with ethyl acetate (3 × 10 mL) to give
thioxo-1,2,4-oxadiazol 25 (71 mg, 63%) as a white solid, mp 110.6−
bromide mediated annulation40 with sodium azide that
delivered tetrazole 19.
Condensation of 37 with 2-(n-butylamino)-2-oxoacetic
acid15 gave rise to oxamide 42. Following the same sequence
of transformations as described above, 42 was converted into
nitrile 45. Condensation of hydroxyimine 46, obtained by
addition41 of hydroxylamine to 45, with 1,1′-(thiocarbonyl)-
diimidazole yielded thioxo-1,2,4-oxadiazol 25 as a crystalline
solid.
1
110.8 °C. TLC: MeOH/CH2Cl2 (1:9), Rf ∼ 0.55. H NMR (400
MHz) δ 8.90 (br s, NH, 1H), 7.52 (br s, NH, 2H), 5.28−5.40 (m,
2H), 3.20−3.40 (m, 4H), 2.59 (t, J = 7.5 Hz, 2H), 1.98−2.10 (m, 4H),
1.21−1.70 (m, 16H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz) δ
160.12, 160.08, 153.31, 130.62, 129.46, 39.93, 39.85, 31.35, 29.33,
28.94, 28.89, 28.68, 27.02, 26.84, 26.69, 23.96, 20.23, 13.90. HRMS
calcd for C19H35N4O4S [M + 1]+ 415.2379, found 415.2372.
EXPERIMENTAL SECTION
■
General Methods and Materials. Final compounds were judged
≥95% pure by HPLC using a Zorbax Eclipse C18 column (250 mm ×
4.6 mm; Agilent) connected to an Agilent 1200 API/LC-MS using
acetonitrile/water combinations as eluent unless otherwise noted.
Nuclear magnetic resonance (NMR) spectra were recorded on Varian
300, 400, or 500 spectrometers at operating frequencies of 300/400/
500 MHz (1H) or 75/100/125 MHz (13C) in CDCl3 with TMS as
12-(tert-Butyldiphenylsilyloxy)dodec-5(Z)-en-1-ol (35).
NaBH4 (82 mg, 2.28 mmol) was added in portions with vigorous
stirring to a room temperature solution of Ni(OAc)2·4H2O (567 mg,
2.28 mmol) in absolute ethanol (20 mL) under a hydrogen
atmosphere (1 atm). After 15 min, freshly distilled ethylenediamine
(0.30 mL, 4.56 mmol) was added to the black suspension, followed
after a further 15 min by a solution of 12-(tert-butyldiphenylsilyloxy)-
dodec-5-yn-1-ol39 (34) (4.0 g, 9.16 mmol) in absolute EtOH (10 mL).
After 1 h, the reaction mixture was diluted with Et2O (20 mL) and
passed through a small bed of silica gel. The bed was rinsed with
another portion of Et2O (5 mL). The combined ethereal filtrates were
concentrated under reduced pressure to afford alcohol 35 (3.85 g,
96%) as a colorless oil sufficiently pure to be used directly in the next
1
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). High resolution mass spectra (HRMS) were obtained
at UT-Arlington using a Shimadzu IT-TOF mass spectrometer or at
the Medical College of Wisconsin by Prof. Kasem Nithipatikom.
Infrared (IR) spectra were obtained using a PerkinElmer Spectrum
1000 Fourier transform spectrometer. Melting points were measured
using an OptiMelt from Stanford Research Systems 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 KMnO4 or phosphomolybdic
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 Na2SO4 and filtered prior to removal of all volatiles under
reduced pressure. Chromatographic purifications utilized preparative
1
step. TLC: EtOAc/hexanes (3:7), Rf ∼ 0.46. H NMR (300 MHz) δ
7.64−7.68 (m, 4H), 7.34−7.42 (m, 6H), 5.42−5.28 (m, 2H), 3.63 (t, J
= 6.4 Hz, 4H), 2.08−1.96 (m, 4H), 1.50−1.60 (m, 4H), 1.40−1.24 (m,
10H), 1.04 (s, 9H). 13C NMR (100 MHz) δ 135.81, 134.40, 130.61,
129.71, 129.60, 127.80, 64.21, 63.14, 32.78, 32.60, 29.98, 29.27, 27.42,
27.14, 27.10, 26.08, 25.92, 19.48. HRMS calcd for C28H43O2Si [M +
1]+ 439.3032, found 439.3027.
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1-tert-Butyldiphenylsilyloxy-12-azidododec-7(Z)-ene (36).
Diisopropyl azodicarboxylate (DIAD; 1.46 mL, 7.35 mmol) was
added dropwise to a −20 °C solution of PPh3 (2.10 g, 8.0 mmol) in
dry THF (45 mL) under an argon atmosphere. After 10 min, a
solution of 12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-en-1-ol (35)
(3.20 g, 7.35 mmol) in dry THF (10 mL) was added dropwise.
After 30 min, the mixture was warmed to 0 °C and diphenylphos-
phoryl azide (1.58 mL, 7.35 mmol) was added dropwise. After stirring
4 h at rt, the reaction mixture was quenched with water (150 mL) and
extracted with EtOAc (2 × 100 mL). The combined organic extracts
were washed with brine (100 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by SiO2 column
chromatography eluting with 4% EtOAc/hexane to afford azide 36
(2.45 g, 72%). TLC: EtOAc/hexanes (1:9), Rf ∼ 0.55. 1H NMR (400
MHz) δ 7.64−7.68 (m, 4H), 7.34−7.42 (m, 6H), 5.28−5.42 (m, 2H),
3.70 (t, J = 5.8 Hz, 2H), 3.27 (t, J = 6.3 Hz, 2H), 1.96−2.10 (m, 4H),
1.24−1.64 (m, 12H), 1.04 (s, 9H). 13C NMR (100 MHz) δ 135.84,
134.41, 130.93, 129.75, 129.12, 127.83, 64.22, 51.62, 32.81, 29.93,
29.30, 28.68, 27.46, 27.14, 27.02, 26.90, 25.96, 19.49. IR (neat) 2930,
2783, 2331, 2097, 1106 cm−1. HRMS calcd for C28H42N3OSi [M + 1]+
464.3097, found 464.3099.
solution of 1-(12-hydroxydodec-5(Z)-enyl)-3-n-pentylurea (39) (0.43
g, 1.38 mmol) in CH2Cl2 (20 mL). After 2 h at room temperature, the
reaction mixture was concentrated in vacuo and the residue was
purified via SiO2 column chromatography to give 1-(12-bromododec-
5(Z)-enyl)-3-n-pentylurea (40) (0.43 g, 83%) as a solid, mp 46.7−46.8
1
°C. TLC: EtOAc/hexanes (2:3), Rf ∼ 0.60. H NMR (300 MHz) δ
5.22−5.42 (m, 2H), 4.40 (br s, 2H), 3.42 (t, J = 9.3 Hz, 2H), 3.10−
3.20 (m, 4H), 1.98−2.10 (m, 4H), 1.80−1.90 (m, 2H), 1.25−1.55 (m,
16H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz) δ 159.51,
130.14, 129.69, 40.48, 40.39, 34.20, 32.96, 30.34, 29.67, 29.36, 28.58,
28.25, 27.31, 27.27, 27.17, 22.68, 14.26. HRMS calcd for
C18H36BrN2O [M + 1]+ 375.2011, found 375.2014.
1-(12-Cyanododec-5(Z)-enyl)-3-n-pentylurea (41). A mixture
of potassium cyanide (0.23 g, 3.54 mmol) and 1-(12-bromododec-
5(Z)-enyl)-3-n-pentylurea (40) (0.90 g, 2.40 mmol) was stirred in
DMSO (5 mL) at room temperature. After 12 h, the reaction mixture
was diluted with water (20 mL) and extracted with ethyl acetate (2 ×
50 mL). The combined organic extracts were washed with water (2 ×
25 mL), brine (25 mL), dried (Na2SO4), and passed through a silica
gel column to give nitrile 41 (0.62 g, 81%) as a colorless solid, mp 56−
57 °C. TLC: EtOAc/hexanes (2:3), Rf ∼ 0.45. 1H NMR (300 MHz) δ
5.29−5.40 (m, 2H), 4.27 (br s, −NH, 2H), 3.10−3.20 (m, 4H), 2.34
(t, J = 7.0 Hz, 2H), 1.98−2.08 (m, 4H) 1.24−1.70 (m, 18H), 0.89 (t, J
= 7.0 Hz, 3H). 13C NMR (125 MHz) δ 159.41, 129.94, 129.86,
120.14, 40.45, 40.35, 30.30, 29.50, 29.33, 28.70, 28.51, 27.26, 27.16,
25.47, 22.66, 17.28, 14.24. IR (neat) 2930, 2281, 2184, 2042, 1936,
1613, 1197, 1042 cm−1. HRMS calcd for C19H36N3O [M + 1]+
322.2858, found 322.2867.
1-tert-Butyldiphenylsilyloxy-12-aminododec-7(Z)-ene (37).
Triphenylphosphine (1.18 g, 4.50 mmol) was added to a stirring
solution of 1-tert-butyldiphenylsilyloxy-12-azidododec-7(Z)-ene (36)
(1.90 g, 4.10 mmol) in THF (12 mL) containing 10 drops of
deionized water. After 12 h, the reaction mixture was diluted with
CH2Cl2 (10 mL), dried, and concentrated in vacuo to give amine 37
(1.36 g, 76%) as a viscous, colorless oil that was used directly in the
next reaction without further purification. TLC: MeOH/CH2Cl2
N1-n-Butyl-N2-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-
enyl)oxalamide (42). A mixture of 2-(n-butylamino)-2-oxoacetic
acid15 (0.40 g, 2.70 mmol), 1-tert-butyldiphenylsilyloxy-12-amino-
dodec-7(Z)-ene (37) (1.20 g, 2.70 mmol), 1-hydroxybenzotriazole
(HOBt; 0.44 g, 3.30 mmol), and [1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride] (EDCI: 0.63 g, 3.30 mmol) in dry
DMF (5 mL) was stirred at room temperature overnight. The reaction
mixture was quenched with water (30 mL) and extracted into ethyl
acetate (3 × 20 mL). The combined organic extracts were washed with
water (2 × 10 mL) and brine (10 mL), dried, and concentrated in
vacuo. The residue was purified by SiO2 column chromatography to
give N1-n-butyl-N2-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)-
oxalamide (42) (1.10 g, 73%). TLC: EtOAc/hexanes (2:3), Rf ∼
1
(1:4), Rf ∼ 0.25. H NMR (400 MHz) δ 7.62−7.68 (m, 4H), 7.32−
7.40 (m, 6H), 5.30−5.40 (m, 2H), 3.63 (t, J = 5.2 Hz, 2H), 2.62 (t, J =
4.8 Hz, 2H), 1.92−2.06 (m, 4H), 1.40−1.58 (m, 4H), 1.20−1.40 (m,
8H), 1.03 (s, 9H). 13C NMR (100 MHz) δ 135.79, 134.37, 130.42,
129.70, 127.78, 64.19, 42.28, 33.44, 32.77, 29.93, 29.28, 27.40, 27.21,
27.10, 25.92, 19.44. HRMS calcd for C28H44NOSi [M + 1]+ 438.3192,
found 438.3186.
1-(12-(tert-Butyldiphenylsilyloxy)dodec-5(Z)-enyl)-3-n-pen-
tylurea (38). A solution of 1-tert-butyldiphenylsilyloxy-12-amino-
dodec-7(Z)-ene (37) (1.32 g, 3.0 mmol) in THF (5 mL) was added
dropwise to a stirring solution of n-pentyl isocyanate (0.386 mL, 3.0
mmol) in THF (10 mL). After 3 h stirring at room temperature, all
volatiles were removed under reduced pressure and the residue was
purified by SiO2 column chromatography eluting with 20% EtOAc/
hexane to afford urea 38 (1.26 g, 76%) as a viscous oil. TLC: EtOAc/
hexanes (2:3), Rf ∼ 0.40. 1H NMR (300 MHz) δ 7.60−7.70 (m, 4H),
7.35−7.42 (m, 6H), 5.28−5.42 (m, 2H), 5.16 (br s, −NH, 2H), 3.65
(t, J = 6.5 Hz, 2H), 3.08−3.20 (m, 4H), 1.96−2.08 (m, 4H), 1.22−
1.60 (m, 18H), 1.02 (s, 9H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100
MHz) δ 159.23, 135.80, 134.24, 130.52, 129.74, 129.49, 127.82, 64.22,
40.62, 40.54, 32.80, 30.33, 29.95, 29.37, 29.32, 27.46, 27.34, 27.18,
27.11, 25.97, 22.71, 19.46, 14.29. HRMS calcd for C34H55N2O2Si [M +
1]+ 551.4033, found 551.4032.
1
0.55. H NMR (400 MHz) δ 8.05 (br s, −NH, 2H), 7.66−7.74 (m,
4H), 7.32−7.42 (m, 6H), 5.30−5.42 (m, 2H), 3.67 (t, J = 3.9 Hz, 2H),
3.31 (q, J = 5.2 Hz, 4H), 1.96−2.10 (m, 4H), 1.50−1.64 (m, 6H),
1.22−1.44 (m, 10H), 1.06 (s, 9H), 0.92 (t, J = 7.8 Hz, 3H). 13C NMR
(100 MHz) δ 160.33, 135.80, 134.35, 130.73, 129.74, 129.20, 127.83,
64.17, 39.89, 39.69, 32.79, 31.48, 29.94, 29.29, 29.07, 27.46, 27.23,
27.14, 27.0, 25.96, 20.29, 19.46, 13.96. HRMS calcd for C34H53N2O3Si
[M + 1]+ 565.3826, found 565.3824.
N1-n-Butyl-N2-(12-hydroxydodec-5(Z)-enyl)oxalamide (43).
N1-n-Butyl-N2-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)-
oxalamide (42) (1.20 g, 2.12 mmol) was desilylated as described above
for 39 to give N1-n-butyl-N2-(12-hydroxydodec-5(Z)-enyl)oxalamide
(43) (0.568 g, 82%) as a colorless solid, mp 102.8−102.9 °C. TLC:
1-(12-Hydroxydodec-5(Z)-enyl)-3-n-pentylurea (39). A mix-
ture of 1-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)-3-n-penty-
lurea (38) (1.12 g, 2.0 mmol) and tetra-n-butylammonium fluoride
(2.20 mL of 1 M soln in THF, 2.2 mmol) in dry THF (10 mL) was
stirred at room temperature under an argon atmosphere for 12 h and
then evaporated to dryness in vacuo. The residue was dissolved in
EtOAc (50 mL) and washed with water (30 mL), brine (30 mL),
dried, and evaporated in vacuo. Purification of the residue via SiO2
column chromatography gave alcohol 39 (0.56 g, 89%) as a colorless
1
EtOAc/hexanes (7:3), Rf ∼ 0.55. H NMR (400 MHz) δ 7.69 (br s,
2H), 5.20−5.35 (m, 2H), 3.56 (t, J = 4.2 Hz, 2H), 3.26 (q, J = 5.6 Hz,
4H), 2.17 (br s, 1H), 1.95−2.02 (m, 4H), 1.44−1.56 (m, 6H), 1.20−
1.40 (m, 10H), 0.87 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz) δ
160.15, 130.66, 129.21, 62.98, 39.80, 39.63, 32.93, 31.39, 29.77, 29.18,
28.95, 27.26, 27.0, 26.88, 25.80, 20.18, 13.85. HRMS calcd for
C18H35N2O3 [M + 1]+ 327.2648, found 327.2648.
solid, mp 63.7−63.8 °C. TLC: EtOAc/hexanes (7:3), Rf ∼ 0.30. H
N1-(12-Bromododec-5(Z)-enyl)-N2-n-butyloxalamide (44).
N1-n-Butyl-N2-(12-hydroxydodec-5(Z)-enyl)oxalamide (43) (330 mg,
1.0 mmol) was brominated as described above for 40 to give N1-(12-
bromododec-5(Z)-enyl)-N2-n-butyloxalamide (44) (330 mg, 84%) as
a white solid, mp 46.0−46.3 °C. TLC: EtOAc/hexanes (3:2), Rf ∼
0.55. 1H NMR (400 MHz) δ 7.79 (br s, −NH, 1H), 7.77 (br s, −NH,
1H), 5.20−5.32 (m, 2H), 3.32 (t, J = 6.4 Hz, 2H), 3.22 (q, J = 7.2 Hz,
4H), 1.90−2.00 (m, 4H), 1.72−1.82 (m, 2H), 1.42−1.56 (m, 4H),
1.20−1.40 (m, 10H), 0.85 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz) δ
1
NMR (300 MHz) δ 5.25−5.42 (m, 2H), 4.48 (br s, −NH, 2H), 3.64
(d, J = 6.5 Hz, 2H), 3.08−3.20 (m, 4H), 1.96−2.14 (m, 4H), 1.22−
1.60 (m, 18H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz) δ
159.26, 130.23, 129.62, 63.72, 40.33, 40.29, 32.92, 30.30, 30.26, 29.74,
29.35, 29.13, 27.26, 27.20, 27.13, 25.82, 22.69, 14.27. HRMS calcd for
C18H37N2O2 [M + 1]+ 313.2855, found 313.2857.
1-(12-Bromododec-5(Z)-enyl)-3-n-pentylurea (40). CBr4 (0.55
g, 1.66 mmol) and PPh3 (0.43 g, 1.66 mmol) were added to a 0 °C
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Journal of Medicinal Chemistry
Article
160.17, 160.15, 130.40, 129.34, 39.77, 39.59, 34.12, 32.93, 31.40,
29.62, 29.0, 28.54, 27.25, 27.24, 27.0, 26.91, 20.18, 13.85. HRMS calcd
for C18H34BrN2O2 [M + 1]+ 389.1804, found 389.1809.
and assigned all rights to the Medical College of Wisconsin and
the University of Texas Southwestern Medical Center.
N1-n-Butyl-N2-(12-cyanododec-5(Z)-enyl)oxalamide (45). N1-
(12-Bromododec-5(Z)-enyl)-N2-n-butyloxalamide (44) (250 mg,
0.642 mmol) was treated with potassium cyanide as described above
for 41 to give N1-n-butyl-N2-(12-cyanododec-5(Z)-enyl)oxalamide
(45) (168 mg, 78%) as a colorless solid, mp 83.0−83.3 °C. TLC:
ACKNOWLEDGMENTS
■
Financial support provided in part by NIH (GM32178,
DK38226, HL83297) and the Robert A. Welch Foundation
(GL 625910).
1
EtOAc/hexanes (3:2), Rf ∼ 0.35. H NMR (400 MHz) δ 7.45 (br s,
−NH, 2H), 5.30−5.40 (m, 2H), 3.34 (q, J = 8.6 Hz, 4H), 2.32 (t, J =
7.6 Hz, 2H), 1.98−2.08 (m, 4H), 1.30−1.68 (m, 16H), 0.92 (t, J = 7.2
Hz, 3H). 13C NMR (100 MHz) δ 160.03 (2C), 130.03, 129.08,
120.10, 39.88, 39.42, 31.22, 29.40, 28.82, 28.60, 28.42, 27.07, 27.06,
26.82, 25.54, 20.06, 17.01, 13.80. HRMS calcd for C19H34N3O2 [M +
1]+ 336.2651, found 336.2650.
ABBREVIATIONS USED
■
14,15-EET, cis-14,15-epoxyeicosa-5(Z),8(Z),11(Z)-trienoic
acid; sEH, soluble epoxide hydrolase
N1-(13-Amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N2-n-bu-
tyloxalamide (46). To a suspension of N1-n-butyl-N2-(12-cyanodo-
dec-5(Z)-enyl)oxalamide (45) (420 mg, 1.29 mmol) in MeOH/H2O
(4:1; 12 mL) was added H2NOH·HCl (228 mg, 3.28 mmol) and
Na2CO3 (344 mg, 3.25 mmol).41 The reaction mixture was heated at
60 °C for 18 h then cooled to room temperature, and all volatiles were
removed in vacuo. The residue was diluted with water (30 mL) and
extracted into ethyl acetate (2 × 25 mL). The combined organic
extracts were washed with water (2 × 10 mL) and brine (10 mL),
dried, and purified via silica gel column chromatography to give N1-
(13-amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N2-n-butyloxalamide
(46) (287 mg, 62%) as a colorless solid, 116.3−116.4 °C. TLC:
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MeOH/CH2Cl2 (1:4), Rf ∼ 0.20. H NMR (CD3OD, 400 MHz) δ
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<
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and copies of the 1H/13C NMR
spectra for all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 214-648-2406. Fax: 214-648-6455. E-mail: j.falck@
utsouthwestern.edu.
■
Notes
The authors declare the following competing financial
interest(s): JRF, JDI, and WBC authored patents describing
the synthesis and clinical uses of the analogues described herein
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