Synthesis and evaluation of fatty acid amides on the N-oleoylethanolamide-like activation of peroxisome proliferator activated receptor α

Article abstract of DOI:10.1248/cpb.c14-00881

A series of fatty acid amides were synthesized and their peroxisome proliferator-activated receptor α (PPAR-α) agonistic activities were evaluated in a normal rat liver cell line, clone 9. The mRNAs of the PPAR-α downstream genes, carnitine-palmitoyltransferase-1 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) as PPAR-α agonistic activities. We prepared nine oleic acid amides. Their PPAR-α agonistic activities were, in decreasing order, N-oleoylhistamine (OLHA), N-oleoylglycine, Oleamide, N-oleoyltyramine, N-oleoylsertonin, and Olvanil. The highest activity was found with OLHA. We prepared and evaluated nine N-acylhistamines (N-acyl-HAs). Of these, OLHA, C16:0-HA, and C18:1Δ⁹-trans-HA showed similar activity. Activity due to the different chain length of the saturated fatty acid peaked at C16:0-HA. The PPAR-α antagonist, GW6471, inhibited the induction of the PPAR-α downstream genes by OLHA and N-oleoylethanolamide (OEA). These data suggest that N-acyl-HAs could be considered new PPAR-α agonists.

Full text of DOI:10.1248/cpb.c14-00881

278
Chem. Pharm. Bull. 63, 278–285 (2015)
Vol. 63, No. 4
Regular Article
Synthesis and Evaluation of Fatty Acid Amides on the
N-Oleoylethanolamide-Like Activation of Peroxisome Proliferator
Activated Receptor α
Koichi Takao,* Kaori Noguchi, Yosuke Hashimoto, Akira Shirahata, and Yoshiaki Sugita
Faculty of Pharmaceutical Sciences, Josai University; 1–1 Keyaki-dai, Sakado, Saitama 350–0295, Japan.
Received December 24, 2014; accepted January 30, 2015
A series of fatty acid amides were synthesized and their peroxisome proliferator-activated receptor
α (PPAR-α) agonistic activities were evaluated in a normal rat liver cell line, clone 9. The mRNAs of the
PPAR-α downstream genes, carnitine-palmitoyltransferase-1 and mitochondrial 3-hydroxy-3-methylglutaryl-
CoA synthase, were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) as
PPAR-α agonistic activities. We prepared nine oleic acid amides. Their PPAR-α agonistic activities were, in
decreasing order, N-oleoylhistamine (OLHA), N-oleoylglycine, Oleamide, N-oleoyltyramine, N-oleoylsertonin,
and Olvanil. The highest activity was found with OLHA. We prepared and evaluated nine N-acylhistamines
(N-acyl-HAs). Of these, OLHA, C16:0-HA, and C18:1Δ9-trans-HA showed similar activity. Activity due to
the different chain length of the saturated fatty acid peaked at C16:0-HA. The PPAR-α antagonist, GW6471,
inhibited the induction of the PPAR-α downstream genes by OLHA and N-oleoylethanolamide (OEA). These
data suggest that N-acyl-HAs could be considered new PPAR-α agonists.
Key words N-oleoylhistamine; fatty acid amide; N-oleoylethanolamide; peroxisome proliferator-activated
receptor-α; clone 9 cell
The obesity epidemic continues to spread throughout the and N-oleoylvanillylamine (Olvanil). The compounds were
world, increasing the need for efficient therapies to combat synthesized by the condensation of oleoylchloride (2), derived
obesity. We are conducting ongoing investigations into new from oleic acid (1) and oxalyl chloride, with the corresponding
leads targeting peroxisome proliferator activated receptor α biogenic amines: histamine, tyramine, serotonine, dopamine,
(PPAR-α). PPAR-α is a nuclear receptor and a key regulator putrescine, spermine, glycine and vanillylamine, respectively
of lipid metabolism and energy balance in mammals. Thus, (Chart 1). OLPut and OLSpm were synthesized by the conden-
PPAR-α agonists such as the ethanolamides of fatty acids1–5) sation of the oleoylchloride with the Boc-protected polyamines
may have potential as antiobesity or antihyperlipidemic drugs. (3 and 4) followed by a deprotection step. Satisfactory yields
The ethanolamides of different long-chain fatty acids were obtained in all cases.
constitute a class of naturally occurring lipid molecules that
Evaluation of PPAR-α Agonist Activity Clone 9 cells, a
are collectively referred to as N-acylethanolamides (NAEs). normal rat liver cell line, were used for evaluating the candi-
NAEs exhibit a wide variety of biological activities, depend- date compounds as PPAR-α agonists. Cell viability of Clone 9
ing on their acyl chains, by binding to and activating specific cells, determined by the trypan blue dye exclusion assay, was
receptors.5) For example, N-palmitoylethanolamide (PEA) was not reduced during 48h incubation in the presence of OEA,
reported to act as an anti-inflammatory and analgesic,6) and N- up to a concentration of 25µM; however, a 50µM solution of
oleoylethanolamide (OEA) to act as an appetite-suppressant1); OEA was cloudy. Incubation of Clone 9 cells with increasing
both actions are believed to be due to PEA, OEA acting on amounts of OEA led to a concentration-dependent increase
PPAR-α.
in mRNA levels of the PPAR-α downstream genes, CPT-1
In this study, fatty acid amides of endogenous fatty acids and mHMG-CoA Syn (data not shown). Thus, the agonist
and various biogenic amines were synthesized and evaluated activities of the candidate compounds were compared at 25µM
for their OEA-like activity to PPAR-α. To evaluate PPAR-α concentration. Under these conditions, N-arachidonoylethanol-
activation, we analyzed the mRNA levels of selected PPAR-α amide (AEA: anandamide) had no effect on the expression of
downstream genes such as mitochondrial 3-hydroxy-3-meth- the downstream genes, while PEA provided similar expression
ylglutaryl-CoA synthase (mHMG-CoA Syn) and carnitine- levels to OEA (Figs. 2A, B). These results were consistent
palmitoyltransferase-1 (CPT-1) in Clone 9 rat hepatocyte cells, with those reported previously using another assay system.1,3)
according to the report7) that these genes responded similarly The synthesized oleic acid amides were examined for PPAR-α
against PPAR-α agonist in human and rat hepatocyte cells.
agonist activity by using our method (Figs. 3A, B). OLHA,
OLGly, oleamide, OLTA, OL-5-HT, and Olvanil exhibited ag-
onistic activities, with OLHA showing the most potent activ-
Results and Discussion
Chemical We prepared nine oleic acid amides (chemi- ity (Fig. 3B). In contrast, OLDA, OLPut, and OLSpm caused
cal structures as listed in Fig. 1): OEA, N-oleoylhista- cell death (data not shown).
mine (OLHA), N-oleoyltyramine (OLTA), N-oleoylserotonine
We further prepared nine OLHA analogs (N-acylhistamines:
(OL-5-HT), N-oleoyldopamine (OLDA), N-oleoylputrescine N-acyl-HAs, Fig. 1) : N-octanoylhistamine (C8:0-HA), N-cap-
(OLPut), N-oleoylspermine (OLSpm), N-oleoylglycine (OLGly) roylhistamine (C10:0-HA), N-didecylhitamine (C12:0-HA), N-
*To whom correspondence should be addressed. e-mail: ktakao@josai.ac.jp
© 2015 The Pharmaceutical Society of Japan

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279
Fig. 1. Chemical Structures of the Fatty Acid Amides Used in This Study
tetradecylhistamine (C14:0-HA), N-palmitoylhistamine (C16:0- similar to C16:0-HA, whereas the activities of the linoleoyl
HA), N-stearoylhistamine (C18:0-HA), N-elaidoylhistamine and arachidonoyl derivatives were weak. These results were
(C18:1Δ9-trans-HA), N-linoleylhistamine (C18:2Δ9,12-cis-HA), consistent with the reports of N-acylethanolamides.1,8) Hista-
and N-arachidonoylhistamine (C20:4Δ5,8,11,14-cis-HA) and mine showed no activity, equivalent to the DMSO blank.
evaluated PPAR-α agonistic activity. Saturated fatty acid
We then examined the effect of a PPAR-α antagonist
histamine amides resulted in the expression of PPAR-α down- (GW6471)9) on the levels of OLHA-induced PPAR-α down-
stream genes, depending on acyl chain length (from C8:0 to stream gene mRNA levels in Clone 9 cells. As shown in Figs.
C18:0), with the maximum expression occurring in the pres- 5A and B, GW6471 significantly and incrementally inhibited
ence of C16:0 (Figs. 4A, B). Unsaturated fatty acid amides, the levels of CPT-1A and mHMG-CoA Syn mRNA, indicat-
OLHA and C18:1Δ9-trans-HA, showed a high level of activity, ing competition of OLHA or OEA with GW6471 at PPAR-α.

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Reagents and conditions: (a) (COCl)2, CH2Cl2; (b) Biogenic amine (RNH2), base; (c) Et3N, CH2Cl2; (d) TFA and then 2M HCl–MeOH (1:1).
Chart 1. Protocol for the Synthesis of Oleic Acid Amides
Fig. 2. Effects of NAEs on the mRNA Levels of CPT-1A (A) and
mHMG-CoA Syn (B) in Clone 9 Cells
Fig. 3. Effects of Fatty Acid Amides on the mRNA Levels of CPT-1A
(A) and mHMG-CoA Syn (B) in Clone 9 Cells
Clone 9 cells were treated with 25µM NAE. The mRNA levels were determined
by real-time RT-PCR analysis using the β-actin mRNA level for normalization.
Values are the mean and range calculated by the ΔΔCt method (n=3–6). *p<0.01
was compared with the DMSO control.
Clone 9 cells were treated with 25µM fatty acid amide. The mRNA levels
were determined by real-time RT-PCR analysis using the β-actin mRNA level
for normalization. Values are the mean and range calculated by the ΔΔCt method
(n=3–6). *p<0.01 was compared with the DMSO control.
This is the first time to report OLHA has PPAR-α agonistic
activity.
This study suggested that N-acyl-HAs could be as a new
member of the PPAR-α agonist. Many groups are attempting
Experimental
Chemistry All reagents and solvents were purchased
to develop OEA-like compounds to be used as new antiobesity from commercial sources. Analytical thin-layer chromatogra-
and antihyperlipidemic drugs. N-Acyl-HAs, such as OLHA, phy was performed on silica-coated plates (silica gel 60F-254,
have potential as a lead compound in these efforts. However, Merck) and visualized under UV light. Column chromatogra-
further investigation on another species PPAR-α agonistic phy was carried out using silica gel (Wakogel C-200, Wako
activity, as well as proper in vivo model needed to evaluate as Pure Chemical Industries, Ltd., Osaka, Japan). All melt-
potential drug.
ing points were determined using a Yanagimoto micro-hot

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281
Fig. 4. Effects of N-Acylhistamines on the mRNA Levels of CPT-1A (A) and mHMG-CoA Syn (B) in Clone 9 Cells
Clone 9 cells were treated with 25µM fatty acid histamine amide. The mRNA levels were determined by real-time RT-PCR analysis using the β-actin mRNA level for
normalization. Values are the mean and range calculated by the ΔΔCt method (n=3–6). *p<0.01 was compared with the DMSO control.
1
1
stage and are uncorrected. H-NMR spectra were recorded 82%; Colorless amorphous; mp 130–132°C; H-NMR (CDCl3,
on a Varian 400-MR spectrometer using tetramethylsilane 400MHz) δ: 7.58 (1H, d, J=1.0Hz, H-2″), 6.82 (1H, d,
as the internal standard (s=singlet, d=doublet, t=triplet, J=1.0Hz, H-5″), 6.37 (1H, brs, NH), 3.54 (2H, q, J=6.4Hz,
m=multiplates and br=broad). MS spectra were measured H-2′), 2.82 (2H, t, J=6.4Hz, H-3′), 2.17 (2H, t, J=7.6Hz,
using a JEOL JMS-700 spectrometer.
H-2), 1.60 (2H, m, H-3), 1.34–1.20 (8H, m, CH2), 0.87 (3H,
General Procedure for Preparation of N-Acylhista- t, J=7.0Hz, H-8); 13C-NMR (CDCl3, 100MHz) δ: 173.6 (C,
mines (N-Acyl-HAs) A solution of fatty acid acyl chlo- C-1), 134.7 (CH, C-2″), 39.1 (CH2, C-2′), 36.9 (CH2, C-2), 31.7
ride (1.0mmol), purchased from Tokyo Chemical Industory (CH2), 29.2 (CH2), 29.0 (CH2), 27.0 (CH2, C-3′), 25.9 (CH2,
(Tokyo, Japan), in N,N-dimethylformamide (DMF) (2mL) was C-3), 22.6 (CH2), 14.0 (CH3, C-18); high resolution-mass
added dropwise to a suspension of histamine dihydrochlolide spectrum (HR-MS) m/z Calcd for C13H23N3O (M+): 237.1841;
(2.0mmol) and Et3N (8mmol) in DMF (5mL) cooled in an ice Found: 237.1833.
bath. In some cases (i.e., for the C18:1, C18:2 and C20:4 ana-
N-[2-(1H-Imidazol-4-yl)ethyl]decanamide
(C10:0-HA)
1
logues), the acyl chlorides were prepared by reacting the free Yield 97%; Colorless amorphous; mp 127–129°C; H-NMR
fatty acids with oxalyl chloride (5 eq, CH2Cl2, r.t., 3h). The (CDCl3, 400MHz) δ: 7.58 (1H, d, J=1.0Hz, H-2″), 6.83 (1H,
reaction mixture was stirred for 5h at room temperature. Ice- d, J=1.0Hz, H-5″), 6.35 (1H, brs, NH), 3.55 (2H, q, J=6.4Hz,
water was added to the mixture and the reaction mix was ex- H-2′), 2.81 (2H, t, J=6.4Hz, H-3′), 2.18 (2H, t, J=7.6Hz,
tracted with CHCl3. The organic layer was dried over Na2SO4 H-2), 1.60 (2H, m, H-3), 1.34–1.20 (12H, m, CH2), 0.87 (3H,
and the solvent was evaporated under reduced pressure. The t, J=6.9Hz, H-10); 13C-NMR (CDCl3, 100MHz) δ: 173.6 (C,
residue was purified by silica gel column chromatography C-1), 134.7 (CH, C-2″), 39.1 (CH2, C-2′), 36.9 (CH2, C-2),
(CHCl3 :MeOH:aq. NH3=20:1:0.5) to give the corresponding 31.8 (CH2), 29.4 (CH2), 29.3 (CH2), 29.3 (2C, CH2), 27.0 (CH2,
N-acylhistamine.
C-3′), 25.8 (CH2, C-3), 22.7 (CH2), 14.1 (CH3, C-18); HR-MS
N-[2-(1H-Imidazol-4-yl)ethyl]octanamide (C8:0-HA) Yield m/z Calcd for C15H27N3O (M+): 265.2154; Found: 265.2146.

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6.32 (1H, brs, NH), 3.55 (2H, q, J=6.3Hz, H-2′), 2.81 (2H,
t, J=6.3Hz, H-3′), 2.16 (2H, t, J=7.6Hz, H-2), 1.60 (2H, m,
H-3), 1.34–1.20 (24H, m, CH2), 0.88 (3H, t, J=7.0Hz, H-16);
13C-NMR (CDCl3-CD3OD, 100MHz) δ: 174.3 (C, C-1), 134.6
(CH, C-2″), 39.2 (CH2, C-2′), 36.6 (CH2, C-2), 31.9 (CH2),
29.62 (3C, CH2), 29.59 (2C, CH2), 29.57 (CH2), 29.5 (CH2),
29.3 (2C, CH2), 29.2 (CH2), 26.6 (CH2, C-3′), 25.7 (CH2, C-3),
22.6 (CH2), 14.0 (CH3, C-18); HR-MS m/z Calcd for C21H39N3O
(M+): 349.3093; Found: 349.3081.
N-[2-(1H-Imidazol-4-yl)ethyl]octadecanamide (C18:0-HA)
1
Yield 72%; Colorless amorphous; mp 128–129°C; H-NMR
(CDCl3, 400MHz) δ: 7.58 (1H, d, J=1.2Hz, H-2″), 6.83 (1H,
d, J=1.0Hz, H-5″), 6.32 (1H, brs, NH), 3.55 (2H, q, J=6.3Hz,
H-2′), 2.81 (2H, t, J=6.2Hz, H-3′), 2.16 (2H, t, J=7.6Hz,
H-2), 1.60 (2H, m, H-3), 1.34–1.20 (28H, m, CH2), 0.88 (3H, t,
J=6.9Hz, H-18); 13C-NMR (CDCl3-CD3OD, 100MHz) δ: 174.3
(C, C-1), 134.7 (CH, C-2″), 39.3 (CH2, C-2′), 36.6 (CH2, C-2),
31.9 (CH2), 29.64 (5C, CH2), 29.60 (2C, CH2), 29.58 (CH2),
29.5 (CH2), 29.3 (2C, CH2), 29.2 (CH2), 26.7 (CH2, C-3′), 25.7
(CH2, C-3), 22.6 (CH2), 14.0 (CH3, C-18); HR-MS m/z Calcd
for C23H43N3O (M+): 377.3406; Found: 377.3397.
(Z)-N-[2-(1H-Imidazol-4-yl)ethyl]-9-octadecenamide
(C18:1-Δ9-cis-HA: OLHA) Yield 84%; Colorless amorphous;
mp 93–95°C; 1H-NMR (CDCl3, 400MHz) δ: 7.58 (1H, d,
J=1.0Hz, H-2″), 6.83 (1H, d, J=1.0Hz, H-5″), 6.32 (1H, brs,
NH), 5.34 (2H, m, H-9, -10), 3.55 (2H, q, J=6.4Hz, H-2′),
2.82 (2H, t, J=6.4Hz, H-3′), 2.16 (2H, t, J=7.6Hz, H-2), 2.01
(4H, m, H-8, -11), 1.61 (2H, m, H-3), 1.38–1.20 (20H, m, CH2),
0.88 (3H, t, J=7.0Hz, H-18); 13C-NMR (CDCl3, 100MHz)
δ: 173.6 (C, C-1), 134.7 (CH, C-2″), 130.0 (CH, C-9 or -10),
129.7 (CH, C-9 or -10), 39.2 (CH2, C-2′), 36.9 (CH2, C-2), 31.9
(CH2), 29.75 (CH2), 29.70 (CH2), 29.5 (CH2), 29.30 (2C, CH2),
29.26 (CH2), 29.24 (CH2), 29.1 (CH2), 27.21 (CH2, C-8 or -11),
27.17 (CH2, C-8 or -11), 26.9 (CH2, C-3′), 25.8 (CH2, C-3),
Fig. 5. Effects of GW6471 (PPAR-α Antagonist) on OEA- or OLHA-
Induced mRNA Levels of CPT-1A (A) and mHMG-CoA Syn (B) in
Clone 9 Cells
Clone 9 cells were treated with 25µM OEA or OLHA in the absence or presence
of 10µM GW6471. The mRNA levels were determined by real-time RT-PCR analy-
sis using the β-actin mRNA level for normalization. Values are the mean and range
calculated by the ΔΔCt method (n=3–6). † p<0.01 was compared with GW6471
treated cells and untreated cells.
N-[2-(1H-Imidazol-4-yl)ethyl]dodecanamide
(C12:0-HA) 22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C23H41N3O
Yield 71%; Colorless amorphous; mp 118–120°C; H-NMR (M+): 375.3250; Found: 375.3240.
1
(CDCl3, 400MHz) δ: 7.58 (1H, d, J=1.0Hz, H-2″), 6.83 (1H,
(E)-N-[2-(1H-Imidazol-4-yl)ethyl]-9-octadecenamide
d, J=1.0Hz, H-5″), 6.36 (1H, brs, NH), 3.55 (2H, q, J=6.3Hz, (C18:1-Δ9-trans-HA) Yield 88%; Colorless amorphous;
1
H-2′), 2.82 (2H, t, J=6.3Hz, H-3′), 2.16 (2H, t, J=7.6Hz, mp 117–119°C; H-NMR (CDCl3, 400MHz) δ: 7.58 (1H, d,
H-2), 1.60 (2H, m, H-3), 1.34–1.20 (16H, m, CH2), 0.88 (3H, J=1.0Hz, H-2″), 6.83 (1H, d, J=1.0Hz, H-5″), 6.31 (1H, brs,
t, J=6.9Hz, H-12); 13C-NMR (CDCl3, 100MHz) δ: 173.6 (C, NH), 5.38 (2H, m, H-9, -10), 3.55 (2H, q, J=6.4Hz, H-2′),
C-1), 134.7 (CH, C-2″), 39.1 (CH2, C-2′), 36.9 (CH2, C-2), 2.81 (2H, t, J=6.4Hz, H-3′), 2.16 (2H, t, J=7.6Hz, H-2), 1.95
31.9 (CH2), 29.60 (CH2), 29.59 (CH2), 29.5 (CH2), 29.34 (CH2), (4H, m, H-8, -11), 1.60 (2H, m, H-3), 1.38–1.20 (20H, m, CH2),
29.32 (CH2), 29.27 (CH2), 27.0 (CH2, C-3′), 25.8 (CH2, C-3), 0.88 (3H, t, J=7.0Hz, H-18); 13C-NMR (CDCl3, 100MHz)
22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C17H31N3O δ: 173.5 (C, C-1), 134.7 (CH, C-2″), 130.4 (CH, C-9 or -10),
(M+): 293.2467; Found: 239.2462.
N-[2-(1H-Imidazol-4-yl)ethyl]tetradecanamide (C14:0-HA) (CH2), 32.5 (CH2), 31.9 (CH2), 29.64 (CH2), 29.58 (CH2), 29.47
Yield 71%; Colorless amorphous; mp 124–126°C; H-NMR (CH2), 29.30 (CH2), 29.28 (CH2), 29.22 (CH2), 29.20 (CH2),
130.2 (CH, C-9 or -10), 39.1 (CH2, C-2′), 36.9 (CH2, C-2), 32.6
1
(CDCl3, 400MHz) δ: 7.58 (1H, d, J=1.0Hz, H-2″), 6.83 (1H, 29.18 (CH2), 29.0 (CH2), 27.0, (CH2, C-3′) 25.7 (CH2, C-3),
d, J=1.0Hz, H-5″), 6.32 (1H, brs, NH), 3.55 (2H, q, J=6.2Hz, 22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C23H41N3O
H-2′), 2.82 (2H, t, J=6.2Hz, H-3′), 2.16 (2H, t, J=7.6Hz, (M+): 375.3250; Found: 375.3248.
H-2), 1.60 (2H, m, H-3), 1.34–1.20 (20H, m, CH2), 0.88 (3H,
(9Z,12Z)-N-[2-(1H-Imidazol-4-yl)ethyl]-9,12-octadecadien-
t, J=7.0Hz, H-14); 13C-NMR (CDCl3, 100MHz) δ: 173.6 (C, amide (C18:2-Δ9,12-cis-HA) Yield 94%; pale yellow oily
1
C-1), 134.6 (CH, C-2″), 39.1 (CH2, C-2′), 36.9 (CH2, C-2), 31.9 solid; H-NMR (CDCl3, 400MHz) δ: 7.58 (1H, d, J=1.0Hz,
(CH2), 29.67 (CH2), 29.64 (2C, CH2), 29.61 (CH2), 29.5 (CH2), H-2″), 6.82 (1H, d, J=1.0Hz, H-5″), 6.34 (1H, brs, NH),
29.4 (2C, CH2), 29.3 (CH2), 26.9 (CH2, C-3′), 25.8 (CH2), 22.7 5.35 (4H, m, H-9, -10, -12, -13), 3.54 (2H, q, J=6.1Hz,
(CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C19H35N3O H-2′), 2.82 (2H, t, J=6.1Hz, H-3′), 2.77 (2H, m, H-11), 2.16
(M+): 321.2780; Found: 321.2782.
(2H, t, J=7.6Hz, H-2), 2.05 (4H, m, H-8, -14), 1.60 (2H, m,
N-[2-(1H-Imidazol-4-yl)ethyl]hexadecanamide (C16:0-HA) H-3), 1.40–1.20 (16H, m, CH2), 0.89 (3H, t, J=7.0Hz, H-18);
Yield 74%; Colorless amorphous; mp 127–129°C; H-NMR 13C-NMR (CDCl3, 100MHz) δ: 173.6 (C, C-1), 136.1 (C, br,
1
(CDCl3, 400MHz) δ: 7.58 (1H, s, H-2″), 6.83 (1H, s, H-5″), C-4″), 134.7 (CH, C-2″), 130.2 (CH, C-9 or -10), 130.0 (CH,

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C-9 or -10), 128.0 (CH, C-12 or -13), 127.9 (CH, C-12 or -13), similar to that previously reported.10)
116.1 (CH, br, C-5″), 39.2 (CH2, C-2′), 36.9 (CH2, C-2), 31.5
N-(2-Hydroxyethyl)hexadecanamide (PEA) Yield 95%;
(CH2), 29.6 (CH2), 29.3 (CH2), 29.26 (CH2), 29.23 (CH2), 29.1 Colorless amorphous; mp 102–103°C (lit. 99–100°C10));
(CH2), 27.2 (2C, CH2, C-8, -14), 26.9 (CH2, C-3′), 25.7 (CH2, 1H-NMR (CDCl3, 400MHz) δ: 5.94 (1H, brs, NH), 3.73 (2H,
C-3), 25.6 (CH2, C-11), 22.6 (CH2), 14.1 (CH3, C-18); HR-MS q, J=4.7Hz, OCH2), 3.43 (2H, dt, J=5.7, 4.7Hz, NCH2), 2.73
m/z Calcd for C23H39N3O (M+): 373.3093; Found: 373.3089.
(1H, m, OH), 2.20 (2H, t, J=7.6Hz, H-2), 1.68–1.58 (4H, m,
(5Z,8Z,11Z,14Z)-N-[2-(1H-Imidazol-4-yl)ethyl]-5,8,11,14- CH2), 1.35–1.20 (22H, m, CH2), 0.88 (3H, t, J=7.0Hz, H-16);
eicosatetraenamide (C20:4-Δ5,8,11,14-cis-HA) Yield 74%; pale 13C-NMR (CDCl3, 100MHz) δ: 174.6, (C, C-1), 62.6 (CH2,
1
yellow oily solid; H-NMR (CDCl3, 400MHz) δ: 7.58 (1H, OCH2), 42.5 (CH2, NCH2), 36.7 (CH2, C-2), 31.9 (CH2), 29.68
d, J=1.0Hz, H-2″), 6.82 (1H, d, J=1.0Hz, H-5″), 6.37 (1H, (2C, CH2), 29.66 (CH2), 29.64 (2C, CH2), 29.61 (CH2), 29.5
brs, NH), 5.37 (8H, m, H-5, -6, -8, -9, -11, -12, -14, -15), 3.54 (CH2), 29.34 (2C, CH2), 29.27 (CH2), 25.7 (CH2), 22.7 (CH2),
(2H, q, J=6.1Hz, H-2′), 2.86–2.75 (8H, m, H-7, -10, -13, -3′), 14.1 (CH3, C-18); HR-MS m/z Calcd for C18H37NO2 (M+):
1
2.18 (2H, t, J=7.6Hz, H-2), 2.14–2.00 (4H, m, H-4, -16), 1.70 299.2824; Found: 299.2836. The H-NMR spectrum was simi-
(2H, m, H-3), 1.40–1.22 (6H, m, H-17, -18, -19), 0.89 (3H, t, lar to that previously reported.10)
J=7.0Hz, H-20); 13C-NMR (CDCl3, 100MHz) δ: 173.4 (C,
Synthesis of Oleic Acid Amides (OLDA, OLTA,
C-1), 135.5 (C, br, C-4″), 134.6 (CH, C-2″), 130.5, 129.1, 128.7, OL-5-HT and Olvanil) According to the general proce-
128.6, 128.2, 128.1, 127.8, 127.5 (CH, C-5, -6, -8, -9, -11, -12, dure for the preparation of N-acylhistamines, oleoyl chloride
-14 or -15), 116.1 (CH, br, C-5″), 39.2 (CH2, C-2′), 36.1 (CH2, and the corresponding amine (1.2eq) were treated with Et3N
C-2), 31.5 (CH2), 29.3 (CH2), 27.2 (CH2), 26.8 (CH2), 26.7 (4eq), and the crude product was purified by silica gel column
(CH2, C-3′), 25.62 (CH2, C-3), 25.60 (2C, CH2), 25.57 (CH2), chromatography (hexane–AcOEt) to give the corresponding
22.6 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C25H39N3O oleic acid amide.
(M+): 397.3093; Found: 397.3087.
(Z)-N-[2-(3,4-Dihydroxyphenyl)ethyl]-9-octadecenamide
General Procedure for the Preparation of N-Acyletha- (OLDA) Yield 97%; Colorless amorphous; mp 58–61°C;
nolamides (NAEs) A solution of fatty acid oleoyl chloride 1H-NMR (CDCl3, 400MHz) δ: 7.66 (1H, brs, OH), 6.81
(1.0mmol) in CH2Cl2 (2mL) was added dropwise to a solution (1H, d, J=8.0Hz, H-5″), 6.75 (1H, d, J=2.0Hz, H-2″), 6.57
of ethanolamine (10mmol) in CH2Cl2 (10mL) cooled in an ice (1H, dd, J=8.0, 2.0Hz, H-6″), 6.02 (1H, brs, OH), 5.63
bath. The reaction mixture was stirred for 1h at room tem- (1H, brt, J=5.8Hz, NH), 5.34 (2H, m, H-9, -10), 3.48 (2H,
perature, then extracted with CHCl3. The organic layer was td, J=7.1, 5.8Hz, H-2′), 2.70 (2H, t, J=7.1Hz, H-3′), 2.15
dried over Na2SO4 and the solvent was evaporated under re- (2H, t, J=7.6Hz, H-2), 1.99 (4H, m, H-8, -11), 1.58 (2H, m,
duced pressure. The residue was purified by silica gel column H-3), 1.38–1.20 (20H, m, CH2), 0.88 (3H, t, J=6.9Hz, H-18);
chromatography (CHCl3 :MeOH=30:1) or by recrystallization 13C-NMR (CDCl3, 100MHz) δ: 174.5, (C, C-1), 144.3 (C, C-3″
(AcOEt–hexane) to give N-acylethanolamide.
or -4″), 143.2 (C, C-3″ or -4″), 130.4 (C, C-1″), 130.0 (CH,
(5Z,8Z,11Z,14Z)-N-(2-Hydroxyethyl)-5,8,11,14-eicosatetra- C-9 or -10), 129.7 (CH, C-9 or -10), 120.4 (CH, C-6″), 115.3
1
enamide (AEA) Yield 92%; Colorless oil; H-NMR (CDCl3, (CH, C-2″ or -5″), 115.1 (CH, C-2″ or -5″), 41.0 (CH2, C-2′),
400MHz) δ: 5.90 (1H, brs, NH), 5.37 (8H, m, H-5, -6, -8, 36.8 (CH2, C-2), 34.9 (CH2, C-3′), 31.9 (CH2), 29.8 (CH2),
-9, -11, -12, -14, -15), 3.73 (2H, t, J=4.6Hz, OCH2), 3.43 (2H, 29.7 (CH2), 29.5 (CH2), 29.4 (CH2), 29.32 (CH2), 29.31 (CH2),
dt, J=5.7, 4.6Hz, NCH2), 2.86–2.76 (6H, m, H-7, -10, -13), 29.19 (CH2), 29.15 (CH2), 29.10 (CH2), 27.2 (CH2, C-8 or
2.60 (1H, brs, OH), 2.22 (2H, t, J=7.6Hz, H-2), 2.16–2.02 -11), 27.1 (CH2, C-8 or -11), 25.7 (CH2, C-3), 22.7 (CH2), 14.1
(4H, m, H-4, -16), 1.73 (2H, m, H-3), 1.40–1.22 (6H, m, H-17, (CH3, C-18); HR-MS m/z Calcd for C26H43NO3 (M+): 417.3234;
-18, -19), 0.88 (3H, t, J=7.0Hz, H-20); 13C-NMR (CDCl3, Found: 417.3230.
100 MHz) δ: 174.3 (C, C-1), 130.5, 129.0, 128.8, 128.6, 128.2,
(Z)-N-[2-(4-Hydroxyphenyl)ethyl]-9-octadecenamide
128.1, 127.8, 127.5 (CH, C-5, -6, -8, -9, -11, -12, -14 or -15), (OLTA) Yield 90%; Colorless amorphous; mp 72–74°C;
62.4 (CH2, OCH2), 42.4 (CH2, NCH2), 35.9 (CH2, C-2), 31.5 1H-NMR (CDCl3, 400MHz) δ: 7.03 (2H, d, J=8.4Hz, H-2″,
(CH2), 29.3 (CH2), 27.2 (CH2), 26.6 (CH2), 25.6 (2C, CH2), -6″), 6.78 (2H, d, J=8.4Hz, H-3″, -5″), 5.60 (1H, brs, OH),
25.5 (CH2), 22.6 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd 5.44 (1H, brt, J=5.9Hz, NH), 5.34 (2H, m, H-9, -10), 3.48
for C22H37NO2 (M+): 347.2824; Found: 347.2824. The 1H-NMR (2H, td, J=6.9, 5.9Hz, H-2′), 2.74 (2H, t, J=6.9Hz, H-3′), 2.12
spectrum was similar to that previously reported.10)
(Z)-N-(2-Hydroxyethyl)-9-octadecenamide
(2H, t, J=7.6Hz, H-2), 2.00 (4H, m, H-8, -11), 1.58 (2H, m,
(OEA) Yield H-3), 1.38–1.20 (20H, m, CH2), 0.88 (3H, t, J=7.0Hz, H-18);
96%; Colorless amorphous; mp 64–65°C (lit. 75–76°C10)); 13C-NMR (CDCl3, 100MHz) δ: 173.7 (C, C-1), 155.0 (C, C-4″),
1H-NMR (CDCl3, 400MHz) δ: 5.92 (1H, brs, NH), 5.34 130.0 (CH, C-9 or -10), 129.73 (CH, C-9 or -10), 129.70 (C,
(2H, m, H-9, -10), 3.73 (2H, q, J=4.6Hz, OCH2), 3.43 (2H, C-1″), 129.70 (CH, C-2″, -6″), 115.6 (CH, C-3″, -5″), 40.8 (CH2,
dt, J=5.6, 4.6Hz, NCH2), 2.66 (1H, m, OH), 2.21 (2H, t, C-2′), 36.8 (CH2, C-2), 34.8 (CH2, C-3′), 31.9 (CH2), 29.8
J=7.6Hz, H-2), 2.01 (4H, m, H-8, -11), 1.68–1.58 (4H, m, (CH2), 29.7 (CH2), 29.5 (CH2), 29.3 (CH2), 29.22 (CH2), 29.20
CH2), 1.38–1.20 (18H, m, CH2), 0.88 (3H, t, J=7.0Hz, H-18); (CH2), 29.1 (CH2), 27.2 (CH2, C-8 or -11), 27.1 (CH2, C-8 or
13C-NMR (CDCl3, 100MHz) δ: 174.6 (C, C-1), 130.0 (CH, C-9 -11), 25.7 (CH2, C-3), 22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z
or -10), 129.7 (CH, C-9 or -10), 62.4 (CH2, OCH2), 42.4 (CH2, Calcd for C26H43NO2 (M+): 401.3294; Found: 401.3292.
NCH2), 36.7 (CH2, C-2), 31.9 (CH2), 29.8 (CH2), 29.7 (CH2),
(Z)-N-[2-(5-Hydroxy-1H-indol-3-yl)ethyl]-9-octadecenamide
29.5 (CH2), 29.3 (2C, CH2), 29.2 (2C, CH2), 29.1 (CH2), 27.2 (OL-5-HT) Yield 82%; Colorless amorphous; mp 80–82°C;
(CH2, C-8 or -11), 27.1 (CH2, C-8 or -11), 25.7 (CH2, C-3), 22.7 1H-NMR (CDCl3, 400MHz) δ: 7.93 (1H, brs, OH), 7.23 (1H,
(CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C20H39NO2 d, J=8.7Hz, H-7″), 7.04 (1H, d, J=2.4Hz, H-4″), 7.00 (1H,
1
(M+): 325.2981; Found: 325.2972. The H-NMR spectrum was d, J=2.3Hz, H-2″), 6.80 (1H, dd, J=8.7, 2.4Hz, H-6″), 5.56

284
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Vol. 63, No. 4 (2015)
(1H, br t, J=5.7Hz, NH), 5.34 (2H, m, H-9, -10), 3.58 (2H, (10mL) cooled in an ice bath. The reaction mixture was
td, J=6.8, 5.7Hz, H-2′), 2.90 (2H, t, J=6.8Hz, H-3′), 2.12 stirred for 3h at room temperature. Ice-water was added to the
(2H, t, J=7.6Hz, H-2), 2.00 (4H, m, H-8, -11), 1.58 (2H, m, mixture and the mixture was extracted with CHCl3. The or-
H-3), 1.38–120 (20H, m, CH2), 0.88 (3H, t, J=6.9Hz, H-18); ganic layer was dried over Na2SO4 and the solvent was evapo-
13C-NMR (CDCl3, 100MHz) δ: 173.8, (C, C-1), 150.1 (C, rated under reduced pressure. The residue was passed once
C-5″), 131.4 (C, C-3a″), 130.0 (CH, C-9 or -10), 129.8 (CH, C-9 through a short silica gel column (hexane:AcOEt=2:1) and
or -10), 128.0 (C, C-7a″), 123.0 (CH, C-2″), 112.3 (CH, C-4″ or the solvent was evaporated. The residue was treated with TFA
-7″), 112.1 (C, C-3″), 111.9 (CH, C-4″ or -7″), 103.2 (CH, C-6″), followed by 2M HCl–MeOH (1:1) to give the corresponding
39.7 (CH2, C-2′), 36.8 (CH2, C-2), 31.9 (CH2), 29.76 (CH2), crude oleic acid amide hydrochloride salt.
29.71 (CH2), 29.5 (CH2), 29.32 (CH2), 29.31 (CH2), 29.25 (2C,
(Z)-N-(4-Aminobutyl)-9-octadecenamide
(OLPut) The
CH2), 29.15 (CH2), 27.22 (CH2, C-8 or -11), 27.18 (CH2, C-8 or crude compound obtained by general procedure was puri-
-11), 25.8 (CH2, C-3), 25.4 (CH2, C-3′), 22.7 (CH2), 14.1 (CH3, fied by silica gel colum chromatography (CHCl3–MeOH–aq.
C-18); HR-MS m/z Calcd for C28H44N2O2 (M+): 440.3403; NH3=20:1:0.5) to give the title compound.
Found: 440.3388.
Yield 68% (for 2 steps); Colorless solid; mp 90–92°C;
(Z)-N-[2-(4-Hydroxy-3-methoxyphenyl)methyl]-9-octa- 1H-NMR (CDCl3, 400MHz) δ: 5.82 (1H, brs, NH), 5.34
decenamide (Olvanil) Yield 89%; Colorless amorphous; (2H, m, H-9, -10), 3.26 (2H, q, J=6.4Hz, NCH2), 2.72 (2H,
mp 42–43°C; 1H-NMR (CDCl3, 400MHz) δ: 6.87 (1H, d, t, J=6.7Hz, NCH2), 2.15 (2H, t, J=7.6Hz, H-2), 2.00 (4H,
J=8.0Hz, H-5″), 6.82 (1H, d, J=1.9Hz, H-2″), 6.77 (1H, dd, m, H-8 and H-11), 1.65–1.45 (6H, m, CH2), 1.38–1.20 (20H,
J=8.0, 1.9Hz, H-6″), 5.64 (1H, brs, NH), 5.60 (1H, brs, OH), m, CH2), 0.88 (3H, t, J=7.0Hz, H-18); 13C-NMR (CDCl3,
5.34 (2H, m, H-9, -10), 4.36 (2H, d, J=5.6Hz, NCH2), 2.19 100 MHz) δ: 173.1 (C, C-1), 130.0 (CH, C-9 or -10), 129.7 (CH,
(2H, t, J=7.6Hz, H-2), 2.00 (4H, m, H-8, -11), 1.66 (2H, m, C-9 or -10), 41.6 (CH2, NH2CH2), 39.2 (CH2, NHCH2), 36.9
H-3), 1.38–1.20 (20H, m, CH2), 0.88 (3H, t, J=7.0Hz, H-18); (CH2, C-2), 31.9 (CH2), 30.6 (CH2), 29.7 (CH2), 29.5 (CH2),
13C-NMR (CDCl3, 100MHz) δ: 172.9, (C, C-1), 146.7 (C, C-3″ 29.3 (3C, CH2), 29.2 (CH2), 29.17 (CH2), 29.13 (CH2), 27.2
or -4″), 145.1 (C, C-3″ or -4″), 130.3 (C, C-1″), 130.0 (CH, (CH2, C-8 or -11), 27.1 (CH2, C-8 or -11), 27.0 (CH2), 25.8
C-9 or -10), 129.7 (CH, C-9 or -10), 120.8 (CH, C-6″), 114.4 (CH2, C-3), 22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd
(CH, C-5″), 110.7 (CH, C-2″), 55.9 (CH3, OMe), 43.5 (CH2, for C22H44N2O (M+): 352.3454; Found: 352.3447.
C-2′), 36.8 (CH2, C-2), 31.9 (CH2), 29.8 (CH2), 29.7 (CH2),
(Z)-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
29.5 (CH2), 29.31 (CH2), 29.28 (CH2), 29.25 (CH2), 29.1 (CH2), 9-octadecenamide (OLSPm) The crude compound obtained
27.2 (CH2, C-8 or -11), 27.1 (CH2, C-8 or -11), 25.8 (CH2, C-3), by general procedure was recrystallized from aqueous MeOH
22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Calcd for C26H43NO3 to give the title compound (trihydrochloride salt).
(M+): 417.3243; Found: 417.3241.
Synthesis of
Yield 64% (for 2 steps); Colorless amorphous; 1H-NMR
N-[(9Z)-1-Oxo-9-octadecenyl]glycine (D2O, 400MHz) δ: 5.21 (2H, m, H-9, -10), 3.12 (2H, t,
(OLGly) Oleoyl chloride (2.0mmol) was added dropwise to J=6.7Hz, NCH2), 3.08–2.90 (10H, m, NCH2), 2.10 (2H,
an aqueous solution containing glycine sodium salt. The solu- t, J=7.6Hz, H-2), 2.00–1.62 (12H, m, CH2), 1.42 (2H, m,
tion was kept between pH 9–12.5 by the simultaneous addition CH2), 1.24–1.05 (20H, m, CH2), 0.72 (3H, t, J=7.0Hz, H-18);
of a 10% aqueous NaOH solution, and the temperature was 13C-NMR (D2O, 100MHz) δ: 176.0 (C, C-1), 129.8 (CH, C-9
maintained below 35°C. After stirring for 1h, the solution was or -10), 129.4 (CH, C-9 or -10), 47.1 (CH2), 45.4 (CH2), 44.6
acidified with 30% H2SO4 to below pH 4.5 and extracted with (CH2), 36.5 (CH2), 36.2 (CH2), 35.9 (CH2), 31.8 (CH2), 29.69
AcOEt. The organic layer was washed with water and then (CH2), 29.67 (CH2), 29.5 (CH2), 29.27 (CH2), 29.23 (CH2), 29.1
dried over Na2SO4. The solvent was evaporated under reduced (CH2), 27.2 (CH2), 27.1 (CH2), 25.8 (CH2), 25.6 (CH2), 23.8
pressure. The residue was purified by silica gel column chro- (CH2), 23.0 (CH2), 22.9 (CH2), 22.5 (CH2), 13.8 (CH2, C-18);
matography (hexane:AcOEt=1:1) to give the title compound HR-MS m/z Calcd for C28H58N4O (M+): 466.4611; Found:
1
in 57% yield.
466.4621. The H-NMR spectrum was similar to that previ-
Colorless amorphous; mp 93–94°C (lit. 92–93°C11)); ously reported.12)
1H-NMR (CDCl3, 400MHz) δ: 6.15 (1H, brt, J=5.2Hz, NH),
Evaluation of PPAR-α Agonist Activity Clone 9 cells
5.35 (2H, m, H-9, -10), 4.08 (2H, d, J=5.2Hz, NCH2), 2.27 and Ham’s F12 medium were purchased from DS Pharma Bio-
(2H, t, J=7.6Hz, H-2), 2.01 (4H, m, H-8, H-11), 1.63 (2H, m, medical. Penicillin–streptomycin stabilized solution, trypsin,
CH2), 1.38–1.20 (20H, m, CH2), 0.88 (3H, t, J=6.9Hz, H-18); ethylenediamine tetraacetic acid disodium salt, oleic acid, and
13C-NMR (CDCl3, 100MHz) δ: 174.7, (C, C=O), 172.7 (C, GW6471 were purchased from Sigma-Aldrich Japan. Oleamide
C=O), 130.0 (CH, C-9 or -10), 129.7 (CH, C-9 or -10), 41.5 was purchased from KANTO Chemicals. Fetal bovine serum
(CH2, NCH2), 36.3 (CH2, C-2), 31.9 (CH2), 29.8 (CH2), 29.7 was purchased from Nichirei Bioscience. An RNeasy Mini Kit
(CH2), 29.5 (CH2), 29.3 (2C, CH2), 29.2 (CH2), 29.16 (CH2), was purchased from QIAGEN. A Prime Script RT Reagent
29.11 (CH2), 27.2 (CH2, C-8 or -11), 27.1 (CH2, C-8 or -11), Kit (Perfect Real Time), SYBR Premix Ex Taq (Tli RNaseH
25.5 (CH2, C-3), 22.7 (CH2), 14.1 (CH3, C-18); HR-MS m/z Plus), and loading buffer were purchased from TaKaRa Bio
Calcd for C20H37NO3 (M+): 339.2773; Found: 339.2758. The (Shiga, Japan). All other reagents were of research grade.
1H- and 13C-NMR spectra were similar to that previously re-
ported.11)
Cell Culture and Treatment of the Tested Compounds
Clone-9 rat hepatocytes were routinely cultured in Ham’s F12
medium in 10cm dishes at 37°C and 5% CO2 in the presence
Procedure for Preparation of OLSPm and OLPut
A
solution of fatty acid oleoyl chloride (2.4mmol) in CH2Cl2 of 1% penicillin/streptomycin with 10% fetal bovine serum.
(2mL) was added dropwise to a solution of Boc-protected The cells used for experiments were passages 25–55. For the
polyamine12,13) (2.0mmol) and Et3N (8mmol) in CH2Cl2 experiments, the Clone 9 cells (105 cells/10cm dish) were cul-

Vol. 63, No. 4 (2015)
Chem. Pharm. Bull.
285
tured for 72h, then the sub-confluent cells were incubated in
Conflict of Interest The authors declare no conflict of
serum-free medium at 37°C for 24h before the addition of the interest.
test compounds. For treatment with GW6471, a PPAR-α antag-
onist,9) 10 µM GW6471 was added with the tested compounds.
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