Inhibition of oleamide hydrolase catalyzed hydrolysis of the endogenous sleep-inducing lipid cis-9-octadecenamide

Article abstract of DOI:10.1021/ja954064z

Oleamide (1, cis-9-octadecenamide) is a naturally occurring brain constituent that has been shown to accumulate and disappear under conditions of sleep deprivation and sleep recovery, respectively. Synthetic 1 has been found to induce sleep in a structurally specific manner at nanomolar quantities. Hydrolysis of 1 by an enzyme (oleamide hydrolase) present in the cell membrane rapidly degrades oleamide to oleic acid (cis-9-octadecenoic acid). Such observations suggest 1 may constitute a prototypical member of a class of fatty acid primary amide biological signaling molecules in which the diversity and selectivity of function are derived from the length of the alkane chain as well as the position, stereochemistry, and degree of unsaturation. A series of inhibitors of oleamide hydrolase were designed and prepared which were expected to derive their properties through interactions with the putative active site cysteine residue within oleamide hydrolase. This approach yielded a series of rapid, selective, and highly potent inhibitors (K(i) = 13 μM to 1 nM) which in addition to their potential therapeutic value may serve as useful tools to define the biological role of oleamide.

Full text of DOI:10.1021/ja954064z

5938
J. Am. Chem. Soc. 1996, 118, 5938-5945
Inhibition of Oleamide Hydrolase Catalyzed Hydrolysis of the
Endogenous Sleep-Inducing Lipid cis-9-Octadecenamide
Jean E. Patterson, Ian R. Ollmann, Benjamin F. Cravatt, Dale L. Boger,*
Chi -Huey Wong,* and Richard A. Lerner*
Contribution from the Department of Chemistry, The Scripps Research Institute,
10666 North Torrey Pines Road, La Jolla, California 92037
ReceiVed December 4, 1995X
Abstract: Oleamide (1, cis-9-octadecenamide) is a naturally occurring brain constituent that has been shown to
accumulate and disappear under conditions of sleep deprivation and sleep recovery, respectively. Synthetic 1 has
been found to induce sleep in a structurally specific manner at nanomolar quantities. Hydrolysis of 1 by an enzyme
(oleamide hydrolase) present in the cell membrane rapidly degrades oleamide to oleic acid (cis-9-octadecenoic acid).
Such observations suggest 1 may constitute a prototypical member of a class of fatty acid primary amide biological
signaling molecules in which the diversity and selectivity of function are derived from the length of the alkane chain
as well as the position, stereochemistry, and degree of unsaturation. A series of inhibitors of oleamide hydrolase
were designed and prepared which were expected to derive their properties through interactions with the putative
active site cysteine residue within oleamide hydrolase. This approach yielded a series of rapid, selective, and highly
potent inhibitors (Ki ) 13 µM to 1 nM) which in addition to their potential therapeutic value may serve as useful
tools to define the biological role of oleamide.
Oleamide (1, cis-9-octadecenamide) is a naturally occurring
brain constituent that has been shown to accumulate and
disappear under conditions of sleep deprivation and sleep
recovery, respectively.1-3 In a structurally specific manner, 1
has been shown to induce physiological sleep in animals at
nanomolar quantities when injected intravascularly.1 In an effort
to isolate a regulatory agent responsible for controlling endog-
enous concentrations of 1, an integral membrane protein,
oleamide hydrolase, was found to catalyze the hydrolytic
degradation of oleamide to give oleic acid (cis-9-octadecenoic
acid) and ammonia (Figure 1), neither of which demonstrate
somnolescent activity.1 To further explore and define the roles
of 1 as a prototypical member of a new class of biological
signaling agents and oleamide hydrolase as a potentially
important factor in its regulation, we describe the preparation
and evaluation of a series of potent transition-state mimetic and
mechanism-based oleamide hydrolase inhibitors, 2-22.
Oleamide hydrolase could be inhibited by phenylmethane-
sulfonyl fluoride, 4,4′-dithiodipyridyl disulfide (a potent disul-
fide-forming reagent), and HgCl2 (IC50 ) 700 nM, Ki,app ) 37
nM), but not 1 mM EDTA, suggesting that a thiol is intimately
involved in the catalytic process and that the enzyme may be a
cysteine amidase or possibly a serine amidase with an active
site cysteine residue. A variety of tight binding or irreversible
inhibitors of serine and cysteine proteases have been described.
These include irreversible inhibitors such as halomethyl
ketones,4-8 Michael acceptors,9 epoxides,10 O-acylhydroxyl-
Figure 1. Oleamide hydrolase catalyzes the hydrolysis of 1, a
biological signaling agent with sleep-inducing properties, to produce
oleic acid and ammonia.
amines,11 and diazomethyl ketones,12 as well as reversible
transition state mimetic inhibitors13 such as ketones,14 alde-
hydes,15 cyclopropenones,16 and electron-deficient carbonyl
compounds such as trifluoromethyl ketones,17-20 R-keto acid
derivatives,21-28 and tricarbonyl compounds.29 Only one pos-
(7) Rauber, P.; Angliker, H.; Walker, B.; Shaw, E. Biochem. J. 1986,
239, 633-640.
(8) Angliker, H.; Wikstrom, P.; Rauber, P.; Shaw, E. Biochem. J. 1987,
241, 871-875.
(9) Hanzlik, R. P.; Thompson, S. A. J. Med. Chem. 1984, 27, 711-712.
(10) Parkes, C.; Kembhavi, A. A.; Barret, A. J. Biochem. J. 1985, 230,
509-516.
(11) Bro¨mme, D.; Schierhorn, A.; Kirschke, H.; Wiederanders, B.; Barth,
A.; Fittkau, S.; Demuth, H.-U. Biochem. J. 1989, 263, 861-866.
(12) Green, G. D. J.; Shaw, E. J. Biol. Chem. 1981, 256, 1923-1928.
(13) Mehdi, S. Bioorg. Chem. 1993, 21, 249-259.
(14) Bro¨mme, D.; Bartels, B.; Kirschke, H.; Fittkau, S. J. Enzyme Inhib.
1989, 3, 13-21.
(15) Westerik, J. O.; Wolfenden, R. J. Biol. Chem. 1972, 247, 8195-
8197.
(16) Ando, R.; Morinaka, Y.; Tokuyama, H.; Isaka, M.; Nakamura, E.
J. Am. Chem. Soc. 1993, 115, 1174-1175.
(17) Wolfenden, R. Annu. ReV. Biophys. Bioeng. 1976, 5, 271.
(18) Gelb, M. H.; Svaren, J. P.; Abeles, R. H. Biochemistry 1985, 24,
1813-1817.
(19) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760-3767.
(20) Koutek, B.; Prestwich, G. D.; Howlett, A. C.; Chin, S. A.; Salehani,
D.; Akhavan, N.; Deutsch, D. G. J. Biol. Chem. 1994, 269, 22937-22940.
(21) Li, Z.; Patil, G. S.; Golubski, Z. E.; Hori, H.; Tehrani, K.; Foreman,
J. E.; Eveleth, D. D.; Bartus, R. T.; Powers, J. C. J. Med. Chem. 1993, 36,
3472-3480.
(22) Harbeson, S. L.; Abelleira, S. M.; Akiyama, A.; Barrett, R.; Carroll,
R. M.; Straub, J. A.; Tkacz, J. N.; Wu, C.; Musso, G. F. J. Med. Chem.
1994, 37, 2918-2929.
(23) Peet, N. P.; Burkhart, J. P.; Angelastro, M. R.; Giroux, E. L.; Mehdi,
S.; Bey, P.; Kolb, M.; Neises, B.; Schirlin, D. J. Med. Chem. 1990, 33,
394-407.
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
(1) Cravatt, B. F.; Prospero-Garcia, O.; Siuzdak, G.; Gilula, N. B.;
Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506-
1509.
(2) Lerner, R. A.; Siuzdak, G.; Prospero-Garcia, O.; Henriksen, S. J.;
Boger, D. L.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A 1994, 91, 9505-
9508.
(3) Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996,
118, 580-590.
(4) Kettner, C.; Shaw, E. Biochemistry 1978, 17, 4778-4784.
(5) Kettner, C.; Shaw, E. Thromb. Res. 1979, 14, 969-973.
(6) Giordano, C.; Gallina, C.; Ottaviano, V.; Consalvi, V.; Scandurra,
R. Eur. J. Med. Chem. 1992, 27, 865-873.
S0002-7863(95)04064-9 CCC: $12.00 © 1996 American Chemical Society

Hydrolysis of the Endogenous Sleep-Inducing Lipid
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 5939
Figure 3. pH-rate dependence of oleamide hydrolase cleavage of 1,
with the fit showing apparent active site pKa values of 5.4, 9.7, and
10.3. The rate maximum occurs at pH 10.0.
the result of the hydrolysis of 100 µM oleamide (∼20Km) by a
membrane bound preparation of oleamide hydrolase. The Km
of oleamide was found to be 5 ( 2 µM. Inhibition constants
were determined by the Dixon method (Figure 2). Subject to
solubility limitations, all inhibitors that were tested were able
to achieve 100% inhibition at high concentrations, and no
inhibitor exhibited polymodal inhibition behavior characteristic
of two or more separate active sites with different Ki values.
Since the likelihood of two or more different enzymes binding
twenty-two separate inhibitors with nearly identical affinity is
remote, this strongly suggests that a single enzyme in the
preparation is responsible for greater than 90% of the observed
oleamide hydrolase activity.
The rate of enzyme-catalyzed oleamide hydrolysis was found
to be pH dependent (Figure 3) with apparent active site pKa
values of 5.4, 9.7, and 10.3. Our unusual pH-rate dependence
profile, oleamide Km, and inhibition results for PMSF are
consistent with results by Maurelli,30,31 suggesting that the
oleamide hydrolase presented here (from rat liver membrane
fractions) and the anandamide amidohydrolase (from mouse
neuroblastoma cell culture membrane fractions) may be the same
enzyme, subject to interspecies variation. However, in the
absence of sequence data or purified enzyme, the latter of which
is often difficult to achieve with integral membrane proteins,
this remains to be proven. However, our results are quite
different from those of another report of anandamide amidohy-
rolase activity which exhibits rate maxima at pH 6 and 8,32 so
there is also evidence to support a many-enzyme model of fatty
acid amide hydrolysis in ViVo. The inhibitors were assayed at
pH 10.0, the pH at which oleamide hydrolase activity is at its
maximum under our assay conditions.
Figure 2. (a) Dixon plot of the activity of 6 against oleamide hydrolase
catalyzed degradation of 1, (b) Effect of 11 against oleamide hydrolase.
(c) Lineweaver-Burke plot of competitive oleamide hydrolase inhibi-
tion by 12.
The most potent inhibitors (Table 1) possess an electrophilic
carbonyl group capable of reversibly forming a (thio)hemiacetal
or (thio)hemiketal to mimic the transition state of a serine or
cysteine protease catalyzed reaction (Figure 4). The relative
potencies of the inhibitors were found to follow the expected
electrophilic character of the reactive carbonyl cumulating in
the tight binding R-keto ethyl ester 8 (1.4 nM) and the
trifluoromethyl ketone inhibitor 12 (1.2 nM). A similar
correlation between carbonyl electrophilicity and binding con-
stant has been observed in inhibitors of insect juvenile hormone
esterase33 and anandaminase.20 However, the most electrophilic
member of the set, the tricarbonyl inhibitor 11, bound relatively
sibly specific inhibitor of oleamide hydrolase has been reported
(IC50 ) 3 µM at [S] ) 0.26Km),30 and only one report of an
investigation of inhibitors of related fatty acid amidases has been
disclosed to date.20
The potency of the inhibitors was determined using an ion
selective electrode to measure the production of ammonia as
(24) Angelastro, M. R.; Mehdi, S.; Burkhart, J. P.; Peet, N. P.; Bey, P.
J. Med. Chem. 1990, 33, 11-13.
(25) Ocain, T. D.; Rich, D. H. J. Med. Chem. 1992, 35, 451-456.
(26) Yuan, W.; Wong, C.-H.; Haeggstro¨m, J. Z.; Wetterholm, A.;
Samuelsson, B. J. Am. Chem. Soc. 1992, 114, 6552-6553.
(27) Patel, D. V.; Rielly-Gauvin, K.; Ryono, D. E.; Free, C. A.; Smith,
S. A.; Petrillo, E. W. J. Med. Chem. 1993, 36, 2431-2447.
(28) Mun˜oz, B.; Giam, C.-Z.; Wong, C.-H. Bioorg. Med. Chem. 1994,
2, 1085-1090.
(31) Mackenzie, N. E.; Grant, S. K.; Scott, A. I.; Malthouse, J. P. G.
Biochemistry 1986, 25, 2293-2298.
(29) Wasserman, H. H.; Ennis, D. S.; Power, P. L.; Ross, M. J.; Gomes,
B. J. Org. Chem. 1993, 58, 4785-4787.
(30) Maurelli, S.; Bisogno, T.; De Petrocellis, L.; Di Luccia, A.; Marino,
G.; Di Marzo, V. FEBS Lett. 1995, 377, 82-86.
(32) Desarnaud, F.; Cadas, H.; Piomelli, D. J. Biol. Chem. 1995, 270,
6030-6035.
(33) Linderman, R. J.; Jamois, E. A.; Roe, R. M. ReV. Pestic. Toxicol.
1991, 1, 261-9.

5940 J. Am. Chem. Soc., Vol. 118, No. 25, 1996
Table 1. Binding Affinities
Patterson et al.
Figure 4. (a) A common intermediate found in papain and other
cysteine or serine proteases53 involving the formation of a (thio)-
hemiacetal during the catalysis of peptide hydrolysis. (b) The possible
mode of action for inhibitors 3-15.
studies of elastase34 and R-chymotrypsin35 and kinetic studies
of a series of serine proteases19 bound to peptidyl trifluoromethyl
ketones. Also, though the R-keto amides 6 and 7 are likely to
exist at least in part as the sp2 keto species in solution, R-keto
amides have been observed in protease active sites to be
completely sp3.19 Similarly, aldehydes in the active site of the
cysteine protease papain bind as thiohemiacetals.31,36 The
hypothesis that these inhibitors bind as (thio)hemiketals rather
than gem-diols (hydrated ketones) is further supported by the
poor inhibiton of oleamide hydrolase by 16, 17, and 18, despite
their structural similarity to the gem-diol.
We note that though electrophilicity of the reactive carbonyl
seems to play a large role in dictating the affinity with which
these inhibitors bind to oleamide hydrolase, there are likely other
factors which also exert influence on the affinity of these
compounds for oleamide hydrolase. While the aldehydes 4 and
R-keto amide 7 appear to be equally electrophilic, the R-keto
amide binds more tightly, suggesting that there are additional
favorable interactions being made between the enzyme and the
amide functionality, possibly an additional hydrogen bond(s).
Similarly, the R-keto ester 8 and the trifluoromethyl ketone 12
bind equally tightly despite the higher electrophilicity of the
trifluoromethyl ketone.
poorly at 150 nM. This behavior may be the result of
destabilizing steric interactions between the bulky tert-butyl ester
and the enzyme or may be in part due to the sp2 character at
C-3, which is uncharacteristic of the natural substrate.
The extent of hydration and the relative electrophilic character
of the inhibitor carbonyls could be easily and accurately assessed
by NMR analysis, and they were found to follow the expected
trends (e.g., 11 > 12 > 8 > 6 g 4). The central carbonyl of
the tricarbonyl inhibitor 11 was fully hydrated upon preparation
and characterization. The remaining agents were isolated and
characterized as their carbonyl structures without hydration
including the reactive trifluoromethyl ketones. 1H NMR and
13C NMR were used to establish and quantitate the addition of
CD3OD or D2O to the electrophilic carbonyl in CD3OD and
acetone-d6, respectively (Table 2). Representative of these
trends, 11 and 12 were fully converted to their hemiacetals in
CD3OD, and the remaining agents showed diminished hemi-
acetal formation consistent with their expected electrophilic
character: 11 (100%), 12 (100%), 8 (75%), 6 (48%), and 4
(47%).
Interestingly, aldehyde 5 which incorporates a carbonyl at a
position analogous to C-2 of oleamide was found to bind 5 times
more tightly than 4, which incorporates the aldehyde carbonyl
in the position analogous to the C-1 of oleamide. This was
also observed with the R-keto ester series of inhibitors, where
the incorporation of the electrophilic carbonyl at C-2 vs C-1 of
oleamide (8 vs 9) resulted in a 6-fold increase in binding affinity.
This distinction was not seen in the R-keto amides or trifluo-
romethyl ketones. In these inhibitor classes the placement of
the electrophilic carbonyl at the C-2 vs C-1 of oleamide position
provided equally effective inhibitors. This suggests the pos-
sibility of subtly different binding modes for carbonyl positional
analogs of C-1 versus C-2 of oleamide.
These studies also reveal that oleamide hydrolase displays
an approximately 10-fold preference for fatty acid inhibitors
which contain a cis double bond stereochemistry at the 9 position
similar to the natural substrate. This trend is seen most clearly
in the trifluoromethyl ketone series where the cis double bond
While the trifluoromethyl ketones 12, 13, 14, and 15 exist in
aqueous solution almost entirely as hydrates, these compounds
are thought to bind to the enzyme as reversible covalent
enzyme-inhibitor hemiketal complexes as shown in structural
Table 2. Electrophilic Carbonyl Hydration Studies, Diagnostic NMR Signals
agent
1H or 13C NMR signal
CDCl3 (δ)
CD3OD (δ)
2.42, 1.56
9.69 (53%), 4.46 (47%)
204.7, 99.9
2.81 (52%), 1.75 (48%)
165.2, 176.2
200.0, 100.1
2.81 (25%), 1.78 (75%)
162.5, 172.4
195.8, 100.0
- (0%), 1.65 (100%)
97.3
acetone-d6 (δ)
2.41
9.71
202.5
2.83
163.4
200.0
2.79
162.1
acetone-d6 + D2O (δ)a
4
1H (R-CH2)
2.40
9.73
202.8
2.89
161.9
198.6
2.81
161.3
194.8
2.68
no change
no change
no change
no change
no change
no change
no change
no change
no change
2.82 (9%), 1.60 (91%)
93.8
1H (CHO)
13C (CdO)
1H (R-CH2)
13C (1CdO)
13C (2CdO)
1H (R-CH2)
13C (1CdO)
13C (2CdO)
1H (R-CH2)
13C (CdO)
13C (CF3)
6
8
195.1
12
2.87 (91%), 1.75 (9%)
192.3, 94.2
116.5, 125.2
191.6
115.6
125.2
125.0
a7% D2O in acetone-d6.

Hydrolysis of the Endogenous Sleep-Inducing Lipid
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 5941
Scheme 1
Scheme 2
of EDCI, 0.2 equiv of DMAP, CH2Cl2, 25 °C, 19 h) provided
22. Treatment of 21 with anhydrous 1 N HCl-EtOAc (25 °C,
10 min, 92%) cleanly provided 3. The aldehyde 438 along with
the dimethyl acetal 1639 could be prepared directly from oleic
acid as described. Trapping of the enolate derived from oleic
acid (LDA, THF) with CCl4 or O2 provided 2040 and 18,41
respectively, which were converted to the corresponding primary
amides 19 and 17 via acid chloride generation (3 equiv of
(COCl)2, CH2Cl2, 25 °C, 3 h) and condensation with aqueous
NH4OH.
The C-18 oleic acid based R-keto amide 6 and R-keto ester
8 bearing an electrophilic carbonyl at the position analogous to
C-2 of oleamide rather than C-1 were prepared by oxidation of
the R-hydroxy amide 17 (PDC) and R-hydroxy acid 18 (Dess-
Martin) followed by ethyl ester formation (Scheme 1). The
corresponding R-keto esters 9 and 10, bearing an electrophilic
carbonyl at the position analogous to C-1 of oleamide were
prepared directly from the corresponding 18-carbon carboxylic
acids, oleic and stearic acids, employing a modified Dakin-
West reaction21,42 (Scheme 1). The R-keto amide 7 of similar
length was prepared by one-carbon extension of oleic acid
available through Wolff rearrangement of 21 (catalytic AgOBz,
CH3OH, 25 °C, 2.5 h, 82%) to provide the methyl ester 23.
Hydrolysis of the methyl ester followed by conversion of the
C-19 carboxylic acid 24 to the R-keto amide followed the
approach detailed for 6 (Scheme 1).
containing 12 is bound approximately an order of magnitude
more tightly than the trans double bond containing 14 or the
saturated derivative 15.
Most of the potential irreversible inhibitors (3, 19-21)
demonstrated no measurable time dependent inhibitory activity
over the first 15 min of incubation at concentrations up to their
solubility limits. The chloromethyl ketone 3 gave time inde-
pendent but moderate inhibition (Ki ) 0.7 µM) which is
consistent with the formation of a reversible (thio)hemiketal
between the putative active site cysteine and the ketone,37 or a
reversible and noncovalent enzyme-inhibitor complex. The
presence of an adjacent chloro substituent augments the elec-
trophilicity of the carbonyl, favoring nucleophilic attack.
2-Chlorooleic acid (20; Ki ) 0.3 µM) also appeared to bind
reversibly, with its binding mode possibly similar to that of oleic
acid (Ki ) 6 µM). Diazomethyl ketone 21 bound more weakly
(Ki ) 18 µM).
Inhibitor Synthesis. Many of the inhibitors were prepared
from oleic acid by known procedures or adaption of known
procedures (Scheme 1). Reaction of the acid chloride derived
from oleic acid (3 equiv of (COCl)2, CH2Cl2, 25 °C, 3 h) with
hydroxylamine or diazomethane provided 2 and 21, and direct
condensation of oleic acid with hydrazine (1.1 equiv; 2.2 equiv
The trifluoromethyl ketone inhibitors 12, 13, which incor-
porates the electrophilic carbonyl at the C-2 position of a C-18
lipid containing a 9-cis-olefin, 14, and 15 were prepared in one
operation by conversion of the corresponding carboxylic acids
to their respective acid chlorides and subsequent treatment with
TFAA-pyridine43 (6 equiv/8 equiv, Et2O, 0.75-2 h, 54-79%)
(Scheme 2). Oxidative cleavage of R-hydroxy acid 18 (Pb-
(OAc)4, 1.1 equiv, 25 °C, benzene, 50 mL) yielded aldehyde 5.
This was further oxidized (NaClO2) to give acid 27 which was
used to prepare 13.
(38) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43,
2480-2482.
(39) Marx, M. H.; Wiley, R. A.; Satchell, D. G.; Maguire, M. H. J. Med.
Chem. 1989, 32, 1319-1322.
(34) Takahasi, L. H.; Radhakrishnan, R.; Rosenfeld, R. E.; Meyer, E.
F.; Trainor, D. A.; Stein, M. J. Mol. Biol. 1988, 201, 423-428.
(35) Liang, T.-C.; Abeles, R. H. Biochemistry 1987, 26, 7603-7608.
(36) Schultz, R. M.; Cheerva, A. C. FEBS Lett. 1975, 50, 47-49.
(37) Bell, R. P. AdV. Phys. Org. Chem. 1966, 4, 1-29 and references
therein.
(40) Snider, B. B.; Kulkarni, Y. S. J. Org. Chem. 1987, 52, 307-310.
(41) Konen, D. A.; Silbert, L. S.; Pfeffer, P. E. J. Org. Chem. 1975, 40,
3253-3258.
(42) Buchanan, G. L. Chem. Soc. ReV. 1988, 17, 91-109.
(43) Boivin, J.; El Kaim, L.; Zard, S. Z. Tetrahedron Lett. 1992, 33,
1285-1288.

5942 J. Am. Chem. Soc., Vol. 118, No. 25, 1996
Patterson et al.
Chromatography (SiO2, 2 × 13 cm, 1-5% EtOAc-hexane gradient
elution) afforded 5 (68 mg, 67%) as a clear oil. Spectral properties
agree with those described in the literature.46,47
Scheme 3
2-Oxo-9(Z)-octadecenamide (6). A solution of 17 (8 mg, 0.027
mmol, 1 equiv) in anhydrous DMF (0.13 mL) under Ar was treated
with PDC (51 mg, 0.13 mmol, 5 equiv), and the reaction mixture was
stirred for 1 h at 25 °C. The crude reaction was treated with H2O (2
mL), and the aqueous layer was extracted with Et2O (4 × 2 mL). The
organic layers were dried (Na2SO4), filtered, and concentrated in vacuo.
Chromatography (SiO2, 1 × 3 cm, 20-66% EtOAc-hexane gradient
elution) afforded 6 (6 mg, 70%) as a white solid and some recovered
The tricarbonyl inhibitor 11 was prepared following the
procedures detailed by Wasserman.44 Treatment of the acid
chloride derived from palmitic acid with tert-butyl (tri-
phenylphosphoranylidene)acetate (29) in the presence of bis-
(trimethylsilyl)acetamide (BSA) followed by oxone oxidation
provided 11 (Scheme 3).
In summary, several potent inhibitors of the enzyme oleamide
hydrolase, responsible for the hydrolysis of an endogenous sleep-
inducing lipid (1, cis-9-octadecenamide), have been developed,
providing insights into the mechanism of the enzyme and the
fundamental basis for the development of agents for the control
and regulation of sleep. One inhibitor, 6, also induces sleep in
a dose dependent manner analogous to oleamide (1) itself and
may function both as an inhibitor of oleamide hydrolase and as
an agonist of 1. The behavior of 6 and related effects of the
inhibitors described herein will be described in due time. Work
is in progress to prepare the pure enzyme for further specificity
and inhibition studies.
1
starting material (2 mg, 26%). Data for 6: mp 85-86 °C; H NMR
(CDCl3, 400 MHz) δ 6.79 (br, 1H), 5.47 (br, 1H), 5.37-5.28 (m, 2H),
2.89 (t, 2H, J ) 7.4 Hz), 2.02-1.93 (m, 4H), 1.59 (p, 2H, J ) 7.2
Hz), 1.39-1.24 (m, 20H), 0.86 (t, 3H, J ) 6.8 Hz); 13C NMR (CDCl3,
100 MHz) δ 198.6, 161.9, 130.1, 129.6, 36.5, 31.9, 29.7, 29.5(2),
29.3(2), 28.9(2), 27.2, 27.1, 23.1, 22.7, 14.1; IR (film) νmax 3391, 2915,
2850, 1716, 1668, 1470, 1400, 1108 cm-1; FABHRMS (NBA-CsI)
m/z 428.1547 (C18H33NO2 + Cs+ requires 428.1566).
2-Oxo-10(Z)-nonadecenamide (7). A solution of 26 (42 mg, 0.14
mmol, 1 equiv) in anhydrous CH2Cl2 (2.8 mL) at 25 °C was treated
with o-Ph(CO2)I(OAc)3 (174 mg, 0.41 mmol, 3 equiv), and the reaction
mixture was stirred for 1.5 h. The mixture was treated with 10%
aqueous NaOH (30 mL), and the aqueous layer was extracted with
EtOAc (3 × 30 mL). The organic layers were dried (Na2SO4), filtered,
and concentrated in vacuo. Chromatography (SiO
2, 1.5 × 13 cm, 10-
20% EtOAc-hexane gradient elution) afforded 7 (24 mg, 57%) as a
1
white solid: mp 69-70 °C; H NMR (CDCl3, 400 MHz) δ 6.82 (br,
1H), 5.68 (br, 1H), 5.36-5.28 (m, 2H), 2.88 (t, 2H, J ) 7.4 Hz), 1.98
(m, 4H), 1.58 (p, 2H, J ) 7.0 Hz), 1.28-1.24 (m, 20H), 0.85 (t, 3H,
J ) 6.9 Hz); 13C NMR (CDCl3, 100 MHz) δ 198.7, 162.0, 130.0, 129.7,
36.5, 31.9, 29.74, 29.66, 29.5, 29.3(2), 29.2, 29.1, 29.0, 27.2, 27.1,
23.1, 22.7, 14.1; IR (film) νmax 3395, 3217, 2922, 2850, 1718, 1672,
1601, 1469, 1406, 1115 cm-1; FABHRMS (NBA-NaI) m/z 332.2570
(C19H35NO2 + Na+ requires 332.2565).
Experimental Section
The agents 1,1 4,38 15,20 16,39 18,41 20,40 and 2945 were prepared as
detailed.
N-Hydroxy-9(Z)-octadecenamide (2). Oleic acid (250 µL, 0.79
mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (4 mL) and cooled
to 0 °C under N2. Oxalyl chloride (2 M in CH2Cl2, 1.2 mL, 2.4 mmol,
3 equiv) was added slowly. The solution was warmed to 25 °C and
allowed to stir for 3 h in the dark. The solvent was removed in vacuo
and the flask cooled to 0 °C. Excess hydroxylamine in EtOAc (the
hydrochloride salt was extracted into EtOAc from a 50% NaOH solution
before use) was added slowly. The solvent was removed in vacuo,
and chromatography (SiO2, 1.5 × 13 cm, 33-66% EtOAc-hexane
gradient elution) afforded 2 as a white solid (104 mg, 45%): mp 61-
Ethyl 2-Oxo-9(Z)-octadecenoate (8). A solution of 18 (102 mg,
0.34 mmol, 1 equiv) in anhydrous CH2Cl2 (1.1 mL) at 25 °C under N2
was treated with o-Ph(CO2)I(OAc)3 (287 mg, 0.68 mmol, 2 equiv) and
stirred for 1 h. The reaction mixture was treated with 10% aqueous
NaOH (20 mL) and extracted with EtOAc (3 × 20 mL). The organic
layers were dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was dissolved in anhydrous CH2Cl2 (1.5 mL) and cooled to 0
°C under N2. Oxalyl chloride (2 M in CH2Cl2, 0.5 mL, 1.0 mmol, 3
equiv) was added slowly. The reaction mixture was warmed to 25 °C
and was stirred in the dark for 3 h before the solvent was removed in
vacuo and absolute EtOH (5 mL) was added. Chromatography (SiO2,
2 × 10 cm, 1-5% EtOAc-hexane) afforded 8 (36 mg, 33%) as a
clear oil: 1H NMR (CDCl3, 400 MHz) δ 5.37-5.27 (m, 2H), 4.29 (q,
2H, J ) 7.2 Hz), 2.81 (t, 2H, J ) 7.3 Hz), 1.98 (m, 4H), 1.61 (p, 2H,
J ) 7.1 Hz), 1.36-1.24 (m, 21H), 0.86 (t, 3H, J ) 6.8 Hz); 13C NMR
(CDCl3, 100 MHz) δ 194.8, 161.3, 130.1, 129.6, 62.4, 39.3, 31.9, 29.8,
29.5(2), 29.3(2), 28.92, 28.86, 27.2, 27.1, 22.9, 22.7, 14.1, 14.0; IR
(film) νmax 2925, 2854, 1729, 1462, 1260, 1056 cm-1; FABHRMS
(NBA-CsI) m/z 457.1706 (C20H36O3 + Cs+ requires 457.1719).
Ethyl 2-Oxo-10(Z)-nonadecenoate (9). A solution of oleic acid
(100 µL, 0.32 mmol, 1 equiv) in anhydrous THF (0.2 mL) at 25 °C
under Ar was treated with DMAP (4 mg, 0.03 mmol, 0.1 equiv),
anhydrous pyridine (77 µL, 0.95 mmol, 3 equiv), and ethyl oxalyl
chloride (71 µL, 0.64 mmol, 2 equiv). The reaction mixture was stirred
for 24 h before additional DMAP (46 mg, 0.37 mmol, 1.1 equiv),
pyridine (80 µL, 0.95 mmol, 3 equiv), ethyl oxalyl chloride (80 µL,
0.64 mmol, 2 equiv), and THF (0.5 mL) were added. The reaction
mixture was stirred at 25 °C for an additional 24 h and then was warmed
to 40 °C for 48 h before the solvent was concentrated in vacuo.
Chromatography (SiO2, 2 × 13 cm, 0-10% EtOAc-hexane) afforded
9 (46 mg, 43%) as a clear oil: 1H NMR (CDCl3, 400 MHz) δ 5.36-
5.28 (m, 2H), 4.29 (q, 2H, J ) 7.1 Hz), 2.80 (t, 2H, J ) 7.3 Hz), 1.99
(m, 4H), 1.60 (m, 2H), 1.36-1.20 (m, 23H), 0.85 (t, 3H, J ) 6.8 Hz);
13C NMR (CDCl3, 100 MHz) δ 194.8, 161.2, 130.0, 129.7, 62.4, 39.3,
1
62 °C; H NMR (CD3OD, 400 MHz) δ 5.28-5.20 (m, 2H), 2.00-
1.91 (m, 6H), 1.50 (p, 2H, J ) 6.8 Hz), 1.22-1.19 (m, 20H), 0.80 (t,
3H, J ) 6.9 Hz); 13C NMR (CD3OD, 100 MHz) δ 173.0, 130.9, 130.8,
33.8, 33.1, 30.9(2), 30.6, 30.5, 30.4, 30.33(2), 30.26, 30.19, 28.2, 26.8,
23.8, 14.5; IR (film) νmax 3276, 2999, 2917, 2849, 1665, 1621, 1463,
1428, 1117, 1067, 968 cm-1; FABHRMS (NBA-NaI) m/z 320.2577
(C18H35NO2 + Na+ requires 320.2565).
1-Chloro-10(Z)-nonadecen-2-one (3). A sample of 21 (347 mg,
1.13 mmol, 1 equiv) was treated with 1 M HCl in EtOAc (4.0 mL, 4.0
mmol, 3.5 equiv) for 10 min at 25 °C before the mixture was
concentrated in vacuo. Chromatography (SiO2, 3 × 13 cm, 5%
EtOAc-hexane) afforded 3 (328 mg, 92%) as a clear oil: 1H NMR
(CD3OD, 400 MHz) δ 5.29-5.21 (m, 2H), 4.18 (s, 2H), 2.48 (t, 2H, J
) 7.3 Hz), 1.93 (m, 4H), 1.50 (p, 2H, J ) 7.1 Hz), 1.31-1.21 (m,
20H), 0.81 (t, 3H, J ) 6.8 Hz); 13C NMR (CD3OD, 100 MHz) δ 204.5,
130.9, 130.8, 49.3, 40.3, 33.1, 30.9, 30.8, 30.6, 30.5, 30.40, 30.37, 30.19,
30.17(2), 28.1, 24.6, 23.8, 14.5; IR (film) νmax 2925, 2854, 1722, 1463,
1403, 1260, 1101, 796, 723 cm-1; FABHRMS (NBA) m/z 315.2468
(C19H35OCl + H+ requires 315.2455).
8(Z)-Heptadecenal (5). A solution of 18 (120 mg, 0.40 mmol, 1
equiv) in anhydrous benzene (1.6 mL) at 25 °C under N2 was treated
with Pb(OAc)4 (197 mg, 0.44 mmol, 1.1 equiv), and the reaction
mixture was stirred for 50 min. Water (2 mL) was added, and the
aqueous layer was extracted with EtOAc (6 × 2 mL). The organic
layers were dried (Na2SO4), filtered, and concentrated in vacuo.
(44) Wasserman, H. H.; Ennis, D. S.; Blum, C. A.; Rotello, V. M.
Tetrahedron Lett. 1992, 33, 6003-6006.
(45) Cooke, M. P.; Burman, D. L. J. Org. Chem. 1982, 47, 4955-4963.
(46) Doleshall, G. Tetrahedron Lett. 1977, 381-382.
(47) Kemp, T. R. J. Am. Oil Chem. Soc. 1975, 52, 300-302.

Hydrolysis of the Endogenous Sleep-Inducing Lipid
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 5943
31.9, 29.7, 29.6, 29.5, 29.3(2), 29.2, 29.0, 28.9, 27.2, 27.1, 22.9, 22.7,
1,1,1-Trifluoro-10(E)-nonadecen-2-one (14). A solution of elaidic
acid (204 mg, 0.72 mmol, 1 equiv) in anhydrous CH2Cl2 (3.5 mL) was
cooled to 0 °C under N2 and treated with oxalyl chloride (2 M in CH2-
Cl2, 1.1 mL, 2.2 mmol, 3 equiv). The reaction mixture was warmed
to 25 °C and stirred for 3 h before the solvent was removed in vacuo.
Anhydrous Et2O (5 mL), trifluoroacetic anhydride (0.6 mL, 4.3 mmol,
6 equiv), and anhydrous pyridine (0.23 mL, 2.8 mmol, 4 equiv) were
added at 25 °C, and the solution was stirred for 1 h before being cooled
to 0 °C. The mixture was treated with H2O (30 mL), and the aqueous
layer was extracted with EtOAc (3 × 30 mL). The organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo. Chromatography
(SiO2, 2 × 13 cm, 1% Et3N in 5-10% EtOAc-hexane gradient elution)
afforded 14 (190 mg, 79%) as a clear oil: 1H NMR (CDCl3, 400 MHz)
δ 5.41-5.31 (m, 2H), 2.68 (t, 2H, J ) 7.3 Hz), 1.94 (m, 4H), 1.65 (p,
2H, J ) 6.9 Hz), 1.28-1.24 (m, 20H), 0.86 (t, 3H, J ) 6.6 Hz); 13C
NMR (CDCl3, 100 MHz) δ 191.5 (q, J ) 35 Hz), 130.6, 130.1, 115.6
(q, J ) 291 Hz), 36.3, 32.6, 32.5, 31.9, 29.7, 29.5(2), 29.3, 29.2, 29.1,
28.8, 28.7, 22.7, 22.4, 14.0; IR (film) νmax 2925, 2855, 1765, 1466,
1208, 1152, 967, 709 cm-1; FABHRMS (NBA-NaI) m/z 334.2475
(C19H33OF3 - H+ requires 334.2484).
14.1, 14.0; IR (film) νmax 2925, 2854, 1730, 1465, 1260, 1059 cm-1
;
FABHRMS (NBA-CsI) m/z 471.1875 (C21H38O3 + Cs+ requires
471.1888).
Ethyl 2-Oxo-nonadecanoate (10). A solution of stearic acid (101
mg, 0.36 mmol, 1 equiv) in anhydrous THF (0.2 mL) at 25 °C under
Ar was treated with DMAP (4 mg, 0.03 mmol, 0.1 equiv), anhydrous
pyridine (85 µL, 1.1 mmol, 3 equiv), and ethyl oxalyl chloride (79 µL,
0.71 mmol, 2 equiv). The reaction mixture was stirred for 24 h before
the solvent was concentrated in vacuo. Chromatography (SiO2, 2 ×
13 cm, 5-10% EtOAc-hexane) afforded 10 (35 mg, 30%) as a white
solid: mp 43-44 °C; 1H NMR (CDCl3, 400 MHz) δ 4.29 (q, 2H, J )
7.2 Hz), 2.80 (t, 2H, J ) 7.4 Hz), 1.60 (p, 2H, J ) 7.2 Hz), 1.35 (t,
3H, J ) 7.1 Hz), 1.33-1.23 (m, 28H), 0.86 (t, 3H, J ) 6.8 Hz); 13C
NMR (CDCl3, 100 MHz) δ 194.8, 161.2, 62.4, 39.3, 31.9, 29.7(7),
29.6, 29.40, 29.35, 29.28, 28.9, 23.0, 22.7, 14.1, 14.0; IR (film) νmax
2916, 2848, 1733, 1472, 1463, 723 cm-1; FABHRMS (NBA-NaI)
m/z 363.2885 (C21H40O3 + Na+ requires 363.2875).
tert-Butyl 3-Oxo-2,2-dihydroxyoctadecanoate (11). A solution of
28 (161 mg, 0.26 mmol, 1 equiv) in THF-H2O (2:1; 3 mL) was treated
with Oxone (249 mg, 0.41 mmol, 1.6 equiv), and the reaction mixture
was stirred at 25 °C for 7 h. Water (30 mL) was added, and the aqueous
layer was extracted with EtOAc (3 × 30 mL). The organic layers were
combined, dried (Na2SO4), filtered, and concentrated in vacuo. Chro-
matography (SiO2, 2 × 15 cm, 10-20% EtOAc-hexane gradient
elution) afforded 11 (65 mg, 64%) as a white solid: mp 49-51 °C; 1H
NMR (DMSO-d6, 400 MHz) δ 6.96 (s, 2H), 2.17 (t, 2H, J ) 7.4 Hz),
1.49-1.38 (m, 11H), 1.22 (s, 24H), 0.84 (t, 3H, J ) 6.8 Hz); 13C NMR
(DMSO-d6, 100 MHz) δ 205.6, 174.5, 94.2, 81.5, 35.6, 33.6, 31.3,
29.0(3), 28.9, 28.8, 28.72, 28.70, 28.53, 28.46, 27.4(2), 24.5, 22.9, 22.1,
13.9; IR (film) νmax 3440, 2914, 2849, 1728, 1471, 1371, 1260, 1122,
831, 718 cm-1; FABHRMS (NBA-NaI) m/z 409.2925 (C22H42O5 +
Na+ requires 409.2930).
1,1,1-Trifluoro-10(Z)-nonadecen-2-one (12). Oleic acid (100 µL,
0.32 mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (1.5 mL) and
cooled to 0 °C under N2. Oxalyl chloride (2 M in CH2Cl2, 0.47 mL,
0.94 mmol, 3 equiv) was added slowly. The reaction mixture was
warmed to 25 °C and was stirred in the dark for 3 h before the solvent
was removed in vacuo. Anhydrous Et2O (2.2 mL), trifluoroacetic
anhydride (270 µL, 1.9 mmol, 6 equiv), and pyridine (0.2 mL, 2.5
mmol, 8 equiv) were added at 25 °C, and the solution was stirred for
45 min before being cooled to 0 °C. The reaction was quenched with
the addition of H2O (30 mL), and the aqueous layer was extracted with
CH2Cl2 (3 × 30 mL). The organic layers were dried (Na2SO4), filtered,
and concentrated in vacuo. Chromatography (SiO2, 1.5 × 13 cm, 1%
Et3N in 5% EtOAc-hexane) afforded 8 (75 mg, 71%) as a clear oil:
1H NMR (CDCl3, 400 MHz) δ 5.37-5.28 (m, 2H), 2.68 (t, 2H, J )
7.3 Hz), 1.98 (m, 4H), 1.65 (p, 2H, J ) 7.1 Hz), 1.29-1.25 (m, 20H),
0.86 (t, 3H, J ) 6.9 Hz); 13C NMR (CDCl3, 100 MHz) δ 191.6 (d, J
) 17 Hz), 130.0, 129.5, 115.6 (q, J ) 145 Hz), 36.3, 31.9, 29.8, 29.6,
29.5, 29.3(2), 29.1, 29.0, 28.7, 27.2, 27.1, 22.7, 22.4, 14.1; IR (film)
2-Hydroxy-9(Z)-octadecenamide (17). A solution of 18 (52 mg,
0.18 mmol, 1 equiv) in anhydrous CH2Cl2 (0.7 mL) cooled to 0 °C
under N2 was treated with oxalyl chloride (2 M in CH2Cl2, 0.22 mL,
0.44 mmol, 3 equiv). The solution was allowed to warm to 25 °C and
stirred for 3 h in the dark. The solvent was removed in vacuo, and the
acid chloride was cooled to 0 °C. The sample was treated with excess
concentrated aqueous NH4OH. Chromatography (SiO2, 1.5 × 10 cm,
66-100% EtOAc-hexane gradient elution) afforded 17 (31 mg, 60%)
1
as a white solid: mp 103-104 °C; H NMR (CDCl3, 400 MHz) δ
6.37 (br, 1H), 5.64 (br, 1H), 5.36-5.28 (m, 2H), 4.12 (t, 1H, J ) 3.8
Hz), 2.66 (br, 1H), 2.02-1.94 (m, 4H), 1.86-1.77 (m, 1H), 1.68-
1.59 (m, 1H), 1.43-1.24 (m, 20H), 0.86 (t, 3H, J ) 6.8 Hz); 13C NMR
(CDCl3, 100 MHz) δ 176.6, 130.0, 129.7, 71.9, 34.8, 31.9, 29.7, 29.6,
29.5, 29.3(2), 29.2, 29.1, 27.2, 27.1, 24.9, 22.7, 14.1; IR (film) νmax
3381, 3289, 2917, 2848, 1637, 1461, 1417, 1331, 1074 cm-1
;
FABHRMS (NBA) m/z 298.2760 (C18H35NO2 + H+ requires 298.2746).
2-Chloro-9(Z)-octadecenamide (19). A solution of 20 (48 mg, 0.15
mmol, 1 equiv) in anhydrous CH2Cl2 (0.7 mL) cooled to 0 °C under
N2 was treated with oxalyl chloride (2 M in CH2Cl2, 0.23 mL, 0.46
mmol, 3 equiv). The solution was allowed to warm to 25 °C and was
stirred for 3 h in the dark before the solvent was removed in vacuo.
The crude acid chloride was cooled to 0 °C and treated with excess
concentrated aqueous NH4OH. Chromatography (SiO2, 1.5 × 10 cm,
20-33% EtOAc-hexane gradient elution) afforded 19 (37 mg, 78%)
as a yellow solid: mp 49-50 °C; 1H NMR (CDCl3, 400 MHz) δ 6.49
(br, 1H), 5.92 (br, 1H), 5.36-5.27 (m, 2H), 4.29 (m, 1H), 2.12-1.86
(m, 6H), 1.53-1.16 (m, 20H), 0.85 (t, 3H, J ) 6.9 Hz); 13C NMR
(CDCl3, 100 MHz) δ 171.9, 130.1, 129.6, 60.6, 35.5, 31.9, 29.7, 29.6,
29.5, 29.3(2), 29.0, 28.7, 27.2, 27.1, 25.8, 22.7, 14.1; IR (film) νmax
3383, 3183, 3001, 2921, 2850, 1657, 1465, 1412, 1240, 1100 cm-1
;
ν
max 2926, 2855, 1766, 1467, 1404, 1261, 1208, 1153, 1039, 802, 709
FABHRMS (NBA) m/z 316.2415 (C18H34NOCl + H+ requires 316.2407).
cm-1; ESIMS m/z (M+) 334.
1-Diazo-10(Z)-nonadecen-2-one (21). Oleic acid (1.0 mL, 3.2
mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (15 mL) under
N2. The solution was cooled to 0 °C, and oxalyl chloride (2 M in
CH2Cl2, 4.8 mL, 9.6 mmol, 3 equiv) was added. The reaction mixture
was allowed to warm to 25 °C and was stirred for 3 h in the dark. The
solvent was removed in vacuo before the acid chloride was transferred
to a flask with no ground glass joints and cooled to 0 °C. Excess
diazomethane in Et2O (prepared from N-nitrosomethylurea in 50%
aqueous KOH and drying over KOH pellets) was added. The reaction
was stirred at 0 °C for 1 h before warming to 25 °C overnight. The
solution was diluted with EtOAc (60 mL) and washed with saturated
aqueous NaHCO3 (60 mL) and saturated aqueous NaCl (60 mL). The
organic layer was dried (Na2SO4), filtered, and concentrated in vacuo.
Chromatography (SiO2, 4.0 × 16 cm, 5-10% EtOAc-hexane gradient
elution) afforded 21 (0.89 g, 92%) as a yellow oil: 1H NMR (CD3OD,
400 MHz) δ 5.72 (br, 1H), 5.29-5.21 (m, 2H), 2.23 (m, 2H), 1.94 (m,
4H), 1.50 (p, 2H, J ) 6.9 Hz), 1.23-1.20 (m, 20H), 0.81 (t, 3H, J )
6.9 Hz); 13C NMR (CD3OD, 100 MHz) δ 198.8, 130.9, 130.8, 41.6,
33.1, 30.9, 30.8(2), 30.6, 30.5, 30.4(2), 30.3, 30.2, 28.1(2), 26.5, 23.8,
1,1,1-Trifluoro-9(Z)-octadecen-2-one (13). A solution of 27 (101
mg, 0.38 mmol, 1 equiv) in anhydrous CH2Cl2 (1.8 mL) was cooled to
0 °C under N2 and treated dropwise with oxalyl chloride (2 M in CH2-
Cl2, 0.56 mL, 1.1 mmol, 3 equiv). The reaction mixture was warmed
to 25 °C and stirred for 3 h before the solvent was removed in vacuo.
Anhydrous Et2O (2.5 mL), trifluoroacetic anhydride (0.32 mL, 2.3
mmol, 6 equiv), and anhydrous pyridine (0.12 mL, 1.5 mmol, 4 equiv)
were added at 25 °C, and the solution was stirred for 2 h before being
cooled to 0 °C. The reaction mixture was treated with H2O (30 mL),
and the aqueous layer was extracted with EtOAc (3 × 30 mL). The
organic layers were dried (Na2SO4), filtered, and concentrated in vacuo.
Chromatography (SiO2, 2 × 15 cm, 1% Et3N in 10% EtOAc-hexane)
afforded 13 (65.5 mg, 54%) as a clear oil: 1H NMR (CDCl3, 400 MHz)
δ 5.39-5.26 (m, 2H), 2.69 (t, 2H, J ) 7.2 Hz), 1.99 (m, 4H), 1.66 (m,
2H), 1.35-1.24 (m, 18H), 0.86 (t, 3H, J ) 6.9 Hz); 13C NMR (CDCl3,
100 MHz) δ 191.4, 130.5, 129.1, 115.6 (q, J ) 146 Hz), 36.3, 31.9,
29.7, 29.5, 29.3(3), 29.2, 28.3, 27.2, 26.8, 22.7, 22.3, 14.1; IR (film)
ν
max 2926, 2855, 1765, 1462, 1209, 1154, 1024 cm-1; ESIMS m/z (M
+ Na+) 343.

5944 J. Am. Chem. Soc., Vol. 118, No. 25, 1996
Patterson et al.
14.5; IR (film) νmax 3083, 2924, 2854, 2102, 1644, 1463, 1372, 1144
cm-1; FABHRMS (NBA) m/z 307.2738 (C19H34N2O + H+ requires
307.2749).
1.67 (m, 1H), 1.47-1.24 (m, 22H), 0.86 (t, 3H, J ) 6.8 Hz); 13C NMR
(CDCl3, 100 MHz) δ 179.8, 130.0, 129.7, 70.2, 34.2, 31.9, 29.8, 29.7,
29.5, 29.33, 29.31(2), 29.22, 29.19, 27.20, 27.16, 24.8, 22.7, 14.1; IR
(film) νmax 3512, 2917, 2849, 1704, 1467, 1293, 1274, 1251, 1212,
1143, 1079, 1041, 918, 726, 648 cm-1; FABHRMS (NBA-NaI) m/z
335.2574 (C19H36O3 + Na+ requires 335.2562).
N-Amino-9(Z)-octadecenamide (22). A solution of oleic acid (250
µL, 0.79 mmol, 1 equiv) and hydrazine monohydrate (42 µL, 0.87
mmol, 1.1 equiv) in anhydrous CH2Cl2 (12 mL) under N2 at 0 °C was
treated with EDCI (267 mg, 0.90 mmol, 1.1 equiv) and DMAP (20
mg, 0.16 mmol, 0.21 equiv) before the reaction mixture was allowed
to stir at 25 °C for 7 h. Another portion of EDCI (269 mg, 0.91 mmol,
1.1 equiv) was added, and the reaction was stirred at 25 °C for an
additional 12 h before the solvent was removed in vacuo. Chroma-
tography (SiO2, 3 × 18 cm, 20-100% EtOAc-hexane gradient elution)
afforded 22 (123 mg, 52%) as a white solid: mp 95-96 °C; 1H NMR
(CDCl3, 400 MHz) δ 8.94 (s, 1H), 5.36-5.27 (m, 2H), 2.23 (t, 2H, J
) 7.6 Hz), 1.98 (m, 4H), 1.63 (p, 2H, J ) 7.0 Hz), 1.27-1.24 (m,
20H), 0.86 (t, 3H, J ) 6.7 Hz); 13C NMR (CDCl3, 100 MHz) δ 169.7,
130.0, 129.7, 34.1, 31.9, 29.8, 29.7, 29.5, 29.3(2), 29.23, 29.19, 29.12,
27.21, 27.17, 25.4, 22.7, 14.1; IR (film) νmax 3201, 2917, 2848, 1595,
1410, 1184, 1090, 927, 717, 671 cm-1; FABHRMS (NBA-NaI) m/z
297.2916 (C18H36N2O + H+ requires 297.2906).
Methyl 10(Z)-Nonadecenoate (23). A solution of silver benzoate
(21.8 mg, 0.095 mmol, 0.1 equiv) and anhydrous Et3N (0.19 mL, 1.36
mmol, 1.4 equiv) was added dropwise to a solution of 1-diazo-10(Z)-
nonadecen-2-one (21; 298 mg, 0.97 mmol, 1 equiv) in anhydrous CH3-
OH (1.5 mL) under N2, and the reaction was stirred at 25 °C for 2.5 h.
The reaction mixture was diluted with EtOAc (30 mL) and washed
with 1 N aqueous HCl (30 mL) and saturated aqueous NaHCO3 (30
mL). The organic layers were dried (Na2SO4), filtered, and concentrated
in vacuo. Chromatography (SiO2, 3 × 15 cm, 1-5% EtOAc-hexane
gradient elution) afforded 23 (246 mg, 82%) as a clear oil: 1H NMR
(CDCl3, 400 MHz) δ 5.35-5.27 (m, 2H), 3.63 (s, 3H), 2.27 (t, 2H, J
) 7.6 Hz), 1.97 (m, 4H), 1.59 (p, 2H, J ) 7.3 Hz), 1.26-1.24 (m,
22H), 0.85 (t, 3H, J ) 6.8 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.3,
129.9, 129.8, 51.4, 34.1, 31.9, 29.74, 29.70, 29.5, 29.3(2), 29.2(2),
29.1(2), 27.2(2), 24.9, 22.6, 14.1; IR (film) νmax 2925, 2854, 1744, 1465,
1436, 719 cm-1; FABHRMS (NBA-NaI) m/z 311.2969 (C20H38O2 +
H+ requires 311.2950).
10(Z)-Nonadecenoic Acid (24). A solution of 23 (620 mg, 2.0
mmol, 1 equiv) in THF-CH3OH-H2O (3:1:1; 7 mL) at 25 °C was
treated with LiOH‚H2O (250 mg, 5.96 mmol, 3 equiv), and the reaction
mixture was stirred for 3 h. The reaction mixture was acidified with
the addition of 1 N aqueous HCl (60 mL), and the aqueous layer was
extracted with EtOAc (2 × 60 mL). The organic layers were dried
(Na2SO4), filtered, and concentrated in vacuo. Chromatography (SiO2,
4 × 15 cm, 10-100% EtOAc-hexane gradient elution) afforded 24
(510 mg, 86%) as a pale yellow oil: 1H NMR (CDCl3, 400 MHz) δ
5.37-5.28 (m, 2H), 2.32 (t, 2H, J ) 7.5 Hz), 1.98 (m, 4H), 1.61 (p,
2H, J ) 7.3 Hz), 1.27-1.25 (m, 22H), 0.86 (t, 3H, J ) 6.9 Hz); 13C
NMR (CDCl3, 100 MHz) δ 180.4, 130.0, 129.8, 34.1, 31.9, 29.8, 29.7,
29.5, 29.3(2), 29.2(2), 29.0(2), 27.20, 27.17, 24.6, 22.7, 14.1; IR (film)
νmax 2925, 2854, 1711, 1466, 1412, 1260, 1093, 1019, 938, 801, 722
cm-1; FABHRMS (NBA-NaI) m/z 319.2605 (C19H36O2 + Na+ requires
319.2613). This compound can alternatively be prepared by the method
of Doleshall.48
2-Hydroxy-10(Z)-nonadecenamide (26). A solution of 25 (71 mg,
0.23 mmol, 1 equiv) in anhydrous CH2Cl2 (1.5 mL) under N2 was
cooled to 0 °C and treated dropwise with oxalyl chloride (2 M in CH2-
Cl2, 0.34 mL, 0.68 mmol, 3 equiv). The reaction mixture was allowed
to warm to 25 °C and was stirred for 3 h in the dark. The solvent was
removed in vacuo, the residue was cooled to 0 °C, and excess
concentrated aqueous NH4OH (2 mL) was added. Chromatography
(SiO2, 1.5 × 13 cm, 50-66% EtOAc-hexane gradient elution) afforded
26 (53 mg, 75%) as a white solid: mp 101-102 °C; 1H NMR (CDCl3,
400 MHz) δ 6.36 (br, 1H), 5.65 (br, 1H), 5.36-5.28 (m, 2H), 4.12
(dd, 1H, J ) 7.9 Hz, 8.0 Hz), 1.99 (m, 4H), 1.81 (m, 1H), 1.63 (m,
1H), 1.43-1.24 (m, 22H), 0.86 (t, 3H, J ) 6.9 Hz); 13C NMR (CDCl3,
100 MHz) δ 176.6, 130.0, 129.8, 71.9, 34.8, 31.9, 29.8, 29.7, 29.5,
29.4, 29.3(3), 29.2, 27.20, 27.16, 24.9, 22.7, 14.1; IR (film) νmax 3383,
3290, 2917, 2849, 1644, 1467, 1426, 1331, 1075 cm-1; FABHRMS
(NBA-NaI) m/z 334.2731 (C19H37NO2 + Na+ requires 334.2722).
8(Z)-Heptadecenoic acid (27). A solution of 5 (66 mg, 0.26 mmol,
1 equiv) and 2-methyl-2-butene (1.6 mL, 15.1 mmol, 58 eq) in tBuOH
(6.5 mL) at 25 °C under N2 was treated dropwise with a solution of
NaClO2 (80%, 208 mg, 2.3 mmol, 9 equiv) and NaH2PO4‚H2O (250
mg, 1.8 mmol, 7 equiv) in deionized H2O (2.5 mL). The reaction
mixture was allowed to stir for an additional 15 min before being
concentrated in vacuo. The residue was treated with water (30 mL),
and the aqueous layer was extracted with EtOAc (3 × 30 mL). The
organic layers were dried (Na2SO4), filtered, and concentrated in vacuo.
Chromatography (SiO2, 2 × 13 cm, 10-20% EtOAc-hexane gradient
elution) afforded 27 (66 mg, 95%) as a clear oil. Spectral properties
agree with those described in the literature.49,50
tert-Butyl 3-Oxo-2-(triphenylphosphoranylidene)octadecanoate
(28). A solution of palmitic acid (103 mg, 0.40 mmol, 1 equiv) in
anhydrous CH2Cl2 (2 mL) under N2 was cooled to 0 °C and treated
with oxalyl chloride (2 M in CH2Cl2, 0.6 mL, 1.2 mmol, 3 equiv). The
solution was allowed to stir at 25 °C for 3 h before the solvent was
removed in vacuo. A solution of tert-butyl (triphenylphosphoranylide-
ne)acetate45 (29; 167 mg, 0.44 mmol, 1.1 equiv) and bis(trimethylsilyl)-
acetamide (195 µL, 0.79 mmol, 2 equiv) in anhydrous benzene (3 mL)
at 5 °C was treated dropwise with a solution of the crude acid chloride
in benzene (3 mL). The reaction mixture was allowed to warm to 25
°C and was stirred for 1.5 h before the solvent was removed in vacuo.
Chromatography (SiO2, 2 × 15 cm, 10 -20% EtOAc-hexane gradient
elution) afforded 28 (193 mg, 78%) as a clear oil: 1H NMR (CDCl3,
400 MHz) δ 7.67-7.61(m, 6H), 7.49-7.37 (m, 9H), 2.82 (t, 2H, J )
7.6 Hz), 1.55 (p, 2H, J ) 7.0 Hz), 1.23-1.21 (m, 24H), 1.04 (s, 9H),
0.86 (t, 3H, J ) 6.8 Hz); 13C NMR (CDCl3, 100 MHz) δ 197.9 (d, J
) 6 Hz), 167.3 (d, J ) 13 Hz), 132.9 (d, 6C, J ) 9 Hz), 131.3 (3),
128.4 (d, 6C, J ) 12 Hz), 127.4 (d, 3C, J ) 96 Hz), 78.4, 71.2 (d, J
) 114 Hz), 40.0, 31.9, 29.70(8), 29.66, 29.3, 28.1(3), 25.9, 22.7, 14.1;
IR (film) νmax 3426, 2923, 2852, 1665, 1551, 1438, 1363, 1302, 1173,
1106, 1081, 746, 690 cm-1; FABHRMS (NBA-CsI) m/z 615.3959
(C40H55O3P + H+ requires 615.3967).
2-Hydroxy-10(Z)-nonadecenoic Acid (25). A fresh solution of
LDA was prepared at -55 °C under Ar from diisopropylamine (0.4
mL, 2.9 mmol, 4.5 equiv), and n-BuLi (2.3M, 1.1 mL, 2.5 mmol, 4
equiv) in anhydrous THF (2 mL). A solution of 10(Z)-nonadecenoic
acid (24; 188 mg, 0.63 mmol, 1 equiv) and anhydrous HMPA (0.11
mL, 0.63 mmol, 1 equiv) in THF (0.5 mL) was added dropwise to the
LDA solution at -55 °C. The reaction mixture was allowed to warm
gradually to 25 °C and then was warmed to 50 °C for 30 min. After
the reaction mixture was recooled to 25 °C, O2 was bubbled through
the solution for 20 min. The mixture was treated with 1 N aqueous
HCl (30 mL), and the aqueous layer was extracted with EtOAc (3 ×
30 mL). The organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo. Chromatography (SiO2, 2 × 13 cm, 50-100%
EtOAc-hexane gradient elution) afforded 25 (96 mg, 49%) as a white
Determination of Binding Constants. The potency of the these
compounds against oleamide hydrolysis was evaluated using an ion-
selective ammonia electrode (ATI/Orion) to directly measure ammonia
formation as the product of the reaction. All Ki values except for that
of oleic acid were determined by the Dixon method. (x intercepts of
weighted linear fits of [I] vs 1/rate plots at a constant substrate
concentration were converted to Ki values using the formula Ki ) -Xint
/ [1 + [S]/Km].) The oleic acid Ki was obtained from a nonlinear
weighted least-squares fit of rate vs substrate and inhibitor concentra-
tions. The assays were done with constant stirring in 10 mL of 50
mM CAPS buffer (Sigma) adjusted to pH 10.0, the pH at which the
rate of enzymatically catalyzed oleamide hydrolysis is maximal. In
all cases which involved Dixon analysis, the substrate concentration
1
solid: mp 53-54 °C; H NMR (CDCl3, 400 MHz) δ 5.36-5.28 (m,
2H), 4.24 (dd, 1H, J ) 7.5 Hz, 7.6 Hz), 1.98 (m, 4H), 1.83 (m, 1H),
(48) Doleshall, G. Tetrahedron Lett. 1980, 21, 4183-4186.
(49) Miralle`s, J.; Barnathan, G.; Galonnier, R.; Sall, T.; Samb, A.;
Gaydou, E. M.; Kornprobst, J.-M. Lipids 1995, 30, 459-466.
(50) Couderc, F. Lipids 1995, 30, 691-699.

Hydrolysis of the Endogenous Sleep-Inducing Lipid
J. Am. Chem. Soc., Vol. 118, No. 25, 1996 5945
was 100 µM. The oleic acid Ki was determined over a range of
substrate concentrations from 10 to 100 µM. Substrate and inhibitors
were dissolved in DMSO prior to addition to the 50 mM CAPS buffer,
generating a final DMSO assay concentration of 1.67%. Concentrations
of up to 20% DMSO exhibited only minor effects upon the rate. The
enzyme concentration was adjusted to produce a rate of roughly 0.2
µM/min in the absence of inhibitor and the rate of ammonia production
observed over a period of 7-10 min.
pH and fit with the equation51 below by a weighted nonlinear least-
squares method.52
[H+]3νH E + [H+]2K1νH E + [H+]K1K2νHE + K1K2K3νE
3
2
rate )
[H+]3 + [H+]2K1 + [H+]K1K2 + K1K2K3
K1
K2
K3
H3E y\z H2E + H y\z HE + 2H y\z E + 3H
The enzyme was used as a crude, heterogenous, membrane-
containing preparation from rat liver. Enzyme boiled for 5 min
demonstrated no activity. Within solubility limits, all inhibitors
achieved 100% inhibition of activity at concentrations greater than
100Ki. No detectable activity was found in the absence of added
oleamide. Likewise, only a very minimal rate of ammonia production
from 100 µM oleamide was detected in the absence of enzyme at this
pH. This suggests that the catalytic oleamide hydrolysis activity
observed in this crude enzyme preparation arises from a single protein.
In cases where two pKa values are close together (less than one unit)
there will be substantial mixing of various species of enzyme present
in solution. Under such conditions, manifestation of the theoretical
rate maximum for that species may never actually be observed because
the most active species of enzyme may never reach a high degree of
abundance. It is for this reason that simple graphical methods of
determination of pKa values may disagree with the values of 9.7 and
10.3 pH units presented here.
Liver Plasma Membrane Prep, Large Scale (12-14 Rat Livers).
Twelve to fourteen rat livers were sectioned and placed in 300 mL of
1 mM NaHCO3. The solution of diced liver was strained and washed
with additional 300 mL of 1 mM NaHCO3. Any conspicuous
connective tissue was removed. The liver was transferred to a fresh
800 mL of 1 mM NaHCO3, stirred, and then transferred in 400 mL
aliquots to a blender. Blended liver aliquots were combined and filtered
through eight layers of cheesecloth. This was diluted to 1.0 L with 1
mM NaHCO3 and centrifuged at 6000 rpm for 20 min at 4 °C (Beckman
JA-17 rotor). The supernatant was decanted and the pellets resuspended
in 1 mM NaHCO3, combined, and dounce homogenized. Centrifuga-
tion, decantation, and resuspension/homogenization were repeated to
give a final volume of approximately 90 mL. The homogenate was
added to 2 volume equivalents of 67% sucrose, mixed thoroughly, and
transferred to ultracentrifuge compatible tubes. The tubes were topped
with 30% sucrose and spun at 27 000 rpm for 2 h (SW-28 rotor). The
middle yellow band was removed from the sucrose gradient, combined,
resuspended in 1 mM NaHCO3, and dounce homogenized. The sample
was further centrifuged at 17 000 rpm for 45 min at 4 °C (JA-17 rotor).
The supernatant was removed and the pellets resuspended in 100 mM
Na2CO3, dounce homogenized, and left on ice for 30 min. The solution
was centrifuged at 27 000 rpm for 1 h (SW-28 rotor), the supernatant
was decanted, and the pellet was resuspended in 15 mL of 50 mM
Tris-HCl, pH 7.4, with 1 mM EDTA, and homegenized with a dounce
homogenizer. This material was divided into multiple aliquots and
frozen at -78 °C until use. Each enzyme sample was only frozen
once.
The Km for oleamide was determined as the average Km obtained
from four independent assays. Each independent Km was obtained from
a weighted linear fit of data in a Lineweaver-Burke plot. A fifth
concurring Km was obtained as a result of the determination of oleic
acid inhibition by nonlinear methods. The rate data were fit with the
standard Michaelis-Menten kinetic equation. (The equation for the
rate of Ping Pong Bi Bi kinetics collapses to the simple Michaelis-
Menten-like equation when the concentration of the second substrate,
in this case water, is constant.) In the range 30-100 µM, the reaction
rate has essentially a zero-order dependence on substrate concentration.
Because we have not yet been able to determine the amount of
oleamide hydrolase present in the enzyme sample, we do not present
values for Vmax here. Our inhibition data suggest that the enzyme
concentration is lower than 2 nM, since higher enzyme concentrations
would have caused significant depletion of inhibitor in solution, causing
the apparent Ki to be measured as [E]/2 in the limiting case of [E] .
Ki. Since 1 nM was the lowest inhibition constant measured, [E] < 2
nM.
Error values presented with Ki values should be considered goodness-
of-fit estimates derived from propagation of errors treatment of data.
They are not necessarily an indication of reproducibility. However,
in cases where experiments were repeated, results were within statistical
agreement as predicted by the apparent error values given here.
pH-Rate Dependence. Crude enzyme was added to a solution of
200 µM oleamide (approximately the solubility limit) 1 in 20 mL of
buffer at the appropriate pH, containing 5% DMSO. (Concentrations
of up to 20% DMSO had only a minimal effect on enzyme rates.) A
50 mM sodium citrate/Bis-Tris buffer was used for data points in the
pH 4-9 range. A 50 mM Bis-Tris/CAPS buffer was used for data
points in the 8-11 range. At pH 12, the solution was assumed to be
self-buffering. At periodic time intervals, 1 mL aliquots were removed
and diluted with 9 mL of pH 14 buffer. Ammonia concentrations were
measured with an ion-selective ammonia electrode (Orion) connected
to a 720A meter (Orion), calibrated against known standards. The rate
was obtained from the linear portion of the curve which was fit using
a standard least-squares procedure. These rates were replotted against
Acknowledgment. I.R.O. thanks the Division of Medicinal
Chemistry, American Chemical Society, for a graduate fellow-
ship sponsored by Abbott Laboratories. B.F.C. thanks the
National Science Foundation for a graduate fellowship.
JA954064Z
(51) Fersht, A. Enzyme Structure and Mechanism; W. H. Freeman and
Co.: New York, 1985; pp 157.
(52) Connors, K. A. Binding Constants; Wiley: New York, 1987; pp
385-395.
(53) O’Leary, M. H.; Urberg, M.; Young, A. P. Biochemistry 1974, 13,
2077-2081.