Tetrahydrolipstatin analogues as modulators of endocannabinoid 2-arachidonoylglycerol metabolism

Article abstract of DOI:10.1021/jm800978m

A series of 21 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the formation or hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Three of the novel compounds, i.e., 11, 13, and 15, inhibited 2-AG formation via the diacylglycerol lipase α (DAGLα) with IC₅₀ values lower than 50 nM (IC₅₀ of THL = 1 μM) and were between 23- and 375-fold selective vs 2-AG hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB₁ and CB₂ receptors and anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16, and 18, were slightly more potent than THL against DAGLα and appreciably selective vs MAGL, CB receptors, and FAAH (15-26-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity (IC₅₀ = 0.41 μM), and relatively (～7-fold) selective vs the other targets tested.

Full text of DOI:10.1021/jm800978m

6970
J. Med. Chem. 2008, 51, 6970–6979
Tetrahydrolipstatin Analogues as Modulators of Endocannabinoid 2-Arachidonoylglycerol
Metabolism
Giorgio Ortar,*,† Tiziana Bisogno,‡ Alessia Ligresti,‡ Enrico Morera,† Marianna Nalli,† and Vincenzo Di Marzo*,‡
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza UniVersita` di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy, Endocannabinoid
Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34,
80078 Pozzuoli (Napoli), Italy
ReceiVed August 1, 2008
A series of 21 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the
formation or hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Three of the novel
compounds, i.e., 11, 13, and 15, inhibited 2-AG formation via the diacylglycerol lipase R (DAGLR) with
IC50 values lower than 50 nM (IC50 of THL ) 1 µM) and were between 23- and 375-fold selective vs 2-AG
hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB1 and CB2 receptors and
anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16,
and 18, were slightly more potent than THL against DAGLR and appreciably selective vs MAGL, CB
receptors, and FAAH (15-26-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity
(IC50 ) 0.41 µM), and relatively (∼7-fold) selective vs the other targets tested.
Introduction
Of the two most studied endogenous agonists of CB1 and
a
CB2 cannabinoid receptors (endocannabinoids), anandamide
(AEA),1 and 2-arachidonoylglycerol (2-AG),2 this latter exhibits
the highest abundance in tissues3 and selectivity for CB
receptors.4 As a consequence, pharmacological manipulation of
the metabolism of 2-AG would be expected to have a major
impact on cannabinergic signaling. As with other monoglycer-
ides, 2-AG takes part in several pathways of lipid metabolism
and many biochemical routes may lead to and/or involve it. In
most of the cases when 2-AG exerts its role as a signaling
molecule, 2-arachidonate-containing diacylglycerols (DAGs)
seem to act as direct precursors for 2-AG biosynthesis (Figure
1).5 DAGs are in turn produced, depending on the type of
stimulus, from the hydrolysis of either phosphoinositides (PI),
catalyzed by a PI-selective phospholipase C, or arachidonate-
containing phosphatidic acid (PA), catalyzed by a PA phos-
phohydrolase. DAGs are eventually converted into 2-AG by
the action of two sn-1 selective DAG lipases R and ꢀ (DAGLs).6
Figure 1. Main biosynthetic pathways for 2-AG. Abbreviations: R,
acyl chain of a fatty acid; In, inositol.
2-AG is inactivated by cellular reuptake and subsequent
intracellular enzymatic hydrolytic degradation. Although fatty
Inhibitors of DAGLs might produce a blockade of 2-AG
amide hydrolase (FAAH), the enzyme responsible of the
signaling with pharmacological consequences parallel (but not
hydrolysis of AEA, catalyzes also 2-AG hydrolysis, a monoa-
identical) to those of cannabinoid receptor antagonists, while
cylglycerol lipase (MAGL) is suggested to play a more
inhibitors of MAGL could offer a rational therapeutic approach
important role in this respect.5 Additional metabolic pathways
to a variety of diseases in which elevation of endocannabinoid
that may be important both in terminating the effects of 2-AG
levels represents an adaptive reaction to re-establish normal
and in the production of biologically active metabolites involve
homeostasis when this is pathologically perturbed.7
oxidative mechanisms catalyzed by lipoxygenases, cyclooxy-
genases, and P-450 monooxygenases.5
Several compounds have been screened as possible MAGL
inhibitors, but they either lack selectivity over FAAH as in the
case of R-methyl-1-arachidonoylglycerol, O-2203, and O-22048
* To whom correspondence should be addressed. For G.O.: phone, +39-
or, even when selective vs FAAH, as in the case of arachi-
donoylmaleimide and URB602,9 they have not been tested on
DAGLs. On the other hand, only recently, two potent and
selective inhibitors of the 2-AG biosynthesis, fluorophosphonates
O-3841 and O-3640, have been described.10 They showed an
excellent selectivity for DAGLR over a series of other proteins
of the endocannabinoid system, but were less potent than
tetrahydrolipstatin (THL). This is a mammalian lipase inhibitor
derived from lipstatin, a lipid produced by Streptomyces
6-49913612; fax, +39-6-491491; E-mail, giorgio.ortar@uniroma1.it. For
V.D.: phone, +39-081-8675093; fax, +39-081-8041770; E-mail,
vdimarzo@icmib.na.cnr.it.
†
Sapienza Universita` di Roma.
Institute of Biomolecular Chemistry.
‡
a Abbrevations: THL, tetrahydrolipstatin; 2-AG, 2-arachidonoylglycerol;
AEA, anandamide; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor
2; DAGLR, diacylglycerol lipase R; MAGL, monoacylglycerol lipase;
FAAH, fatty acid amide hydrolase; DDC, N,N′-dicyclohexylcarbodiimide;
DMAP, 4-dimethylaminopyridine; COS-7, transformed African green
monkey kidney fibroblast cells; HEK293, human embryonic kidney cells.
10.1021/jm800978m CCC: $40.75
2008 American Chemical Society
Published on Web 10/03/2008

Tetrahydrolipstatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6971
toxytricini.11 Irreversible inhibition with THL as a consequence
of the formation of a stable covalent binding of the ꢀ-lactone
moiety of the molecule with a catalytic serine hydroxyl group
has been initially reported for pancreatic and gastric lipases,12a,d,e
while, in the case of pancreatic carboxylester lipase, the
inhibition was found to be reversible due to hydrolysis of the
enzyme-THL adduct.12c By virtue of these biological activities,
THL has been widely investigated since the early 1990s as an
antiobesity drug and is currently marketed under the trade name
of Xenical. In 2003, THL was found to potently inhibit also
DAGLR and ꢀ6 and has since been used, despite its nonspeci-
ficity, in most studies investigating the biological roles of 2-AG
and of DAGLs, in particular their involvement in depolarization-
induced suppression inhibition (DSI) or excitation (DSE), two
well-known forms of endocannabinoid-mediated short-term
synaptic plasticity.13 Finally, THL was recently found also to
inhibit one of the minor enzymes catalyzing 2-AG hydrolysis
in the brain, the R/ꢀ hydrolase 12 (ABDH12), although at
concentrations ∼100-fold higher than those required to inhibit
DAGLR.14 By contrast, controversial results have been very
recently reported as to whether THL exerts an antagonistic effect
on CB1 receptors.13a,15
group furnished the corresponding amines 5,16i,n,p,20 21, and
32-38, which were converted, with the exception of 5, into
their N-formyl derivatives 2,21 8,21 11, 12,21and 13,21 15, 17,
and 19 with formic acetic anhydride, while treatment of amines
36 and 37 with acetyl chloride afforded the corresponding
N-acetyl derivatives 14 and 16. Attempts to obtain the above
N-formyl and N-acetyl derivatives by direct coupling of 22 with
the corresponding N-formyl or N-acetyl amino acids resulted
in lower yields.16q
Biological Evaluation. Well established protocols were used
to assess the activity of the novel compounds on MAGL and
DAGLR. The membrane fraction of COS-7 cells overexpressing
DAGLR was used to assess the activity against the hydrolysis
of sn-1-[3H]oleoyl-2-arachidonoylglycerol to [3H]oleic acid and
2-AG. The formation of [3H]oleic acid, measured by scintillation
counting after thin layer chromatography separation of the
reaction products, was used as a measure of DAGL activity.
The cytosolic fraction of COS-7 cells, which contains MAGL
as opposed as the other 2-AG hydrolysing enzymes,14 was used
to measure the rate of 2-[3H]arachidonoylglycerol hydrolysis
to [3H]arachidonic acid and glycerol. The formation of the
tritiated fatty acid, measured by scintillation counting after thin
layer chromatography separation of the reaction products, was
used as a measure of MAGL activity. The inhibitory activity
of the most potent DAGL inhibitor was also evaluated on 2-AG
biosynthesis in intact neuronal cells in order to gain an idea of
its degree of permeation through the cell plasma membrane.
Finally, the specificity of the most active compounds against
either 2-AG formation or hydrolysis vs cannabinoid receptors
was evaluated by binding assays carried out with membranes
prepared from HEK-293 cells overexpressing either the human
recombinant CB1 or CB2 receptors, whereas specificity vs FAAH
was evaluated by using rat brain membranes and [14C]ananda-
mide as substrate.
In view of the considerable interest in the development of
selective and efficacious pharmacological tools for the study
of the functions of all the endocannabinoid enzymes cloned so
far and their targeting in various disorders, we have synthesized
a series of 21 analogues of THL by varying the nature of the
aminoacidic moiety and of the N-substitution and have inves-
tigated the effects of these variations on the activity on MAGL
and DAGLR and their selectivity vs cannabinoid CB1 and CB2
receptors and FAAH.
Chemistry. The THL analogues in Table 1 were prepared
as detailed below and as summarized in Scheme 1. Because of
its biological properties, THL has been the subject of much
synthetic activity and several total syntheses have been re-
ported.16 However, such known processes require generally
more than 10 steps, utilize and/or form unstable intermediates,
and require sometimes complicated purification procedures of
stereoisomers. It was therefore envisioned that the key inter-
mediate for our analogues, the hydroxy lactone 22, could be
most conveniently obtained from the readily available and rather
inexpensive THL itself.17
Results and Discussion
The results of the pharmacological assays shown in Table 1,
and the salient aspects of SARs can be summarized as follows.
Three of the compounds examined, i.e., 11, 13, and 15, inhibited
2-AG formation via the diacylglycerol lipase R (DAGLR) with
IC50 values lower than 50 nM. Under the same assay conditions,
the IC50 of THL was 1 µM, i.e., ∼17-fold higher than previously
published.6 This might be due to the stronger degree of
expression of recombinant DAGLR observed in the transfected
cells used for these experiments with respect to those used for
previous experiments (data not shown). In fact, DAGLR activity
increases with the amounts of protein6 and, when using the same
amount of substrate, higher concentrations of inhibitors might
be required to exert a 50% inhibition in the presence of higher
amounts of enzyme. The three compounds were between 23-
and 375-fold selective vs 2-AG hydrolysis by MAGL as well
as vs cannabinoid CB1 and CB2 receptors and AEA hydrolysis
by FAAH in rat brain membranes. Three other THL analogues,
i.e., 14, 16, and 18, were slightly more potent than THL against
DAGLR and appreciably selective vs MAGL, CB receptors, and
FAAH (15-26-fold). Three other compounds, i.e. 9, 12, and
17, were about as potent as THL against DAGLR but were not
selective vs the other targets tested. Finally, one compound, i.e.,
8, was a potent inhibitor of MAGL-like activity (IC50 ) 0.41
µM) and relatively (∼7-fold) selective vs the other targets tested.
The amino acidic side chain is essentially for DAGLR inhibitory
activity, as demonstrated by the lack of activity of the hydroxy
lactone 22. The N-formyl group is also important for activity.
Its removal or replacement with the more bulky Boc or Cbz
Saponification of THL (1) with KOH in MeOH, followed
by the immediate treatment of the resulting dihydroxy acid
potassium salt with benzyl bromide, afforded benzyl ester 23.
Selective protection of the more accessible C-5 hydroxy group
was carried out with triisopropylsilyl trifluoromethanesulfonate
(TIPSOTf) and sym-collidine.16j Hydrogenolysis of the benzyl
group of 24 and lactonization of the acid 25 with N-phenyl-
bis(trifluoromethanesulfonimide) (PhNTf2) furnished the ꢀ-lac-
tone 26. It is to be noted that our attempts to achieve ꢀ-lactone
ring formation under Adam’s conditions (PhSO2Cl/pyridine)
used in the majority of approaches to the total synthesis of THL
resulted in a rather poor yield (22%) of 26. The removal of the
TIPS protecting group with n-Bu4N+F- and AcOH in THF
provided the hydroxy lactone 22, whose physical and spectral
data were in good agreement with those reported.16i,p,q,r Esteri-
fication of 22 with N-Boc-L-leucine, 4-methylpentanoic acid,
N-acetylglycine, and pyroglutamic acid using DCC and DMAP
afforded compounds 3,18 7, 9, and 18,19 respectively. Similar
coupling of 22 with N-Cbz-protected L-leucine, D-leucine,
glycine, L-phenylalanine, L-alanine, ꢀ-alanine, L-valine, L-
isoleucine, and L-proline gave compounds 4,16h,i,n,q,20 6, 10, 20,
27-31, respectively. Hydrogenolysis of the Cbz protecting

6972 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Ortar et al.
Table 1. Structures and Results of DAGL, MAGL, CB1, CB2, and FAAH Assays of THL (1) and THL Analogues (2-22)a

Tetrahydrolipstatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6973
Table 1. Continued
a Data are means of n ) 4 separate determinations. Standard errors are not shown for the sake of clarity and were never higher than 10% of the means.
Abbreviations: n.t., not tested.
groups leads to inactivation (compare compounds 1, 3, 4, 5,
and 8 with 10). However, replacement with a COCH3 group
was better tolerated. Thus, even though a 13- and a 27-fold
loss of potency was observed with the N-acetyl derivatives 14
and 16 as compared to their N-formyl counterparts 13 and 15,
compounds 14 and 16 are still more potent and selective
DAGLR inhibitors than THL. Interestingly, substitution of CHO
with COCH3 in compound 8 (compound 9) resulted in a 3.5-
fold increase in the activity on DAGLR and, conversely, in a
∼4-fold decrease of the inhibitory potency on MAGL. For
optimal activity on DAGLR, the presence of aliphatic amino
acidic residues, in particular with C-ꢀ branched chains, appears
to be necessary (compare compounds 1, 8, 11-13, and 15).
Especially noteworthy in this respect is the ∼60-fold difference
in the inhibitory potency between THL and compound 15 due
to the different position of a methyl group. On increasing the
distance between N-formylamino and ester groups, activity is
increased ∼5-fold (compare compounds 8 and 12). Rigidifica-
tion of the aliphatic side chain (compare compounds 11 and
17) is acceptable without dramatic loss of activity. The
stereochemistry at C-R has a notable effect on the activity;
indeed, THL turns out to be 5-fold more active on DAGLR
than its epimer 2. All compounds exhibiting activity on DAGLR
show weak or no affinity for cannabinoid receptors. Finally,
none of our compounds, with the exception of 7, 8, 16, and 17,
inhibits significantly FAAH up to concentrations of 50 µM.
The possibility that the newly developed compounds de-
scribed here, like THL,22 poorly penetrate into the systemic
circulation when orally administered, thus limiting their use as
pharmacological tools, deserves some discussion. Previously,
THL has been used to inhibit DAGLR in mostly in vitro
preparations,6,10,13 and its effects on the activity of the enzyme
and 2-AG tissue levels, after systemic administration, and in
particular per os, have not yet been investigated. Intriguingly,
however, THL was previously found to permeate the plasma
membrane as it was capable of inhibiting intracellular DAGLR
activity and, subsequently, the calcium-induced de novo bio-
synthesis of 2-AG, in intact mouse N18TG2 neuroblastoma
cells.10 We found here that, in similar experiments, compound
15, at the concentration of 0.3 µM, behaved in exactly the same
way by inhibiting the ionomycin-induced stimulation of 2-AG
biosynthesis in intact N18TG2 cells. The compound exerted a
57.3 ( 5.8% inhibition of 2-AG levels, which was comparable
to the 79.8 ( 6.9% inhibition observed with THL (1 µM) (means
( SEM, N ) 3). By contrast, compound 8 (1.5 µM) enhanced
2-AG levels by 55.0 ( 4.9%, in agreement with its actions as
rather selective MAGL inhibitor. Although these experiments
suggest that these compounds are plasma membrane permeable,
further specific studies are now required to investigate their
efficacy in vivo after systemic (and particularly oral) administra-
tion and their penetration into the systemic circulation.
Conclusions
A series of THL analogues was prepared and evaluated for
their inhibitory potency on metabolic enzymes of 2-AG and
their affinity for cannabinoid receptors. The influence of the
nature of the aminoacidic moiety and of the N-substitution on
the activity have been explored and led to the identification of
three nanomolar DAGLR inhibitors that substantially exceeded
the potency and selectivity of THL (compounds 11, 13, and
15) and of a potent and relatively selective inhibitor of 2-AG
hydrolysis by MAGL (compound 8). These compounds, and

6974 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Ortar et al.
Scheme 1a
a Reagents and conditions: (a) KOH, MeOH, room temp, 1 h; (b) PhCH2Br, DMF, room temp, 16 h; (c) TIPSOTf, sym-collidine, CH2Cl2, -78 °C, 3 h;
(d) H2, Pd/C, AcOEt, room temp, 2.5 h; (e) PhNTf2, Et3N, CH2Cl2, room temp, 16 h; (f) n-Bu4NF, THF, AcOH, -20 °C, 16 h; (g) R1R2CHCO2H, DCC,
CH2Cl2, 0 °C, 15 min, then 20, DMAP, DMF, room temp, 16 h; (h) H2, Pd/C, THF, room temp, 2.5 h; (i) AcOCHO, Et3N, CH2Cl2, room temp, 15 min; (j)
AcCl, Et3N, CH2Cl2, room temp, 1 h.
Compounds 2,21 3,18 4,16h,i,n,p,20 5,16i,n,p,20 8,21 12,21a 13,21b 18,19
particularly 8, 13, and 15, qualify as useful pharmacological
and 2216i,p,q,r have been described in the literature.
tools for the selective modulation of the metabolism of 2-AG
(2S,3S,5S)-3,5-Dihydroxy-2-hexylhexadecanoic Acid Phenyl-
methyl Ester (23). A solution of THL (1, 6.49 g, 13.1 mmol) in
MeOH (175 mL) was treated with aqueous 1N KOH (28.9 mL,
28.9 mmol) and kept at room temperature for 1 h. The reaction
mixture was evaporated under vacuum, and the residue redissolved
in toluene and evaporated under vacuum three times. The residue
(6.07 g) was dissolved in dry DMF (111 mL) and treated with
benzyl bromide (3.43 mL, 28.9 mmol). After 24 h, the reaction
mixture was diluted with water and extracted with AcOEt. The
organic phase was washed three times with water, dried (Na2SO4),
and evaporated under vacuum. The residue (9.02 g) was chromato-
graphed on silica gel with hexane/AcOEt ) 85/15 as eluent to give
3.03 g (50%) of 23 as an oil; [R]D -1°. IR 3485, 3007, 2928, 2856,
with respect to that of AEA and, given the pharmaceutical
success of THL, might serve as templates for the development
of new therapeutic drugs. On the other hand, their pharmaco-
kinetics still need to be assessed in order to gain a complete
picture of their potential usefulness in the clinic.
Experimental Section
Chemistry. All chemical reagents were commercially available
unless otherwise indicated. Melting points were determined on a
Kofler hot-stage apparatus and are uncorrected. Optical rotations
were measured at 20 °C with a Schmidt-Haensch Polartronic D
polarimeter (1 dm cell) in 1% CHCl3 solutions unless otherwise
indicated. IR spectra were recorded on a Perkin-Elmer 1000 FT-
IR spectrophotometer as CHCl3 solutions unless otherwise indicated.
1H and 13C NMR spectra were obtained on a Bruker Avance 400
or a Varian Gemini 200 spectrometer using CDCl3 as solvent unless
otherwise indicated and with TMS as internal standard. Satisfactory
elemental analyses were obtained for the newly synthesized
compounds (C, H, N (0.4%).
1718, 1456, 1165 cm-1 1H NMR (200 MHz) δ 0.87 (6H, m),
.
1.24-1.64 (32H, m), 2.49 (1H, dt, J ) 5.4, 9.2 Hz), 3.30 (1H, br
s), 3.64 (1H, m), 3.81 (1H, m), 3.97 (1H, m), 5.17 (2H, s), 7.36
(5H, m). 13C NMR (50 MHz) δ 14.02, 14.10, 22.53, 22.70, 25.35,
27.26, 29.01, 29.15, 29.37, 29.64, 29.69, 31.59, 31.94, 38.00, 40.97,
51.78, 66.41, 72.52, 73.48, 126.98, 127.61, 128.35, 128.58, 135.80,
175.08. Anal. (C29H50O4), C, H.

Tetrahydrolipstatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6975
(2S,3S,5S)-2-Hexyl-5-hydroxy-3-[(triisopropylsilyl)oxy]hexa-
decanoic Acid Phenylmethyl Ester (24). A stirred solution of 23
(5.00 g, 10.8 mmol) in dry CH2Cl2 (117 mL) was treated at -78
°C and under N2 with sym-collidine (3.55 mL, 27.0 mmol) and
triisopropylsilyl trifluoromethanesulfonate (4.37 mL, 16.2 mmol).
The mixture was stirred at a -78 °C for 3 h under N2 and then
diluted with water and extracted with CH2Cl2. The organic phase
was washed with 2 N HCl solution and brine until neutral, dried
(Na2SO4), and evaporated under vacuum. The residue (7.80 g) was
chromatographed on silica gel with hexane/AcOEt ) 95/5 as eluent
to give 5.71 g (85%) of 24 as an oil; [R]D +5°. IR 3494, 3025,
m), 2.19 (1H, m), 3.21 (1H, dt, J ) 7.6, 4.0 Hz), 4.24 (1H, m),
4.31 (1H, m), 4.84 (1H, d, J ) 8.4 Hz), 5.00 (1H, m). 13C NMR
(100 MHz) δ 14.02, 14.12, 21.77, 22.53, 22.69, 22.93, 24.86, 25.13,
26.67, 27.68, 28.32, 28.99, 29.35, 29.47, 29.56, 29.62, 29.64, 31.50,
31.92, 34.16, 38.76, 41.54, 52.44, 57.00, 71.94, 74.50, 79.87,
155.41, 170.93, 172.95.
4-Methylpentanoic Acid (1S)-1-[[(2S,3S-3-Hexyl-4-oxo-2-ox-
etanyl]methyl]dodecyl Ester (7). The title compound was prepared
from 4-methylpentanoic acid following the same procedure that
was used for the synthesis of 3 using hexane/AcOEt ) 95/5 as
eluent for the chromatographic purification; yield 68%; oil; [R]D
-17 °. IR 3034, 2958, 2928, 2856, 1818, 1726, 1467, 1181, 1124
1
2928, 2858, 1727, 1465, 1265, 1164, 883 cm-1. H NMR (200
1
cm-1. H NMR (400 MHz) δ 0.88 (6H, m), 0.91 (6H, d, J ) 6.3
MHz) δ 0.88 (6H, m), 1.07 (18H, m), 1.26-1.73 (35H, m), 2.49
(1H, dt, J ) 5.2, 9.4 Hz), 3.21 (1H, d, J ) 5.2 Hz), 3.89 (1H, m),
4.04 (1H, m), 5.14 (1H, d, J ) 12.2 Hz), 5.17 (1H, d, J ) 12.2
Hz), 7.34 (5H, m). 13C NMR (50 MHz) δ 12.91, 14.03, 14.10,
18.18, 18.23, 22.55, 22.70, 24.68, 27.48, 28.93, 29.19, 29.37, 29.63,
29.66, 29.92, 31.64, 31.95, 37.34, 41.33, 51.89, 66.17, 70.89, 72.02,
128.19, 128.27, 128.51, 136.04, 175.04. Anal. (C38H70O4Si), C, H.
(3S,4S)-3-Hexyl-4-[(2S)-2-[(triisopropylsilyl)oxy]tridecyl]oxetan-
2-one (26). A stirred solution of 24 (3.03 g, 4.84 mmol) in AcOEt
(25 mL) was hydrogenated in the presence of 10% Pd/C (518 mg)
at room temperature and atmospheric pressure for 2.5 h. The
suspension was filtered through a short pad of celite, and the filtrate
was evaporated under vacuum to give the hydroxy acid 25, which
was used in the next step without further purification. A stirred
solution of 25 (2.56 g, 4.84 mmol) and TEA (1.35 mL, 9.68 mmol)
in dry CH2Cl2 (43 mL), was treated with N-phenyl-bis(trifluo-
romethanesulfonimide) (2.59 g, 7.26 mmol) at 0 °C, and the mixture
was stirred at room temperature overnight. The reaction mixture
was evaporated under vacuum, and the residue (6.00 g) was
chromatographed on silica gel with hexane/CH2Cl2 ) 85/15 as
eluent to give 1.91 g (77%) of 26 as an oil; [R]D -19°. IR 3027,
Hz), 1.26-1.42 (27H, m), 1.49-1.81 (6H, m), 1.96 (1H, ddd, J )
3.6, 6.0, 12.4 Hz), 2.16 (1H, m), 2.30 (2H, t, J ) 7.6 Hz), 3.21
(1H, dt, J ) 7.6, 7.2 Hz), 4.32 (1H, m), 4.99 (1H, m). 13C NMR
(100 MHz) δ 14.02, 14.12, 22.21, 22.41, 22.53, 22.70, 25.26, 26.72,
27.68, 27.70, 29.00, 29.33, 29.36, 29.48, 29.54, 29.64, 31.51, 31.93,
32.59, 33.80, 38.82, 56.94, 70.75, 74.87, 171.10, 173.60. Anal.
(C28H52O4), C, H.
N-Acetyl-glycine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (9). The title compound was prepared
from N-Acetyl-Gly-OH following the same procedure that was used
for the synthesis of 3 using hexane/AcOEt ) 75/25 as eluent for
the chromatographic purification; yield 39%; mp 55-56°; [R]D
-23°. IR 3438, 3036, 2957, 2929, 2856, 1820, 1740, 1676, 1517,
1
1466, 1382, 1223, 1197 cm-1. H NMR (400 MHz) δ 0.87 (6H,
m), 1.26-1.43 (26H, m), 1.59-1.80 (4H, m), 1.99 (1H, m), 2.04
(3H, s), 2.13 (1H, m), 3.21 (1H, m), 3.97 (1H, dd, J ) 4.9, 18.0
Hz), 4.03 (1H, dd, J ) 5.3, 15.4 Hz), 4.33 (1H, m), 5.09 (1H, m),
6.19 (1H, br s). 13C NMR (100 MHz) δ 14.02, 14.12, 22.53, 22.70,
22.89, 25.20, 26.76, 27.65, 28.97, 29.32, 29.36, 29.45, 29.56, 29.63,
31.49, 31.93, 34.11, 38.87, 41.66, 57.07, 72.74, 75.06, 169.83,
170.28, 170.90. Anal. (C26H47NO5), C, H, N.
1
2928, 2858, 1814, 1465, 1379, 1175, 882 cm-1. H NMR (200
MHz) δ 0.88 (6H, m), 1.07 (18H, m), 1.20-2.01 (35H, m), 3.28
(1H, m), 3.95 (1H, m), 4.51 (1H, dt, J ) 4.6, 7.6 Hz). 13C NMR
(50 MHz) δ 14.01, 14.11, 18.17, 22.52, 22.70, 25.16, 26.73, 27.77,
29.04, 29.28, 29.36, 29.59, 29.65, 29.77, 31.52, 31.94, 36.30, 40.69,
56.85, 69.65, 74.72, 171.84. Anal. (C31H62O3Si), C, H.
5-oxo-L-Proline-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (18).19 The title compound was prepared from
L-pyroglutamic acid following the same procedure that was used
for the synthesis of 3 using hexane/AcOEt ) 6/4 as eluent for the
chromatographic purification; yield 42%; mp 41-43 °C; [R]D -19°.
IR 3437, 3034, 2960, 2929, 2856, 1821, 1737, 1706, 1460, 1413,
(3S,4S)-3-Hexyl-4-[(2S)-2-hydroxytridecyl]oxetan-2-one (22).16i,p,q,r
A stirred solution of 26 (678 mg, 1.3 mmol) in THF (7.2 mL) was
treated with tetrabutylammonium fluoride solution (1 M in THF)
(3.9 mL, 3.9 mmol) and acetic acid (0.076 mL, 1.3 mmol) at -20
°C. The mixture was stirred at -20 °C overnight and then diluted
with water and extracted with AcOEt. The organic phase was
washed with brine until neutral, dried (Na2SO4), and evaporated
under vacuum. The residue (960 mg) was chromatographed on silica
gel with hexane/AcOEt ) 8/2 as eluent to give 428 mg (91%) of
22;16i,p,q,r mp 62-65 °C; [R]D -15°. IR (KBr disks) 3553, 2956,
1392, 1236, 1124 cm-1 1H NMR (400 MHz) δ 0.88 (6H, m),
.
1.25-1.42 (26H, m), 1.59-1.80 (4H, m), 2.01 (1H, m), 2.12 (1H,
m), 2.29-2.47 (4H, m), 3.20 (1H, m), 4.23 (1H, m), 4.32 (1H, m),
5.13 (1H, m), 6.48 (1H, s). 13C NMR (100 MHz) δ 14.01, 14.11,
22.51, 22.69, 24.77, 25.22, 26.79, 27.60, 28.94, 29.21, 29.29, 29.34,
29.46, 29.55, 29.62, 31.48, 31.92, 34.36, 39.20, 55.61, 57.16, 73.07,
75.45, 170.68, 171.85, 177.99.
N-[(Phenylmethoxy)carbonyl]-L-leucine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (4).16h,i,n,p,20 The
title compound was prepared from Cbz-Leu-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 9/1 as eluent for the chromatographic purification; yield 81%;
mp 45-47 °C; [R]D -24°. IR (KBr disks) 3335, 2959, 2918, 2850,
1842, 1731, 1693, 1536, 1285, 1193 cm-1. 1H NMR (400 MHz) δ
0.88 (6H, m), 0.93 (6H, m), 1.27-1.44 (27H, m), 1.48-1.74 (6H,
m), 1.97 (1H, m), 2.16 (1H, m), 3.21 (1H, m), 4.29 (1H, m), 4.36
(1H, m), 5.01 (1H, m), 5.12 (3H, m), 7.34 (5H, m). 13C NMR (100
MHz) δ 14.03, 14.13, 21.70, 22.53, 22.69, 22.94, 24.83, 25.13,
26.69, 27.65, 28.99, 29.31, 29.35, 29.45, 29.55, 29.63, 31.49, 31.92,
34.06, 38.68, 41.60, 52.81, 57.00, 67.02, 72.34, 74.57, 128.08,
128.20, 128.55, 136.26, 155.97, 170.84, 172.55.
1
2924, 2850, 1813, 1470, 1136, 1091, 837 cm-1. H NMR (200
MHz) δ 0.89 (6H, m), 1.29-2.02 (33H, m), 3.32 (1H, ddd, J )
4.0, 6.8, 8.4 Hz), 3.76 (1H, m), 4.47 (1H, dt, J ) 4.1, 6.6 Hz). 13
C
NMR (50 MHz) δ 14.04, 14.13, 17.71, 22.55, 22.70, 25.48, 26.82,
27.84, 28.97, 29.36, 29.54, 29.59, 29.64, 29.66, 31.51, 31.93, 37.68,
41.18, 56.77, 69.27, 76.26, 171,41.
N-[(1,1-Dimethylethoxy)carbonyl]-L-leucine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (3).18 To a stirred
solution of Boc-Leu-OH (462 mg, 2.0 mmol) in dry CH2Cl2 (2
mL) was added DCC (413 mg, 2.0 mmol) at 0 °C, and the mixture
was stirred at 0 °C for 15 min. The solvent was evaporated under
vacuum, and the residue was dissolved in DMF (2 mL) and added
to a solution of 22 (355 mg, 1.0 mmol) and DMAP (15 mg, 0.12
mmol) in DMF (1 mL). The mixture was stirred at room temperature
overnight and then diluted with water and extracted with AcOEt.
The organic phase was washed with brine until neutral, dried
(Na2SO4), and evaporated under vacuum. The residue (2.031 g)
was chromatographed on silica gel with hexane/AcOEt ) 9/1 as
eluent to give 503 mg of 318 (89%) as an oil; [R]D -27°. IR 3444,
N-[(Phenylmethoxy)carbonyl]-D-Leucine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (6). The title com-
pound was prepared from Cbz-D-Leu-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 8/2 as eluent for the chromatographic purification; yield 80%;
mp 30-32 °C; [R]D -6°. IR 3438, 3033, 2959, 2928, 2856, 1819,
1
1721, 1509, 1456, 1263 cm-1. H NMR (400 MHz) δ 0.88 (6H,
3029, 2959, 2929, 2856, 1819, 1711, 1500, 1368, 1162, 1124 cm-1
.
m), 0.95 (6H, d, J ) 6.4 Hz), 1.27-1.53 (27H, m), 1.54-1.62
(3H, m), 1.68-1.76 (3H, m), 2.01 (1H, m), 2.18 (1H, m), 3.22
(1H, m), 4.33 (2H, m), 5.00 (1H, m), 5.11 (3H, m), 7.33 (5H, m).
1H NMR (400 MHz) δ 0.88 (6H, m), 0.96 (6H, d, J ) 6.5 Hz),
1.25-1.31 (27H, m), 1.44 (9H, s), 1.55-1.8 (6H, m), 1.96 (1H,

6976 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Ortar et al.
13C NMR (100 MHz) δ 14.03, 14.12, 21.85, 22.55, 22.69, 22.86,
24.83, 25.22, 26.68, 27.66, 29.02, 29.28, 29.35, 29.47, 29.53, 29.64,
31.50, 31.92, 33.86, 38.54, 41.49, 52.82, 56.99, 67.01, 72.19, 74.44,
128.07, 128.21, 128.55, 136.24, 155.96, 171.09, 172.87. Anal.
(C36H59NO6), C, H, N.
(1H, d, J ) 9.4 Hz), 7.31 (5H, m). 13C NMR (100 MHz) δ 14.02,
14.13, 17.33, 19.28, 22.52, 22.70, 25.16, 26.69, 27.66, 28.97, 29.32,
29.36, 29.45, 29.55, 29.63, 30.95, 31.49, 31.92, 33.95, 38.58, 57.03,
59.32, 67.04, 72.44, 74.46, 128.11, 128.19, 128.55, 136.30, 156.30,
170.89, 171.53. Anal. (C35H57NO6), C, H, N.
N-[(Phenylmethoxy)carbonyl]-glycine-(1S)-1-[[(2S,3S)-3-hexyl-
4-oxo-2-oxetanyl]methyl]dodecyl Ester (10). The title compound
was prepared from Cbz-Gly-OH following the same procedure that
was used for the synthesis of 3 using hexane/AcOEt ) 9/1 as eluent
for the chromatographic purification; yield 65%; mp 32-35 °C;
[R]D -22°. IR 3448, 2957, 2928, 2856, 1820, 1723, 1517, 1466,
1223, 1197 cm-1. 1H NMR (400 MHz) δ 0.88 (6H, m), 1,28-1.42
(26H, m), 1.58-1.77 (4H, m), 1.99 (1H, m), 2.13 (1H, m), 3.19
(1H, m), 3.96 (2H, m), 4.32 (1H, m), 5.09 (1H, m), 5.13 (2H, s),
5.27 (1H, m), 7.32 (5H, m). 13C NMR (100 MHz) δ 14.02, 14.12,
22.52, 22.69, 25.19, 26.73, 27.61, 28.95, 29.30, 29.34, 29.44, 29.54,
29.61, 31.47, 31.91, 34.08, 38.83, 42.96, 57.03, 67.11, 72.67, 74.90,
128.10, 128.22, 128.55, 136.23, 156.25, 169.76, 170.92. Anal.
(C32H51NO6), C, H, N.
N-[(Phenylmethoxy)carbonyl]-L-isoleucine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (30). The title
compound was prepared from Cbz-Ile-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 9/1 as eluent for the chromatographic purification; yield 68%;
mp 42-44 °C; [R]D -16°; IR 3437, 3038, 2960, 2929, 2856, 1820,
1
1722, 1509, 1465, 1335, 1226, 1213, 1197 cm-1. H NMR (200
MHz) δ 0.85-0.97 (12H, m), 1.26 (28H, m), 1.61-1.78 (4H, m),
1.98 (2H, m), 2.15 (1H, m), 3.21 (1H, dt, J ) 7.6, 4.0 Hz), 4.29
(2H, m), 5.00 (1H, m), 5.11 (2H, s), 5.30 (1H, d, J ) 9.0 Hz), 7.32
(5H, m). 13C NMR (50 MHz) δ 11.56, 13.99, 14.09, 15.67, 22.52,
22.69, 24.90, 25.16, 26.71, 27.72, 28.97, 29.34, 29.45, 29.55, 29.63,
31.49, 31.92, 34.00, 37.71, 38.66, 57.13, 58.88, 67.06, 72.46, 74.47,
128.08, 128.19, 128.55, 136.36, 156.14, 170.67, 171.41. Anal.
(C36H59NO6), C, H, N.
N-[(Phenylmethoxy)carbonyl]-L-phenylalanine-(1S)-1-[[(2S,3S)-
3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (20). The title
compound was prepared from Cbz-Phe-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 9/1 as eluent for the chromatographic purification. Yield 80%;
mp 64-66 °C; [R]D -10°. IR (KBr disks) 3338, 2954, 2922, 2852,
N-[(Phenylmethoxy)carbonyl]-L-proline-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (31). The title
compound was prepared from Cbz-Pro-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 85/15 as eluent for the chromatographic purification; yield 96%;
oil; [R]D -48°. IR 2957, 2928, 2856, 1819, 1740, 1698, 1467, 1455,
1
1842, 1723, 1688, 1534, 1290, 1265, 1180 cm-1. H NMR (400
MHz) δ 0.88 (6H, m), 1.26-1.72 (30H, m), 1.85 (1H, m), 2.05
(1H, m), 3.10 (3H, m), 4.14 (1H, m), 4.60 (1H, m), 4.95 (1H, m),
5.08 (2H, s), 5.19 (1H, d, J ) 8.0 Hz), 7.15 (2H, d, J ) 6.8 Hz),
7.24-7.33 (8H, m). 13C NMR (100 MHz) δ 14.03, 14.12, 22.53,
22.69, 25.05, 26.67, 27.61, 28.98, 29.32, 29.36, 29.43, 29.56, 29.63,
31.48, 31.92, 33.92, 38.14, 38.54, 55.09, 56.98, 72.65, 74.40,
127.24, 128.10, 128.23, 128.55, 128.69, 129.27, 130.38, 135.78,
155.65, 170.05, 171.27. Anal. (C39H57NO6), C, H, N.
N-[(Phenylmethoxy)carbonyl]-L-alanine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (27). The title com-
pound was prepared from Cbz-Ala-OH following the same procedure
that was used for the synthesis of 3 using CH2Cl2/AcOEt ) 95/5 as
eluent for the chromatographic purification; yield 78%; mp 36-38
°C; [R]D -23°. IR 3436, 2957, 2929, 2856, 1820, 1720, 1508, 1455,
1337, 1240, 1197, 1062 cm-1. 1H NMR (400 MHz) δ 0.87 (6H, m),
1.25 (26H, m), 1.42 (3H, d, J ) 6.8 Hz), 1.58-1.71 (4H, m), 1.97
(1H, m), 2.13 (1H, m), 3.19 (1H, m), 4.29 (1H, m), 4.35 (1H, m),
5.03 (1H, m), 5.11 (2H, s), 5.30 (1H, m), 7.34 (5H, m). 13C NMR
(100 MHz) δ 14.00, 14.11, 18.49, 22.51, 22.69, 25.13, 26.70, 27.64,
28.95, 29.31, 29.35, 29.44, 29.55, 29.62, 31.48, 31.92, 34.19, 38.83,
49.88, 57.03, 66.93, 72.59, 74.78, 128.09, 128.19, 128.55, 136.30,
155.61, 170.81, 172.55. Anal. (C33H53NO6), C, H, N.
1
1420, 1356, 1229, 1123 cm-1. H NMR (400 MHz) δ 0.87 (6H,
m), 1.19-1.57 (26H, m), 1.67-1.75 (4H, m), 1.83-2.23 (6H, m),
3.13 and 3.18 (1H, 2m), 3.48-3.60 (2H, m), 4.19 and 4.36 (1H,
2m), 4.36 (1H, m), 4.95 and 5.02 (1H, 2m), 5.06 and 5.15 (2H,
2m), 7.30-7.36 (5H, m). 13C NMR (100 MHz) δ 14.02, 14.11,
22.53, 22.69, 24.38, 25.17, 26.66, 27.58, 27.64, 29.00, 29.35, 29.47,
29.56, 29.63, 29.88, 30.84, 31.49, 31.92, 34.19, 34.34, 38.86, 46.49,
47.01, 56.96, 58.97, 59.58, 66.97, 72.00, 74.84, 127.70, 127.79,
127.98, 128.44, 128.50, 136.59, 136.76, 154.85, 170.83, 171.18,
172.23, 172.29. Anal. (C35H55NO6), C, H, N.
L-Leucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (5).16i,n,p,20 A stirred solution of 4 (105 mg,
0.25 mmol) in THF (6 mL) was hydrogenated in the presence of
10% Pd/C (47 mg) at room temperature and atmospheric pressure
for 2.5 h. The suspension was filtered through a short pad of celite
and the filtrate was evaporated under vacuum. The residue was
chromatographed on silica gel with hexane/AcOEt ) 7/3 as eluent
to give 56 mg of 516i,n,p,20 (48%); oil; [R]D -16° (CHCl3, c )
0.5). IR 3503, 3433, 2958, 2928, 2856, 1820, 1728, 1602, 1467,
1126 cm-1. 1H NMR (400 MHz) δ 0.88 (6H, m), 0.95 (6H, t, J )
7.6 Hz), 1.26-1.78 (35H, m), 1.98 (1H, m), 2.17 (1H, m), 3.21
(1H, m), 3.46 (1H, m), 4.30 (1H, m), 5.02 (1H, m). 13C NMR (100
MHz) δ 14.02, 14.12, 21.74, 22.53, 22.70, 23.06, 24.82, 25.25,
26.72, 27.57, 27.69, 29.00, 29.35, 29.48, 29.55, 29.63, 31.50, 31.93,
34.14, 38.86, 44.01, 53.11, 57.01, 71.62, 74.81, 170.88, 175.34.
Anal. (C28H53NO4), C, H, N.
N-[(Phenylmethoxy)carbonyl]-ꢀ-alanine-(1S)-1-[[(2S,3S)-3-
hexyl-4-oxo-2-oxetanyl]methyl]dodecyl Ester (28). The title
compound was prepared from Cbz-ꢀ-Ala-OH following the same
procedure that was used for the synthesis of 3 using hexane/AcOEt
) 85/15 as eluent for the chromatographic purification; yield 81%;
oil; [R]D -22°. IR 3452, 3029, 2929, 2856, 1820, 1723, 1513, 1466,
1
1280, 1236, 1189, 1131 cm-1. H NMR (400 MHz) δ 0.88 (6H,
L-Phenylalanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester Hydrochloride (21). The title
compound was prepared from 20 following the same procedure
that was used for the synthesis of 5 using hexane/AcOEt ) 8/2 as
eluent for the chromatographic purification. The amine was
dissolved in anhydrous Et2O and was treated with gaseous HCl to
give a precipitate of the corresponding hydrochloride; yield 80%;
mp 87-91 °C; [R]D -18° (MeOH, c ) 1). IR (KBr disks) 3314,
3161, 2956, 2922, 2853, 2622, 1819, 1727, 1597, 1523, 1469, 1376,
m), 1.25-1.41 (26H, m), 1.57-1.77 (4H, m), 1.96 (1H, m), 2.10
(1H, m), 2.54 (2H, m), 3.18 (1H, m), 3.47 (2H, m), 4.30 (1H, m),
5.05 (1H, m), 5.09 (2H, s), 5.29 (1H, br s), 7.34 (5H, m). 13C NMR
(100 MHz) δ 14.01, 14.11, 22.51, 22.69, 25.26, 26.74, 27.62, 28.93,
29.34, 29.46, 29.55, 29.62, 31.47, 31.91, 34.22, 34.71, 36.63, 38.95,
57.04, 66.72, 71.70, 75.17, 128.07, 128.11, 128.51, 136.53, 156.30,
170.83, 171.97. Anal. (C33H53NO6), C, H, N.
N-[(Phenylmethoxy)carbonyl]-L-valine-(1S)-1-[[(2S,3S)-3-hexyl-
4-oxo-2-oxetanyl]methyl]dodecyl Ester (29). The title compound
was prepared from Cbz-Val-OH following the same procedure that
was used for the synthesis of 3 using hexane/AcOEt ) 9/1 as eluent
for the chromatographic purification; yield 65%; mp 58-61 °C; [R]D
-18°. IR 3437, 2959, 2929, 2856, 1820, 1723, 1509, 1466, 1346, 1236,
1125 cm-1. 1H NMR (400 MHz) δ 0.88 (9H, m), 0.99 (3H, d, J )
6.7 Hz), 1.26-1.42 (26H, m), 1.59-1.75 (4H, m), 1.97 (1H, m), 2.17
(2H, m), 3.20 (1H, m), 4.28 (2H, m), 5.00 (1H, m), 5.11 (2H, s), 5.32
1
1278, 1125 cm-1. H NMR (400 MHz, CD3OD) δ 0.96 (6H, m),
1.26-1.41 (26H, m), 1.67 (2H, m) 1.77 (2H, m), 2.08 (2H, m),
3.19 (1H, dd, J ) 7.9, 14.2 Hz), 3.34 (2H, m), 4.37 (2H, m), 5.15
(1H, m), 7.34-7.41 (5H, m). 13C NMR (100 MHz, CD3OD) δ
14.41, 14.46, 23.63, 23.74, 26.03, 27.83, 28.66, 30.14, 30.47, 30.57,
30.75, 32.75, 33.08, 34.99, 37.46, 39.48, 55.18, 57.92, 75.98, 76.63,
128.93, 130.18, 130.48, 135.70, 169.87, 172.91. Anal.
(C31H52ClNO4), C, H, N.

Tetrahydrolipstatin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6977
D-Leucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (32). Prepared as above from 6 and used in the
next step without further purification.
Glycine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dode-
cyl Ester (33). Prepared as above from 10 and used in the next
step without further purification.
L-Alanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (34). Prepared as above from 27 and used in
the next step without further purification.
ꢀ-Alanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (35). Prepared as above from 28 and used in
the next step without further purification.
L-Valine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (36). Prepared as above from 29 and used in
the next step without further purification.
L-Isoleucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (37). Prepared as above from 30 and used in the
next step without further purification.
L-Proline-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]meth-
yl]dodecyl Ester (38). Prepared as above from 31 and used in
the next step without further purification.
N-Formyl-D-leucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (2).21 A solution of crude 32 (117 mg,
0.25 mmol) was stirred with formic acetic anhydride (0.24 mL,
3.2 mmol) in dry CH2Cl2 (0.74 mL) at room temperature for 15
min and then diluted with water and extracted with AcOEt. The
organic phase was washed with saturated NaHCO3 and brine until
neutral, dried (Na2SO4), and evaporated under vacuum. The residue
(115 mg) was chromatographed on silica gel with hexane/AcOEt
) 7/3 as eluent to give 82 mg (66%) of 2;21 mp 40-43 °C; [R]D
-4°. IR (KBr disks) 3385, 2958, 2921, 2853, 1842, 1707, 1685,
1671, 1468, 1384, 1291, 1258 cm-1. 1H NMR (400 MHz) δ 0.89
(6H, m), 0.96 (6H, d, J ) 6.0), 1.54-1.78 (33H, m), 2.00 (1H, m),
2.18 (1H, m), 3.23 (1H, m), 4.37 (1H, m), 4.65 (1H, m), 5.03 (1H,
m), 6.19 (1H, d, J ) 8.0), 8.20 (1H, s). 13C NMR (100 MHz) δ
14.03, 14.12, 21.86, 22.53, 22.69, 22.82, 24.87, 25.21, 26.68, 27.64,
28.98, 29.26, 29.34, 29.46, 29.52, 29.63, 31.48, 31.91, 33.83, 38.53,
41.36, 49.69, 56.99, 72.50, 74.60, 160.77, 171.07, 172.27.
N-Formyl-glycine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (8).21 The title compound was prepared
from 33 following the same procedure that was used for the
synthesis of 2 using hexane/AcOEt ) 6/4 as eluent for the
chromatographic purification; yield 21%; oil; [R]D -24° (CHCl3,
c ) 0.5). IR 3428, 2957, 2928, 2856, 1820, 1742, 1690, 1508, 1467,
1379, 1197 cm-1. 1H NMR (400 MHz) δ 0.88 (6H, m), 1.26-1.43
(26H, m), 1.60-1.82 (4H, m), 2.02 (1H, m), 2.13 (1H, m), 3.21
(1H, m), 4.03 (1H, dd, J ) 5.5, 18.4 Hz), 4.13 (1H, dd, J ) 5.8,
18.2 Hz), 4.34 (1H, m), 5.12 (1H, m), 6.24 (1H, br s), 8.26 (1H,
s). 13C NMR (100 MHz) δ 14.00, 14.11, 22.50, 22.67, 25.16, 26.73,
27.60, 28.93, 29.28, 29.33, 29.41, 29.52, 29.60, 31.45, 31.89, 34.07,
38.87, 40.11, 57.04, 73.03, 75.08, 161.05, 169.24, 170.86.
N-Formyl-L-alanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (11). The title compound was prepared
from 34 following the same procedure that was used for the
synthesis of 2 using CH2Cl2/AcOEt ) 9/1 as eluent for the
chromatographic purification; yield 21%; mp 31-32 °C; [R]D -29°.
IR 3423, 2957, 2929, 2856, 1821, 1735, 1686, 1505, 1456, 1380,
1197, 1123 cm-1. 1H NMR (400 MHz) δ 0.88 (6H, m), 1.25-1.35
(26H, m), 1.45 (3H, d, J ) 7.2 Hz), 1.60-1.81 (4H, m), 2.00 (1H,
m), 2.14 (1H, m), 3.22 (1H, m), 4.30 (1H, m), 4.65 (1H, m), 5.07
(1H, m), 6.22 (1H, br s), 8.19 (1H, s). 13C NMR (100 MHz) δ
14.01, 14.11, 18.37, 22.52, 22.70, 25.13, 26.75, 27.65, 28.96, 29.31,
29.35, 29.44, 29.55, 29.63, 31.49, 31.93, 34.22, 38.93, 47.13, 57.12,
73.03, 74.98, 160.39, 170.72, 172.13. Anal. (C26H47NO5), C, H,
N.
cm-1. 1H NMR (400 MHz) δ 0.87 (6H, m), 1.26-1.42 (26H, m),
1.59-1.81 (4H, m), 2.00 (1H, m), 2.11 (1H, m), 2.56 (2H, m),
3.21 (1H, m), 3.58 (2H, m), 4.36 (1H, m), 5.10 (1H, m), 6.31 (1H,
br s), 8.15 (1H, s). 13C NMR (100 MHz) δ 14.02, 14.13, 22.52,
22.69, 25.24, 26.78, 27.60, 28.95, 29.32, 29.35, 29.47, 29.55, 29.63,
31.48, 31.92, 33.50, 34.33, 39.08, 57.09, 72.02, 75.68, 161.31,
170.82, 172.01.
N-Formyl-L-valine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (13).21b The title compound was prepared
from 36 following the same procedure that was used for the
synthesis of 2 using CH2Cl2/AcOEt ) 95/5 as eluent for the
chromatographic purification; yield 57%; mp 43-45 °C; [R]D -24°.
IR 3425, 2959, 2929, 2856, 1821, 1733, 1690, 1505, 1466, 1309,
1195, 1127 cm-1. 1H NMR (400 MHz) δ 0.87 (6H, m), 0.92 (3H,
d, J ) 6.8 Hz), 1.00 (3H, d, J ) 6.8 Hz), 1.25-1.40 (26H, m),
1.62-1.79 (4H, m), 2.01 (1H, dt, J ) 4.7, 15.1 Hz), 2.21 (2H, m),
3.22 (1H, m), 4.29 (1H, m), 4.63 (1H, dd, J ) 4.8, 8.8 Hz), 5.03
(1H, m), 6.18 (1H, d, J ) 8.8 Hz), 8.27 (1H, s). 13C NMR (100
MHz) δ 14.02, 14.13, 17.45, 19.28, 22.52, 22.70, 25.14, 26.73,
27.68, 28.96, 29.33, 29.35, 29.44, 29.55, 29.62, 31.04, 31.49, 31.92,
33.97, 38.65, 55.90, 57.12, 72.87, 74.66, 160.94, 170.75, 171.02.
N-Acetyl-L-valine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (14). To a stirred solution of 36 (162
mg, 0.36 mmol) and TEA (0.20 mL, 1.44 mmol) in dry CH2Cl2 (1
mL) was added acetyl chloride (0.086 mL, 1.21 mmol). The mixture
was stirred at room temperature for 1 h and then diluted with water
and extracted with AcOEt. The organic phase was washed with
brine, dried (Na2SO4) and evaporated under vacuum. The residue
(170 mg) was chromatographed on silica gel with hexane/AcOEt
) 75/25 as eluent to give 112 mg of 14 (68%); mp 62-63 °C;
[R]D -26°. IR 3435, 2959, 2929, 2856, 1821, 1731, 1676, 1509,
1
1466, 1373, 1309, 1124 cm-1. H NMR (400 MHz) δ 0.88-0.93
(9H, m), 0.97 (3H, d, J ) 6.8 Hz), 1.26-1.29 (26H, m), 1.41-1.79
(4H, m), 1.99 (1H, m), 2.05 (3H, s), 2.16 (2H, m), 3.23 (1H, m),
4.30 (1H, m), 4.53 (1H, dd, J ) 5.1, 8.5 Hz), 5.01 (1H, m), 6.10
(1H, d, J ) 8.6 Hz). 13C NMR (100 MHz) δ 14.01, 14.12, 17.70,
19.19, 22.51, 22.69, 23.19, 25.12, 26.71, 27.67, 28.95, 29.34, 29.44,
29.55, 29.61, 31.03, 31.48, 31.91, 33.98, 38.64, 57.10, 57.44, 72.62,
74.78, 170.11, 170.79, 171.49. Anal. (C29H53NO5), C, H, N.
N-Formyl-L-isoleucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (15). The title compound was prepared
from 37 following the same procedure that was used for the
synthesis of 2 using CH2Cl2/AcOEt ) 95/5 as eluent for the
chromatographic purification; yield 56%; mp 57-59 °C; [R]D)
-24°. IR 3425, 3024, 2960, 2929, 2857, 1821, 1732, 1689, 1502,
1
1465, 1197 cm-1. H NMR (400 MHz) δ 0.88-0.97 (12H, m),
1.26-1.29 (28H, m), 1.62-1.78 (4H, m), 1.94-2.03 (2H, m), 2.17
(1H, m), 3.23 (1H, m), 4.29 (1H, m), 4.65 (1H, m), 5.02 (1H, m),
6.29 (1H, d, J ) 8.5 Hz), 8.24 (1H, s). 13C NMR (100 MHz) δ
11.32, 11.53, 14.01, 14.12, 15.59, 22.53, 22.70, 24.94, 25.13, 26.74,
27.69, 28.97, 29.45, 29.55, 29.62, 31.49, 31.92, 33.95, 37.64, 38.64,
55.49, 57.14, 72.84, 74.67, 160.85, 170.74, 170.94. C29H53NO5, C,
H, N.
N-Acetyl-L-isoleucine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (16). The title compound was prepared
from 37 following the same procedure that was used for the
synthesis of 14 using hexane/AcOEt ) 75/25 as eluent for the
chromatographic purification; yield 60%; mp 89-91 °C; [R]D -22°.
IR 3435, 2960, 2929, 2856, 1821, 1731, 1675, 1509, 1465, 1379,
1236, 1197, 1123 cm-1. 1H NMR (200 MHz) δ 0.85-0.97 (12H,
m), 1.26-1.29 (28H, m), 1.60-1.99 (6H, m), 2.04 (3H, s), 2.18
(1H, m), 3.23 (1H, dt, J ) 4.2, 7.5 Hz), 4.29 (1H, m), 4.56 (1H,
dd, J ) 5.0, 8.6 Hz), 5.02 (1H, m), 6.02 (1H, d, J ) 8.6 Hz). 13C
NMR (50 MHz) δ 11.55, 13.99, 14.09, 15.55, 22.52, 22.69, 23.21,
25.14, 26.74, 27.72, 28.96, 29.35, 29.45, 29.55, 29.62, 31.49, 31.93,
34.04, 37.72, 38.71, 56.98, 57.20, 72.68, 74.79, 169.85, 170.66,
171.40. Anal. (C30H55NO5), C, H, N.
N-Formyl-ꢀ-alanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (12).21a The title compound was prepared
from 35 following the same procedure that was used for the
synthesis of 2 using hexane/AcOEt ) 6/4 as eluent for the
chromatographic purification; yield 73%; oil, [R]D -26°. IR 3442,
2957, 2929, 2856, 1821, 1727, 1686, 1508, 1466, 1390, 1231, 1126
N-Formyl-L-proline-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxeta-
nyl]methyl]dodecyl Ester (17). The title compound was prepared
from 38 following the same procedure that was used for the
synthesis of 2 using hexane/AcOEt ) 6/4 as eluent for the

6978 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Ortar et al.
chromatographic purification; yield 47%; mp 49-50 °C; [R]D -66°.
Bands corresponding to [3H]arachidonic acid were cut, and their
radioactivity was counted with a ꢀ-counter.
IR 3523, 2957, 2929, 2856, 1819, 1739, 1667, 1466, 1419, 1379,
1276, 1127, 1090 cm-1
.
1H NMR (400 MHz) δ 0.87 (6H, m),
Assay of DAGLr Inhibitory Activity in Intact Cells. Confluent
mouse N18TG2 neuroblastoma cells, cultured as described10 and
attached to 10 cm Petri dishes, were incubated for 20 min at 37 °C
in 5 mL of medium containing 3 µM ionomycin in the presence of
vehicle or THL (1 µM) or compound 15 (0.3 µM) or compound 8
(1.5 µM). After terminating the incubation by addition of 5 mL of
ice-cold CH3OH, cells were scraped off of the Petri dishes,
transferred to 50 mL Falcon tubes, and extracted three times with
5 mL of CHCl3 for lipid extraction. Lyophilized lipids were then
analyzed for the presence of 2-AG by isotope-dilution liquid
chromatography mass spectrometry as described previously.10
Ionomycin-stimulated levels of 2-AG in N18TG2 cells were 57.6
( 2.7 pmol/mg of extracted lipid. The effects of the inhibitors were
compared by using ANOVA, followed by the Bonferroni’s test.
1.26-1.28 (26H, m), 1.59-1.82 (4H, m), 1.92-2.11 (5H, m), 2.25
(1H, m), 3.19 (1H, m), 3.54 and 3.61 (1H, 2m), 3.64 (1H, m), 4.33
(1H, m), 4.40 (1H, dd, J ) 3.6, 8.4 Hz), 5.03 and 5.11 (1H, 2m),
8.26 and 8.29 (1H, 2s). 13C NMR (100 MHz) δ 14.03, 14.13, 22.53,
22.70, 22.82, 24.09, 25.15, 25.22, 26.68, 26.78, 27.59, 28.98, 29.35,
29.46, 29.58, 29.63, 31.50, 31.92, 34.35, 38.84, 46.33, 56.86, 56.99,
57.14, 58.95, 72.34, 73.21, 75.01, 160.71, 161.63, 171.15, 171.25.
Anal. (C28H49NO5), C, H, N.
N-Formyl-L-phenylalanine-(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-
oxetanyl]methyl]dodecyl Ester (19). The title compound was
prepared from 21 following the same procedure that was used for
the synthesis of 2 using hexane/AcOEt ) 7/3 as eluent for the
chromatographic purification; yield 100%; mp 59-63 °C; [R]D
-20°. IR (KBr disks) 3346, 2955, 2918, 2849, 1844, 1719, 1654,
1
Acknowledgment. We thank Mr. Marco Allara`, Endocan-
nabinoid Research Group, CNR, Italy, for his technical as-
sistance in binding assays.
1522, 1464, 1381, 1280, 1176 cm-1. HNMR (400 MHz) δ 0.88
(6H, m), 1.25-1.43 (26H, m), 1.59 (2H, m), 1.71 (2H, m), 1.89
(1H, dt, J ) 4.4, 14.8 Hz), 2.07 (1H, m), 3.13 (3H, m), 4.17 (1H,
dt, J ) 5.1, 6.9 Hz), 4.90 (1H, q, J ) 7.0 Hz), 4.98 (1H, m), 6.07
(1H, d, J ) 7.5 Hz), 7.17 (2H, d, J ) 6.7 Hz), 7.28 (3H, m), 8.16
(1H, s). 13C NMR (100 MHz) δ 14.03, 14.12, 22.53, 22.69, 25.05,
26.69, 27.61, 28.97, 29.32, 29.35, 29.43, 29.56, 29.62, 31.48, 31.92,
33.93, 37.81, 38.60, 52.11, 57.04, 73.03, 74.56, 127.34, 128.71,
129.26, 135.62, 160.51, 170.77, 170.81. Anal. (C32H51NO5), C,
H, N.
Supporting Information Available: Elemental analysis data for
compounds 5-7, 9-11, 14-17, 19-21, 23, 24, 26-31. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
CB1R and CB2R Binding Assays. The affinity of compounds
for either CB1 or CB2 receptors was evaluated using membranes
from HEK cells transfected with either the human recombinant CB1
or CB2 receptor cDNA and [3H]-(-)-cis-3-[2-hydroxy-4-(1,1-
dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]-
CP-55,940; Kd ) 0.18 nM for CB1 and Kd ) 0.31 nM for CB2) as
the high affinity ligand, as described by the manufacturer (Perkin-
Elmer, Italy). Displacement curves were generated by incubating
drugs with a fixed concentration of [3H]CP-55,940 (0.14 nM for
CB1 and 0.084 nM for CB2 binding assay). In all cases, Ki values
were calculated by applying the Cheng-Prusoff equation to the
IC50 values (obtained by GraphPad) for the displacement of the
bound radioligand by increasing concentrations of the test compounds.
Assay of Fatty Acid Amide Hydrolase (FAAH). The effect
on the enzymatic hydrolysis of AEA was measured by using
membranes prepared from rat brain, incubated with increasing
concentrations of the test compounds and [14C]AEA (2.4 µM) in
50 mM Tris-HCl, pH 9, for 30 min at 37 °C. [14C]Ethanolamine
produced from [14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/CH3OH ) 1/1 (by volume). Data
are expressed as the concentration exerting 50% inhibition of AEA
hydrolysis (IC50), calculated by GraphPad.
Assay of DAGL Activity. Confluent COS-7 cells, overexpressing
the human recombinant DAGLR, were harvested in tris-HCl buffer,
pH 7 and homogenized in a homogenizer (Dounce). The homoge-
nates were centrifuged at 4 °C at 800g (5 min) and then at 10000g
(25 min). The 10000g membrane fraction was incubated in
incubation buffer (Tris-HCl 50 mM) at pH 7.0 at 37 °C for 20
min, with synthetic sn-1-[14C]oleoyl-2-arachidonoylglycerol (1.0
mCi/mmol, 25 µM). After the incubation, lipids were extracted with
2 volumes of CHCl3/MeOH ) 2/1 (by volume) and the extracts
were lyophilized under vacuum. Extracts were fractionated by TLC
on silica on plastic plates using CHCl3/MeOH/NH4OH (85/15/1
by volume) as the eluting system. Bands corresponding to [14C]oleic
acid were cut, and their radioactivity was counted with a ꢀ-counter.
Assay of MAGL Activity. The 10000g cytosolic fraction from
COS-7 cells, which contains high levels of MAGL, was incubated
in Tris-HCl 50 mM, at pH 7.0 at 37 °C for 20 min, with synthetic
2-[3H]arachidonoylglycerol (1.0 mCi/mmol, 25 µM). After the
incubation, lipids were extracted with 2 volumes of CHCl3/MeOH
) 2/1 (by volume) and the extracts were lyophilized under vacuum.
Extracts were fractionated by TLC on silica on plastic plates using
CHCl3/MeOH/NH4OH (85/15/1 by volume) as the eluting system.
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