Synthesis of N-functionalized oleamide derivatives

Article abstract of DOI:10.1016/j.tet.2007.02.074

Oleamide is an interesting compound, which shows various pharmacological activities including the inhibitory effect of gap junction formation. Recently, the studies of the gap junction have been some of the hot topics in biology and its inhibitors have become more useful tools [Cravatt, B. F.; Garcia, O. P.; Siuzdak, G.; Gilula, N. B.; Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506-1509; Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580-590; Guan, X; Cravatt, B. F.; Ehring, G. R.; Hall, J. E.; Boger, D. L.; Lerner, R. A.; Gilula, N. B. J. Cell Biol. 1997, 139, 1785-1792; Boger, D. L.; Patterson, J. E.; Guan, X.; Cravatt, B. F.; Lerner, R. A.; Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815; Ito, A.; Morita, N.; Miura, D.; Koma, Y.; Kataoka, T. R.; Yamasaki, H.; Kitamura, Y.; Kita, Y.; Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. However, many reports suggest that the functionalizations of oleamide to retain its biological activity were difficult [Boger, D. L.; Patterson, J. E.; Guan, X.; Cravatt, B. F.; Lerner, R. A.; Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815; Ito, A.; Morita, N.; Miura, D.; Koma, Y.; Kataoka, T. R.; Yamasaki, H.; Kitamura, Y.; Kita, Y.; Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. The synthesis of the functionalized oleamide derivatives, whose biological activity is not blocked, has become attractive in both the biological and chemical fields. Herein, by linking the fluorophore to the oleamide by alkyl chains, we synthesized the fluorescently tagged oleamide whose biological feature is similar to that of oleamide. Moreover, we also synthesized other bioactive derivatives tagged by other groups such as the sugars and biotin via alkyl chain linkers.

Full text of DOI:10.1016/j.tet.2007.02.074

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Tetrahedron 63 (2007) 3754–3761
Synthesis of N-functionalized oleamide derivatives
Yusuke Ohba,a Yukiko Kanao,a Mayuko Takatsuji,a Motoki Ito,a Norikazu Yabuta,b
Hiroshi Nojimab and Yasuyuki Kitaa,
*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka, 565-0871, Japan
bDepartment of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita,
Osaka, 565-0871, Japan
Received 18 January 2007; revised 16 February 2007; accepted 19 February 2007
Available online 21 February 2007
Abstract—Oleamide is an interesting compound, which shows various pharmacological activities including the inhibitory effect of gap junc-
tion formation. Recently, the studies of the gap junction have been some of the hot topics in biology and its inhibitors have become more useful
tools [Cravatt, B. F.; Garcia, O. P.; Siuzdak, G.; Gilula, N. B.; Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506–1509;
Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580–590; Guan, X; Cravatt, B. F.; Ehring, G. R.; Hall, J. E.; Boger, D.
L.; Lerner, R. A.; Gilula, N. B. J. Cell Biol. 1997, 139, 1785–1792; Boger, D. L.; Patterson, J. E.; Guan, X.; Cravatt, B. F.; Lerner, R. A.; Gilula,
N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810–4815; Ito, A.; Morita, N.; Miura, D.; Koma, Y.; Kataoka, T. R.; Yamasaki, H.; Kitamura, Y.;
Kita, Y.; Nojima, H. Carcinogenesis 2004, 25, 2015–2022]. However, many reports suggest that the functionalizations of oleamide to retain its
biological activity were difficult [Boger, D. L.; Patterson, J. E.; Guan, X.; Cravatt, B. F.; Lerner, R. A.; Gilula, N. B. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 4810–4815; Ito, A.; Morita, N.; Miura, D.; Koma, Y.; Kataoka, T. R.; Yamasaki, H.; Kitamura, Y.; Kita, Y.; Nojima, H.
Carcinogenesis 2004, 25, 2015–2022]. The synthesis of the functionalized oleamide derivatives, whose biological activity is not blocked,
has become attractive in both the biological and chemical fields.
Herein, by linking the fluorophore to the oleamide by alkyl chains, we synthesized the fluorescently tagged oleamide whose biological feature
is similar to that of oleamide. Moreover, we also synthesized other bioactive derivatives tagged by other groups such as the sugars and biotin
via alkyl chain linkers.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, the studies of the gap junction have been some of
the hot topics in biology. For example, many Cx-subtypes
that constitute the gap junction were discovered and many
functions of them were found in the last decade.1–4 Then,
its inhibitors have become more useful tools. The synthesis
of the functionalized oleamide derivatives, whose biological
activity is not blocked, has become attractive in both the bi-
ological and chemical fields. However, many reports suggest
that the derivatizations of oleamide to retain the inhibitory
effect of GJICs were difficult.3,4
Oleamide, an endogenous fatty acid with a primary amide, is
known to accumulate in the cerebrospinal fluid under sleep
deprivation conditions.1 Oleamide induces physiological
sleep in mice by ip or iv injection. Oleamide also inhibits
the gap junction-mediated intercellular communications
(GJICs) without any effect on the calcium wave transmis-
sion.2,3 We also found that it inhibits both connexin
(Cx)43/Cx26-mediated GJICs, a non-selective connexin in-
hibitor.4 Cx belongs to the cell membrane protein family that
constitutes a gap junction and nearly 22 kinds of its subtypes
have already been identified. Connexon, a hexamer of the Cx
molecule, is a pipe-shaped structure and penetrates the cell
membrane and enables the passage of small molecules,
such as ions and proteins with low molecular weight through
cells.5
Now, we report that the fluorescently tagged oleamides,
which inhibit the Cx43-mediated GJICs at a level similar
to oleamide, were synthesized. And various functionalized
oleamide derivatives were also synthesized.
2. Results and discussion
2.1. Design and synthesis of fluorescently tagged
oleamide
Keywords: Gap junction; Oleamide derivatives; Functional groups; Alkyl
linker.
Oleamide has an unsaturated alkyl chain and primary amide.
For derivatization of the oleamide into functionalized
* Corresponding author. Tel.: +81 6 6879 8225; fax: +81 6 6879 8229;
e-mail: kita@phs.osaka-u.ac.jp
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.02.074

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Y. Ohba et al. / Tetrahedron 63 (2007) 3754–3761
3755
analogues, two strategies are available: (1) the derivatization
of its alkyl chain and (2) the introduction of the functional
groups on its amide unit. Boger and co-workers reported
that the oleamide was the most potent inhibitor of GJICs
and the changes in its alkyl chain reduced its activity.3 We
also found that the alkyl chain of the oleamide was critical
for its potent biological activity.4 The oleamide structure is
similar to half of phospholipids consisting of the plasma
membrane. Therefore, the derivatizations of its alkyl chain
may lead to the reduction of its similarity to the phospho-
lipids and loss of its biological activity. As described above,
the functionalizations of oleamide for retaining its biological
activity were difficult.3,4 Thus, we tried to modify its amide
unit by labeling with functional groups.
oleamide via the alkyl chain linkers that show the electron
donating, less bulky, and hydrophobic nature. Since various
fluorescent groups could be tagged by a simple procedure,
we selected diamines as the linker. Two synthetic methodol-
ogies are available: (1) oleamide part is first constructed and
tagged by the dansyl group and (2) the dansyl group is first
linked to the diamine and then the oleamide part is intro-
duced (Scheme 2).
For the first method, the mono-substituted amines including
the oleamide part were obtained. However, their yields were
low because the formations of its dimeric compounds mainly
occurred. Moreover, they were insoluble solids, which were
difficult to react with the dansyl group. Thus, we chose the
latter method. Dansyl chloride (1 equiv) was reacted with
the diamines (3 equiv) to afford the mono-dansylated amines
1. They were easily detected by UVand easy to handle. The
Cn-linked dansyl oleamides were obtained in high yield by
the reaction of 1 with oleic chloride prepared from oleic
acid and oxalyl chloride (Scheme 3).
First, the fluorescent 5-(dimethylamino)naphth-1-yl sulfonyl
(dansyl) group as the functional group was introduced into
the oleamide. We previously reported that the amide part
of oleamide derivatives substituted by the electron withdraw-
ing groups such as acyl and sulfonyl, the aromatic and the
hydrophilic groups, did not inhibit the GJICs.4 Indeed, the
oleamide derivative labeled on its amide part by the dansyl
group,6 namely, the dansyl oleamide, was synthesized. How-
ever, it did not inhibit the Cx43-mediated GJICs (Scheme 1,
Table 1).
2.2. Cell study of fluorescently tagged oleamide
All the Cn-linked dansyl oleamides inhibited the Cx43-
mediated GJICs at a level similar to the oleamide (Table 1).
Among them, the biological activity of the C4/C10-linked
dansyl oleamides was more similar to the oleamide (92%,
87%) than that of the C2-linked dansyl oleamide (67%).
O
O2
S
O
Me
N
H
N
7
7
NH2
7
7
Me
oleamide
2.3. Synthesis and biological evaluation of functionalized
oleamide via the alkyl chain linker
dansyl oleamide (18%)
Scheme 1. Reaction conditions: NaH, DnsCl, DMF, room temperature. Dns
(dansyl)¼5-(dimethylamino)naphth-1-yl sulfonyl.
Based on these results, various functionalized oleamide
derivatives via the alkyl chain linkers were synthesized
(Scheme 4). The oleamide derivatives tagged by the 7-
nitro-2,1,3-benzoxadiazol-4-yl (NBD) group,8 that are
Therefore, a new dansyl oleamide design is necessary. Based
on these results, we decided to attach the dansyl group to the
Table 1. Dye transfer assay of Cn-linked dansyl oleamides using HeLa-Cx437
Agent
Cx43-mediated GJIC scorea
Inhibition rateb (%)
—
Control
5.18ꢀ1.10
O
NH2
2.82ꢀ0.81*
100
7
7
oleamide
O
O2
S
Me
N
H
N
7
7
7
4.77ꢀ0.87
20
Me
dansyl oleamide
O
O2
S
Me
N
H
N
H
N
m
7
Me
Cn-linked dansyl oleamide
C2-linked dansyl oleamide (m¼0)
C4-linked dansyl oleamide (m¼2)
C10-linked dansyl oleamide (m¼8)
3.61ꢀ0.68*
3.17ꢀ0.62*
3.00ꢀ0.68*
67
85
92
*p<0.05; n>20.
Donor and recipient cells were co-cultured in the presence of the agents at a concentration of 40 mM. The standard deviations (SD) are shown as ꢀ.
a
b
The percent inhibition of the transfer of the calcein dye from the donor cells to recipient cells was determined for comparison with the oleamide (100%).

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3756
Y. Ohba et al. / Tetrahedron 63 (2007) 3754–3761
(COCl)2
then
NH2
O
O
H2N
m
(1)
(2)
N
H
NH2
OH
OH
m
7
7
7
7
RCl
oleic acid
O
O
H2N
NHR
(COCl)2
m
Cl
N
H
NHR
7
7
O
7
7
m
Cn-linked fluorescent oleamide
7
7
H2N
NHR
m
oleic acid
EDC HCl, 4-DMAP
Scheme 2. Synthetic methodologies of Cn-linked functional oleamide. R¼functional groups, EDC$HCl¼1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride, 4-DMAP¼4-dimethylamino-pyridine.
O
O2
S
Me
N
H
N
H
N
7
7
m
a)
b)
H2N
NHDns
m
Me
DnsCl
1a (m = 0, 84%)
1b (m = 2, 90%)
1c (m = 8, 83%)
C2-linked dansyl oleamide (m = 0, 66%)
C4-linked dansyl oleamide (m = 2, 91%)
C10-linked dansyl oleamide (m = 8, 87%)
Scheme 3. Reaction conditions: (a) diamines, CH2Cl2, room temperature; (b) oleic chloride, CH2Cl2, room temperature. Dns (dansyl)¼5-(dimethylamino)-
naphth-1-yl sulfonyl.
NO2
O
N
H
N
H
N
O
7
7
m
a)
b)
H2N
NHNBD
m
N
NBDCl
2a (m = 0, 62%)
2b (m = 2, 71%)
2c (m = 8, 61%)
C2-linked NBD oleamide (m = 0, 70%)c
C4-linked NBD oleamide (m = 2, 83%)
C10-linked NBD oleamide (m = 8, 73%)
Scheme 4. Reaction conditions: (a) diamines, NaHCO3, THF/EtOH, room temperature; (b) oleic acid, EDC$HCl, 4-DMAP, DMF, room temperature. NBD¼
7-nitro-2,1,3-benzoxadiazol-4-yl, EDC$HCl¼1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 4-DMAP¼4-(dimethylamino)pyridine; (c)
when 2a was reacted with oleic acid in the presence of EDC$HCl and 4-DMAP, only the diacylated compound was formed. Thus, oleic chloride was used
in this case.
known to show the different lexc (315 nm for dansyl moiety
and 460–480 nm for NBD moiety), were synthesized by
a method similar to the Cn-linked dansyl oleamides. Al-
though the exactly same method failed to give the Cn-linked
NBD oleamides, these were obtained in high yields by the
condensation reaction of 2 with oleic acid in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydro-
chloride (EDC$HCl).
Moreover, the oleamide derivatives labeled by (+)-biotin
or sugar (galactose) via the alkyl chain linkers were also
synthesized by the simple procedure (Scheme 5).
The inhibitory effects of the Cx43-mediated GJICs by these
functionalized oleamides were examined (Table 2). Since
the NBD group has the same excitation spectrum of the
dyes used in the dye transfer assay,4 the functionalized
RO
RO
AcO
AcO
OAc
O
O
O
O
RO
O
b)
N
H
X
9
7
7
RO
9
AcO
3 (X = Br)9
4 (X = N3)
5 (R = Ac, 46% from 3)
6 (R = H, 82%)
a)
c)
O
O
O
NH
H
HN
H
NH
H
HN
NH
H
HN
H
O
O
H
O
d)
f)
n
O
N
4
S
7
7
O
X
4
4CO2H
S
n
H
S
7 (X = Br)
8 (X = N3)
9a (n = 4, 30% from (+)-biotin)
9b (n = 10, 50% from (+)-biotin)
(+)-biotin
e)
Scheme 5. Reaction conditions: (a) NaN3, DMF, 60 ꢁC; (b) PPh3, CH2Cl2, then oleic chloride, CH2Cl2, room temperature; (c) NaOEt, EtOH, room temperature;
(d) 1-bromo-4-butanol or 1-bromo-10-decanol, EDC$HCl, 4-DMAP, DMF, room temperature; (e) NaN3, DMF, 60 ꢁC; (f) (i) Lindlar catalyst, H2 (1 atm),
MeOH, room temperature; (ii) oleic chloride, CH2Cl2, room temperature.

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Y. Ohba et al. / Tetrahedron 63 (2007) 3754–3761
3757
standard at 20–25 ꢁC. IR spectra were recorded by a diffuse
reflectance measurement of samples dispersed in KBr pow-
der. E. Merck silica gel 60 for column chromatography and
E. Merck pre-coated TLC plates, silica gel F254, for prepar-
ative thin-layer chromatography were used.
Table 2. Dye transfer assay of the functionalized oleamide derivatives using
the alkyl linker7
Agent
Cx43-mediated Inhibition
GJIC scorea
rateb (%)
Control
4.34ꢀ0.49
—
O
4.2. Synthetic chemistry
NH2
7
7
2.00ꢀ0.57*
2.76ꢀ0.81*
100
68
oleamide
4.2.1. Synthesis of dansyl oleamide. Under a nitrogen
atmosphere, a solution of NaH (60% dispersion in mineral
oil, 21 mg, 0.53 mmol) in THF (2.5 mL) was cooled to
0 ꢁC, and oleamide (100 mg, 0.36 mmol) in THF (1.0 mL)
was added. The reaction mixture was stirred at room temper-
ature for 30 min and dansyl chloride (94 mg, 0.36 mmol)
was added to the reaction mixture at 0 ꢁC. The reaction mix-
ture was stirred at room temperature for 3 h and poured into
H2O. The aqueous layer was extracted with CH2Cl2. The or-
ganic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. Purification of the residue by column
chromatography (n-hexane/AcOEt, 4/1) gave dansyl ole-
amide (32 mg, 18%) as slightly green oil.
AcO
AcO
OAc
O
O
O
N
H
9
7
7
AcO
5
HO
HO
OH
O
O
O
2.69ꢀ0.85*
71
N
9
7
7
HO
H
6
O
IR (KBr): 3234, 1693, 1454 cmꢂ1 1H NMR (CDCl3,
.
NH
H
O
HN
H
O
300 MHz) d: 0.87 (3H, t, J¼6.2 Hz), 1.11–1.62 (22H, m),
1.91–2.05 (4H, m), 2.21 (2H, t, J¼7.5 Hz), 2.90 (6H, s),
5.29–5.34 (2H, m), 7.19 (1H, d, J¼7.2 Hz), 7.59 (2H, m),
8.21 (1H, d, J¼9.0 Hz), 8.50 (2H, m), 8.62 ppm (1H, d,
J¼8.4 Hz). 13C NMR (CDCl3, 67.8 MHz) d: 171.1, 152.2,
133.2, 132.3, 131.9, 130.0, 129.7, 129.7, 129.5, 128.8,
123.3, 118.0, 115.2, 45.4 (2C), 36.2, 31.9, 29.7, 29.6, 29.5,
29.3 (2C), 29.0, 28.9, 28.6, 27.2, 27.1, 24.2, 22.6,
14.1 ppm. LRMS (FAB) m/z 537 (MNa+): HRMS (FAB)
calcd for C30H46O3N2SNa: 537.3127. Found: 537.3118
(MNa+).
n
O
N
S
4
7
7
H
9
9a (n¼4)
9b (n¼10)
*p<0.05; n>30.
2.72ꢀ0.78*
2.63ꢀ0.73*
69
73
a
Donor and recipient cells were co-cultured in the presence of the agents at
a concentration of 40 mM. The standard deviations (SD) are shown as ꢀ.
The percent inhibition of the transfer of the calcein dye from the donor
cells to recipient cells was determined for comparison with the oleamide
(100%).
b
4.2.2. Synthesis of Cn-linked dansyl oleamide.
oleamides excluding the Cn-linked NBD oleamides were
used in this study. As expected, all the oleamide derivatives
functionalized by the various groups via the alkyl chain
linkers inhibited the Cx43-mediated GJICs at a level similar
to oleamide. These results suggested that the functionaliza-
tions of the oleamide could be accomplished by using the
alkyl chain linkers without its biological activity being
influenced by these groups.
4.2.2.1. C2-linked dansyl oleamide. Under a nitrogen
atmosphere, the solution of 1,2-ethylenediamine (90 mg,
1.50 mmol) in CH2Cl2 (3 mL) was cooled to 0 ꢁC, and dan-
syl chloride (135 mg, 0.50 mmol) in CH2Cl2 (2.0 mL) was
added. The reaction mixture was stirred at room temperature
for 6 h and poured into 10% NaOHaq. The aqueous layer
was extracted with CH2Cl2. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo.
Purification of the residue by column chromatography
(CH2Cl2/MeOH, 2/1) gave 1a (123 mg, 84%).
3. Conclusion
We synthesized the Cn-linked dansyl oleamides whose bio-
logical activities are retained. Especially, the C4/C10-linked
dansyl oleamides show the inhibitory effect of GJICs at
a level similar to the oleamide. Moreover, we found that
other functional groups such as NBD, sugars, and (+)-biotin
could be introduced into the oleamide via alkyl chain linkers
without affecting its biological activity. By the strategy, var-
ious functions would be fused into oleamide. This method
presented here is very useful in various fields.
1H NMR (CDCl3, 270 MHz) d: 2.72 (2H, t, J¼6.5 Hz),
2.89–3.01 (2H, m), 2.89 (6H, s), 7.20 (1H, d, J¼7.3 Hz),
7.55 (2H, m), 8.28 (2H, m), 8.54 ppm (1H, d, J¼8.6 Hz).
Under a nitrogen atmosphere, the solution of oleic acid
(50 mg, 0.18 mmol) in CH2Cl2 (3.4 mL) was cooled to
0 ꢁC, and oxalyl chloride (64 mL, 0.72 mmol) was added.
The reaction mixture was stirred at room temperature for
4 h and concentrated in vacuo to give the residue.
4. Experimental
4.1. General
Under a nitrogen atmosphere, the residue (oleic chloride) in
CH2Cl2 (2.0 mL) was slowly added to the solution of 1a
(44 mg, 0.15 mmol) in CH2Cl2 (1.0 mL). The reaction mix-
ture was stirred at room temperature for 3 h and poured into
H2O. The aqueous layer was extracted with CH2Cl2. The or-
ganic layer was washed with brine, dried over Na2SO4, and
The NMR spectra were measured using 300 MHz or
270 MHz spectrometer with tetramethylsilane as the internal