Discovery of a new class of highly potent inhibitors of acid ceramidase: Synthesis and structure-activity relationship (SAR)

Article abstract of DOI:10.1021/jm301879g

Acid ceramidase (AC) is an intracellular cysteine amidase that catalyzes the hydrolysis of the lipid messenger ceramide. By regulating ceramide levels in cells, AC may contribute to the regulation of cancer cell proliferation and senescence and to the response to cancer therapy. We recently identified the antitumoral agent carmofur (4a) as the first nanomolar inhibitor of intracellular AC activity (rat AC, IC₅₀ = 0.029 μM). In the present work, we expanded our initial structure-activity relationship (SAR) studies around 4a by synthesizing and testing a series of 2,4-dioxopyrimidine-1- carboxamides. Our investigations provided a first elucidation of the structural features of uracil derivatives that are critical for AC inhibition and led us to identify the first single-digit nanomolar inhibitors of this enzyme. The present results confirm that substituted 2,4-dioxopyrimidine-1-carboxamides are a novel class of potent inhibitors of AC. Selected compounds of this class may represent useful probes to further characterize the functional roles of AC.

Full text of DOI:10.1021/jm301879g

Article
pubs.acs.org/jmc
Discovery of a New Class of Highly Potent Inhibitors of Acid
Ceramidase: Synthesis and Structure−Activity Relationship (SAR)
Daniela Pizzirani,†,‡ Chiara Pagliuca,†,‡ Natalia Realini,† Davide Branduardi,⊥ Giovanni Bottegoni,†
Marco Mor,§ Fabio Bertozzi,† Rita Scarpelli,† Daniele Piomelli,*,†,∥ and Tiziano Bandiera*,†
†Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy
⊥Theoretical Molecular Biophysics Group, Max Planck Institute for Biophysics, Max-von-Laue Strasse 3, 60438, Frankfurt am Main,
Germany
§Dipartimento di Farmacia, Universita
̀
degli Studi di Parma, Viale delle Scienze 27/A, I-43124 Parma, Italy
∥Department of Anatomy and Neurobiology, University of CaliforniaIrvine, Irvine, California 92697-4625, United States
S
* Supporting Information
ABSTRACT: Acid ceramidase (AC) is an intracellular
cysteine amidase that catalyzes the hydrolysis of the lipid
messenger ceramide. By regulating ceramide levels in cells, AC
may contribute to the regulation of cancer cell proliferation
and senescence and to the response to cancer therapy. We
recently identified the antitumoral agent carmofur (4a) as the
first nanomolar inhibitor of intracellular AC activity (rat AC,
IC50 = 0.029 μM). In the present work, we expanded our initial
structure−activity relationship (SAR) studies around 4a by
synthesizing and testing a series of 2,4-dioxopyrimidine-1-
carboxamides. Our investigations provided a first elucidation of
the structural features of uracil derivatives that are critical for
AC inhibition and led us to identify the first single-digit nanomolar inhibitors of this enzyme. The present results confirm that
substituted 2,4-dioxopyrimidine-1-carboxamides are a novel class of potent inhibitors of AC. Selected compounds of this class
may represent useful probes to further characterize the functional roles of AC.
colon and melanoma) overexpress AC, which is suggestive of a
role for this enzyme in the resistance to radiotherapy and
chemotherapy.7
INTRODUCTION
■
Sphingosine-containing lipids are essential structural compo-
nents of cell membranes that also serve important signaling
functions in cell migration, cell recognition, and inflammation,
as well as in the control of cell growth and differentiation.1
Among the various groups of sphingolipids, the ceramides,
amides of sphingosine with long-chain fatty acids, have
attracted attention for their roles in the replication and
differentiation of normal and neoplastic cells.2 Stress-related
signals such as tumor necrosis factor α (TNF-α) stimulate the
production of ceramides in cells, which can cause in turn
replicative senescence and apoptosis.3 Thus, enzymes involved
in the biological deactivation of ceramides have emerged as
potential targets for therapeutic manipulation by small
molecules.4
Acid ceramidase (AC) is a ubiquitous cysteine amidase that
operates within the acidic milieu of the lysosome to catalyze the
hydrolysis of ceramides into sphingosine and fatty acid. By
regulating the intracellular levels of ceramides, AC may
influence the survival, growth, and death of tumor cells.5
Consistent with this view, cells that express abnormally high
levels of AC are more resistant than normal cells to
pharmacological induction of programmed cell death.6 Indeed,
several types of cancer (including prostate, head and neck,
Although several AC inhibitors have been described in the
literature, potent small-molecule compounds capable of
inhibiting this enzyme in vivo are still needed.4,7b,8 The
majority of compounds reported so far, including oleoyletha-
nolamide (1, OEA), B-13 (2), D-MAPP (3) (Figure 1),7d,9 and
their derivatives, are structural analogues of ceramide and
therefore suffer from several limitations, including low potency
and insufficient druglikeness. For example, pharmacological
inhibition of AC with 1 has been related to sensitization of
hepatoma cells to daunorubicin-induced cell death, but the low
potency (Ki ≈ 500 μM) and rapid metabolic degradation of this
compound preclude its therapeutic use.10 Compound 2 inhibits
AC activity with a median inhibitory concentration (IC50) of
approximately 10 μM, causes apoptosis in several human cancer
cell lines, and sensitizes prostate tumors to the effects of
radiation.11 Compound 3, which is structurally related to 2,
inhibits both AC and neutral ceramidase activities and triggers
apoptosis in squamous carcinoma cells.12 These results suggest
Received: December 20, 2012
© XXXX American Chemical Society
A
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Figure 1. Structures of a representative ceramide and various AC inhibitors (1−3) reported in the literature.
Figure 2. Illustration of the design strategy for structure−activity relationship studies.
a
Table 1. Reaction Conditions for the Preparation of Compounds 4f−o
entry
R2
method
product (yield, %)
entry
R2
method
product (yield, %)
1
2
3
4
5
CH2CH3
CH2OH
B
C
B
A
A
4f (16)
4g (27)
4h (56)
4i (75)
4j (60)
6
N-methylpiperazine
A
A
A
A
C
4k (17)
4l (49)
4m (19)
4n (62)
4o (13)
7
Ph
I
OCH3
8
N(CH3)Bn
morpholine
9
Br
CN
10
a
Method A: isocyanate, DMAP, pyridine, room temperature, 12 h. Method B: isocyanate, DMSO, 50 °C, 4−12 h. Method C: isocyanate, pyridine,
microwave, 100 °C, 10 min.
that AC blockade may promote or facilitate cancer cell death,
especially when combined with the use of other chemo-
therapeutic agents. The findings also highlight the need to
discover more potent and druglike compounds, which might be
used to investigate further the usefulness of AC as a target for
chemosensitizing drugs.
We recently reported that the 5-fluorouracil (5-FU) releasing
drug carmofur (4a, Figure 2) inhibits AC activity with
nanomolar potency (IC50 for rat AC of 0.029 μM) and
increases intracellular ceramide levels both in vitro and in
vivo.13 These effects are independent of carmofur’s ability to
generate 5-FU and are accompanied by a marked enhancement
in the antiproliferative actions of other antitumoral agents.
Preliminary structure−activity relationship (SAR) studies
revealed that replacement of the fluorine atom of carmofur
with selected chemical groups with different stereoelectronic
properties leads to molecules that retain the ability to inhibit
AC with high potency (e.g., 4e, IC50 = 0.012 μM).13
In the present report, we expanded our SAR studies around
carmofur with the objective of identifying other potent small-
molecule inhibitors for this enzyme. We synthesized a series of
2,4-dioxopyrimidine-1-carboxamides to explore the role of
B
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Scheme 1. Synthesis of 5-Substituted Uracils 5i−l
a
Table 2. Inhibitory Potencies (IC50) of Compounds on Rat AC Activity
a
b
IC50 values are reported as mean values of three or more determinations. IC50 values from ref 13.
different substituents at carbons C5 and C6 and at nitrogen N3
of the uracil ring, as well as the effect of the alkyl side chain on
AC inhibition (Figure 2). In addition, we conducted a
quantum-mechanical study on the new molecules to explore
C
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Scheme 2. Syntheses of 3-Alkyluracil Derivatives 8a,b, 9a−10a
a
Scheme 3. Synthetic Pathways for the Preparation of Uracil Derivatives 15a−19a
a
15a: TEA, DCM. 16a−17a: Py. 18a−19a: Py, DCM.
Scheme 4. Synthesis of 2,4-Dioxopyrimidine-1-carboxamides 20b−24b, 23a, and 23e
the effect of uracil substitution on orbital stabilization energies
and inhibitory potency.
5-iodouracil (5m). This compound was transformed into the
N1-benzhydryl derivative (6m) by treatment with benzhydryl
bromide in the presence of bis(trimethylsilyl)acetamide (BSA)
and a catalytic amount of iodine (I2).16 Subsequent reaction
with phenylboronic acid under standard Suzuki coupling
conditions, followed by removal of the protecting group with
10% triflic acid solution in TFA, afforded the desired 5-
phenyluracil (5l) in good yield.
CHEMISTRY
■
We explored and optimized various methods reported in the
literature for the synthesis of ureas to access differently
substituted 2,4-dioxopyrimidine-1-carboxamides.14 The prep-
aration of most 2,4-dioxopyrimidine-1-carboxamide derivatives
was accomplished by coupling differently substituted uracils
with alkyl isocyanates. Urea formation occurred regioselectively
at position N1 under all conditions employed. Coupling
reactions were conducted in pyridine at room temperature with
a stoichiometric amount of DMAP (method A), in DMSO at
50 °C (method B), or in pyridine under microwave irradiation
at 100 °C (method C). Dimethylaminopyridine was necessary
for acylation reactions that proceeded incompletely or did not
occur in pyridine or DMSO alone.14b
N-Hexyl-5-methylamino-2,4-dioxopyrimidine-1-carboxamide
(4p, Table 2, Scheme S1) and N-hexyl-2,4-dioxohexahydropyr-
imidine-1-carboxamide (7, Table 2, Scheme S2) were obtained
by catalytic hydrogenation from 4i and 4b, respectively. The
N3-substituted-1-carboxamide derivatives 8a,b, 9a−10a were
prepared following a three-step sequence starting from N1-Boc
uracils 11a,b (Scheme 2).17 These compounds were first
reacted with the appropriate alkyl iodides in the presence of
cesium carbonate to give N3-substituted 2,4-dioxopyrimidine-1-
carboxylates. The tert-butyloxycarbonyl group was then
removed with potassium carbonate in methanol to afford 3-
alkyluracils 12a,b, 13a−14a which were then converted into the
corresponding carboxamides 8a,b, 9a−10a following method A.
Syntheses of N3-substituted uracils 15a−19a were accom-
plished with minor modifications of literature procedures, as
reported in Scheme 3, by direct N3-acylation of N1-
carboxamide derivatives. No decomposition of the ureidic
function occurred in agreement with what observed with N1-
alkoxycarbonyluracils.18 Compound 15a was prepared starting
from carmofur (4a) by reaction with isobutyryl chloride in the
presence of triethylamine. The carbamoyl derivatives 16a−17a
We first focused on the synthesis of carboxamide derivatives
differently substituted at position 5. Table 1 shows the reaction
conditions adopted for the coupling of 5-substituted uracils
with n-hexyl isocyanate to afford the compound series 4f−o.
Non-commercially available uracils such as 5-(benzyl(methyl)-
amino)uracil (5i), 5-(morpholin-4-yl)uracil (5j), 5-(4-methyl-
piperazin-1-yl)uracil (5k), and 5-phenyluracil (5l) were
prepared as described in Scheme 1. Compounds 5i−k were
obtained by nucleophilic substitution reaction of the appro-
priate amine on 5-bromouracil (5n) under microwave
irradiation at 120 °C.15 The synthesis of 5-phenyl-1H-
pyrimidine (5l) was accomplished in three steps starting from
D
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were obtained by treatment of 4a with the appropriate
chloroformate in the presence of pyridine as base. The
syntheses of 3-isobutyl carboxylate 18a and 3-benzoyluracil
derivative 19a were carried out by treatment of 4a with isobutyl
chloroformate and benzoyl chloride in pyridine as solvent and
base.
The small series of compounds (20b−24b, 23a, and 23e)
bearing N1-carboxamide alkyl chains of different length was
prepared as shown in Scheme 4. Differently substituted uracils
(5a,b, 5e) were coupled with various alkyl isocyanates following
method A to afford the desired carboxamides in good yields.
Derivative 23a was functionalized at position N3 by reaction
with methyl chloroformate, as previously described for the
preparation of 16a (Scheme 3), to yield methyl 3-
(octylcarbamoyl)-2,6-dioxopyrimidine-1-carboxylate (25a,
Table 4, Scheme S3).
scaffold for the discovery of potent AC inhibitors. Encouraged
by these results, we decided to expand this series of compounds
to elucidate the structural features that may be critical for AC
inhibition.
The new substituted 2,4-dioxopyrimidine-1-carboxamides
were tested for their ability to inhibit the hydrolysis of N-
lauroylceramide by recombinant rat AC (r-AC). IC50 values are
reported in Tables 2−4.
The limited stability of some 2,4-dioxopyrimidine-1-carbox-
amide derivatives in DMSO-d6 prompted us to ask first whether
the compounds were sufficiently stable under the conditions of
our assay to obtain meaningful IC50 values. A set of molecules
representative of the substitution pattern explored in our SAR
analysis was selected and tested for stability in buffer over the
30 min period of the assay (Table S2).20 Most compounds
tested were stable enough to be included in the study except
4o, which was extensively hydrolyzed (22% of the initial
amount remained after 30 min) and was not further
investigated. The 6-substituted derivatives 26 and 30 were
also excluded because of their chemical instability.
An alternative synthetic procedure to access 1-carbamoylur-
acils was adopted for compounds 26 and 27b (Scheme 5). The
Scheme 5. Synthesis of 5,6-Dimethyluracil Derivative 26 and
N-Methylurea 27b
We initially focused our attention on substitutions at position
5 to expand the initial set of derivatives 4a−e (Table 2). In
particular, the potency of 4e (IC50 = 0.012 μM) prompted us to
synthesize the 5-iodo, 5-bromo, and 5-cyano derivatives (4m−
o) in order to further investigate the effect of electron-
withdrawing groups at that position. The introduction of iodine
led to a 5-fold decrease in activity (IC50 = 0.147 μM) compared
to carmofur (4a), while the 5-bromo derivative was equipotent
to the 5-chloro-substituted carboxamide 4d, with an IC50 of
0.051 μM. Next, we introduced a set of electron-donating
groups at position 5 of the uracil ring. The compounds
obtained (4f−h, 4p) were 14- to 100-fold less potent than
carmofur (4a). Replacement of fluorine with an alkyl group was
detrimental for activity. The 5-methyl derivative 4c (IC50 = 1.46
μM) showed a 50-fold decrease in potency compared to
carmofur, whereas a less pronounced decrease was observed
with the 5-ethyl analogue 4f (IC50 = 0.733 μM) and the 5-
hydroxymethyl derivative 4g (IC50 = 0.417 μM). The
introduction of substituents containing a heteroatom directly
bound to the uracil ring led to a considerable loss in potency.
The 5-methoxy derivative 4h showed low micromolar AC
inhibition (IC50 = 1.1 μM), as did the N-methylamino analogue
4p (IC50 = 3.4 μM). Replacing the N-methylamino group with
an N-methylbenzylamino moiety led to a further decrease in
potency (4i, IC50 = 5.5 μM), probably due to increased steric
hindrance. We also introduced heterocyclic and aromatic rings
at position 5. Consistent with the data obtained with analogues
bearing a heteroatom at position 5, introduction of morpholine
or N-methylpiperazine caused a decrease in potency, albeit less
pronounced than that observed with the N-methylamino
derivative 4p, as compounds 4j and 4k yielded IC50 values of
0.875 and 1.4 μM, respectively. Surprisingly, introduction of a
phenyl ring led to the most potent compound within this small
series (4l, IC50 = 0.177 μM), indicating that a planar group is
well accepted even if it is sterically demanding.
5,6-dimethyl substituted derivative 26 was obtained by reaction
of 5,6-dimethyluracil (28) with triphosgene followed by
treatment in situ with hexylamine. The synthesis of the N-
methylurea 27b was accomplished with a similar procedure
starting from the sodium salt 29b of the uracil 5b (Scheme 5).
In the characterization process of the final 2,4-dioxopyr-
imidine-1-carboxamide derivatives, we found that few of them
showed limited stability in DMSO-d6 solution (Table S1),
slowly releasing the corresponding uracil.19 In addition, we
observed that N-hexyl-2,4-dioxo-5,6-dimethylpyrimidine-1-car-
boxamide (26) and N-hexyl-2,4-dioxo-6-chloropyrimidine-1-
carboxamide (30, Scheme S4) decomposed both in solution
and as powder, revealing a marked chemical instability.
RESULTS AND DISCUSSION
■
The objective of the present study was to investigate
substituted 2,4-dioxopyrimidine-1-carboxamides as a novel
class of AC inhibitors. A preliminary SAR study on carmofur
analogues (4b−e, Table 2) bearing substituents with different
stereoelectronic properties at position 5 had shown that an
electron-withdrawing group is crucial to achieve potent
inhibition of AC activity. Accordingly, replacement of fluorine
with electron-withdrawing substituents, such as chlorine and
trifluoromethyl, led to the identification of two novel double-
digit nanomolar AC inhibitors (4d,e). The results obtained
with this small set of compounds suggested that 2,4-
dioxopyrimidine-1-carboxamide might provide a promising
To complete the SAR study around positions 5 and 6, we
examined the role of the C5−C6 double bond. Compound 7,
the dihydro derivative of 4b, showed no inhibitory activity
toward AC, highlighting the functional importance of a fully
conjugated 2,4-dioxopyrimidine-1-carboxamide system.
As the next step in our SAR study, we investigated the effect
of substituents at position N3 of the uracil ring introducing
alkyl, acyl, and carbamoyl groups at this position (Table 3). N3-
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derivative 9a had an IC50 of 0.018 μM, and compound 10a,
bearing a methylcyclopropyl moiety, had an IC50 of 0.061 μM.
Comparable high potencies were obtained with acyl sub-
stituents: 15a, bearing a 2-methylpropanoyl group, and the
benzoyl-substituted 19a had an IC50 value of 0.02 μM. The
effect of the N3-substitution was further explored by
introducing a carbamoyl functionality, as in compounds 16a−
18a. Of particular interest was the result obtained with the
methylcarbamoyl derivative 16a, which showed an IC50 of
0.007 μM. High inhibitory potencies were retained with the
more sterically demanding carbamoyl derivatives 17a and 18a,
which displayed IC50 values of 0.012 and 0.016 μM,
respectively.
Table 3. Inhibitory Potencies (IC50) of Compounds on Rat
AC Activity
a
Next, we examined the role of the alkyl side chain of the N1-
carboxamide function, focusing on the influence of chain
length. We chose the moderately potent 5-unsubstituted
derivative 4b as reference compound to better appreciate
possible changes in potency by variations in the chain length.
Unsubstituted uracil derivatives 20b−24b, where the length of
the aliphatic side chain was progressively increased from four to
nine methylene units, were tested for their inhibitory activity;
results are reported in Table 4. With respect to compound 4b
(IC50 = 0.426 μM), which bears a six-carbon alkyl chain, a
decrease in chain length was associated with a drop in potency,
as N-butyl-2,4-dioxopyrimidine-1-carboxamide 20b (IC50 = 8.5
μM) was 20-fold less potent than 4b. Increasing the length of
the alkyl chain from four to eight methylene units, we observed
progressively higher potencies, with the N-octyl derivative 23b
showing the highest activity (IC50 = 0.283 μM) within this set
of compounds. Further extension of the chain length to nine
methylene units resulted, however, in a loss in potency
(compound 24b, IC50 = 0.464 μM).
The role of the 1-carboxamide NH group was investigated by
preparing the N-methyl derivative 27b (Table 4). This
compound showed no inhibitory activity toward AC.
Finally, we prepared and tested the 2,4-dioxopyrimidine-1-
carboxamides 23a, 23e, and 25a bearing at positions 5, N1, and
N3 the best substituents identified in the previous SAR (Table
4). The combination of the electron-withdrawing fluorine or
trifluoromethyl group at position 5 with the n-octyl chain at N1
led to potent AC inhibitors. The 5-fluoro derivative 23a (IC50 =
0.046 μM) turned out to be 6-fold more potent than the
corresponding unsubstituted analogue 23b, while the 5-
trifluoromethyl compound 23e (IC50 = 0.007 μM) showed a
40-fold improvement in potency compared to 23b. Notably, in
line with our previous observations, carbamoylation of
compound 23a at position N3 delivered the single-digit
nanomolar inhibitor 25a, which displayed an IC50 of 0.004 μM.
The critical importance of a fully conjugated 2,4-dioxopyr-
imidine-1-carboxamide system for displaying AC inhibitory
activity, and the opposite effect on potency of electron-
withdrawing and electron-donating substituents at position 5,
prompted us to undertake a quantum mechanical study on all
the reported compounds to test whether any correlation could
be established between the potency of the compounds and
electronic descriptors of the substituted 2,4-dioxopyrimidine-1-
carboxamide derivatives. The SAR exploration around position
5 found a computational counterpart in the second order
perturbation theory analysis based on the natural bond orbital
(NBO) approach.21 Such technique was useful to understand
how the orbital deviation from the ideal Lewis structure was
modulated by different substituents and its relationship with
activity (a complete list of calculated NBO energies is reported
a
IC50 values are reported as mean values of three or more
determinations.
Methylation was first explored on 5-fluoro and unsubstituted
2,4-dioxopyrimidine-1-carboxamides (4a, 4b) and was found to
be highly beneficial for the activity, resulting in the two double-
digit nanomolar inhibitors 8a (IC50 = 0.013 μM) and 8b (IC50
= 0.053 μM). Notably, an 8-fold increase in potency was
observed for compound 8b vs 4b, and the 5-fluoro derivative 8a
turned out to be equipotent to 4e. Considering that the 5-
unsubstituted derivative 8b was 4-fold less potent than 8a, we
turned our attention to 5-fluoro substituted compounds. The
introduction of progressively bulkier alkyl groups led only to a
slight decrease in inhibitory activity, as the N3-ethyl substituted
F
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the most relevant contributions to the orbitals in close
proximity to N1.
Table 4. Inhibitory Potencies (IC50) of Compounds on Rat
AC Activity
a
We next extended the analysis to N3-substituted derivatives
(8a,b, 9a−10a, 15a−19a; see Figure S2). With the only
exception of compound 19a, the activity of which was lower
than predicted by the orbital stabilization energy, a satisfactory
degree of correlation was maintained between stabilization
energy of the exocyclic N1-CO antibonding orbitals and
inhibitory potency. The squared correlation coefficients
between the stabilization energy of the N1-CO antibonding
orbitals and the potency of derivatives are R2 = 0.52 and R2 =
0.73, including and excluding 19a, respectively (see Figure S2).
It is reasonable to hypothesize that because of the bulky nature
of the benzoyl substituent, the potency of 19a is reduced by a
possible steric clash at the binding site of AC. A more
comprehensive PLS model confirmed that when introducing
N3-substituted derivatives, the overall potency variation is still
mainly accounted for by contributions of the orbitals in close
proximity to N1. However, it is interesting to note that in this
case the orbitals surrounding N3 also play an important role.
As observed with potency, the stability of 5-substitued
compounds is linearly related to the stabilization energy of the
antibonding N1-CO orbital (see Figure S5). For the derivatives
substituted at position 6 or N3, other electronic properties of
the uracil scaffold, besides the stabilization energy, are likely to
be taken into account to explain their stability. Nonetheless, it
appeared that energy values below 45 kcal/mol guarantee
chemical stability. In this light, the most active compounds in
the series could be regarded as the result of an optimal trade-off
between potency and stability.
Compounds 4l, 23b, and 8a, which showed good AC
inhibition and stability, have been tested for their effect on the
proliferation of the human colon adenocarcinoma cell line
SW430. Cell viability was determined after a 72 h treatment
schedule (see Supporting Information). Compounds 4l, 23b,
and 8a decreased SW403 cell viability with EC50 values of 20,
24, and 22 μM, respectively (Table 5).23
CONCLUSIONS
■
We have identified and characterized 2,4-dioxopyrimidine-1-
carboxamides as a novel class of potent small-molecule AC
inhibitors. A systematic SAR study around the uracil scaffold
allowed a first elucidation of critical structural features
associated with AC inhibition. Our study confirmed the
importance of an electron-withdrawing group at position 5 of
the uracil ring to access potent inhibitors, as previously
observed. Additionally, we found that substitution at position
N3 is beneficial for activity, leading to highly potent
compounds, and that a 1-carboxamide alkyl chain of six to
eight methylene units is important to achieve potent AC
inhibition. Finally, our SAR study revealed that the C5−C6
double bond in the uracil ring and unsubstituted nitrogen in the
1-carboxamide moiety are mandatory structural features for
activity. Computational studies provided a theoretical explan-
ation for the potency of 2,4-dioxopyrimidine-1-carboxamides as
AC inhibitors in terms of the electronic properties of the
substituted uracil ring.
a
IC50 values are reported as mean values of three or more
determinations.
in Table S3).22 As illustrated in Figure 3, we observed that the
stabilization energy of the antibonding (non-Lewis) exocyclic
N1-CO orbital linearly correlated with inhibitory potency (R2 =
0.87). In other words, a progressively more stable and
populated antibonding N1-CO orbital designates a compound
that is a more potent AC inhibitor. These results also explained
the importance of the uracil ring conjugated system: in the case
of the saturated derivative 7, the stabilization energy (41.36
kcal/mol) was considerably lower than the energy values
associated with pharmacologically active compounds. Through
a partial least squares (PLS) regression technique (reported in
detail in the Supporting Information), it was possible to
confirm that the potency (pIC50) of the 2,4-dioxopyrimidine-1-
carboxamide 5-substituted derivatives 4a−n and 4p and the
orbital energies of the 39 valence NBOs of the common
scaffold are highly correlated. The insights gathered from this
analysis were in line with the stabilization energy data, assigning
A notable result of this study is the identification of the first
single-digit nanomolar inhibitors (16a, 23e, and 25a) of AC
activity. Selected derivatives in this series may represent useful
probes to characterize the functional roles of AC and assess the
therapeutic potential of AC inhibitors in cancer and other
G
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Figure 3. Correlation between the stabilization energies of the exocyclic N1-CO antibonding orbital and potencies (expressed as pIC50) of the 5-
substituted derivatives. For each compound, the stabilization energy is reported explicitly. Note that no activity data is associated with compound 4o,
which could not be considered in the plot.
phase was 10 mM NH4OAc in MeCN−H2O (95:5) at pH 5.
Electrospray ionization in positive and negative modes was used.
Hydrogenation reactions were performed using H-Cube continuous
hydrogenation equipment (SS-reaction line version), employing
disposable catalyst cartridges (CatCart) preloaded with the required
heterogeneous catalyst. Microwave heating was performed using
Explorer-48 positions instrument (CEM). NMR experiments were run
Table 5. Antiproliferation Activity of Representative
Compounds on SW403 Cells
a
Compound
4l
23b
8a
EC50 (μM)
24
20
22
a
Viability of SW403 cells following a 72 h treatment with the reported
compounds was measured by MTT reduction as described in the
Supporting Information (n=4 for each drug concentration). Results
are shown as EC50 values (μM).
1
on a Bruker Avance III 400 system (400.13 MHz for H and 100.62
MHz for 13C) equipped with a BBI probe and Z-gradients. Spectra
were acquired at 300 K, using deuterated dimethylsulfoxide (DMSO-
d6), deuterated chloroform (CDCl3), or deuterated acetone (acetone-
d6) as solvents. UPLC−MS analyses were run on a Waters ACQUITY
UPLC−MS system consisting of a SQD (single quadrupole detector)
mass spectrometer equipped with an electrospray ionization interface
and a photodiode array detector. PDA range was 210−400 nm.
Analyses were performed on an ACQUITY UPLC HSS T3 C18
column (50 mm × 2.1 mm i.d., particle size 1.8 μm) with a VanGuard
HSS T3 C18 precolumn (5 mm × 2.1 mm i.d., particle size 1.8 μm).
Mobile phase was either 10 mM NH4OAc in H2O at pH 5 adjusted
with AcOH (A) and 10 mM NH4OAc in MeCN−H2O (95:5) at pH 5
(B). Electrospray ionization in positive and negative modes was
applied. All final compounds, 4a−p, 7, 8a,b, 9a−10a, 15a−19a, 20b−
24b, 23a, 23e, 25a, 26, 27b, and 30, showed ≥95% purity by NMR
and UPLC−MS analysis.
disorders in which modulation of ceramide levels is clinically
relevant.
EXPERIMENTAL SECTION
■
a. Chemicals, Materials, and Methods. All the commercial
available reagents and solvents were used as purchased from vendors
without further purification. Dry solvents (pyridine, CH2Cl2, DMSO)
were purchased from Sigma-Aldrich. Automated column chromatog-
raphy purifications were done using a Teledyne ISCO apparatus
(CombiFlash Rf) with prepacked silica gel columns of different sizes
(from 4 to 40 g). Mixtures of increasing polarity of cyclohexane and
ethyl acetate (EtOAc) were used as eluents. Flash column
chromatography was performed manually on prepacked silica
cartridges (2 or 5 g) from Biotage or on glass columns using Merck
silica gel 60 (230−400 mesh) as stationary phase. Purifications by
preparative HPLC−MS were run on a Waters Autopurification system
consisting of a model 3100 single quadrupole mass spectrometer
equipped with an electrospray ionization interface and a 2998
photodiode array detector. HPLC system included a model 2747
sample manager, model 2545 binary gradient module, system fluidic
organizer, and model 515 HPLC pump. The PDA range was 210−400
nm. Purifications were performed on a XBridge Prep C18 OBD column
(100 mm × 19 mm i.d., particle size 5 μm) with a XBridge Prep C18
(10 mm × 19 mm i.d., particle size 5 μm) Guard cartridge. Mobile
General Procedure for the Synthesis of 2,4-Dioxopyrimi-
dine-1-carboxamides 4i−n, 8a,b, 9a−10a, 20b−24b, 23a, and
23e via Method A (Table 1, Schemes 3 and 5). The properly
substituted uracil (1.0 equiv) was dissolved in dry pyridine. DMAP
(1.1 equiv) was added, and the reaction mixture was stirred under
nitrogen atmosphere at room temperature for 30 min. The appropriate
alkyl isocyanate (1.1−1.8 equiv) was then added, and the resulting
mixture was stirred for 12 h. The solvent was evaporated under
reduced pressure, and the crude was purified by silica gel column
chromatography, eluting with a mixture cyclohexane/EtOAc.
5-(Benzyl(methyl)amino)-N-hexyl-2,4-dioxopyrimidine-1-
carboxamide (4i). The reaction was carried out following method A,
H
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Journal of Medicinal Chemistry
Article
dissolving 5i (0.12 g, 0.52 mmol) in dry pyridine (6 mL) and
employing DMAP (0.07 g, 0.57 mmol) and hexyl isocyanate (0.08 mL,
0.57 mmol). The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc 60:40) to afford compound 4i
(0.14 g, 75%) as a white powder. 1H NMR (400 MHz, CDCl3) δ 0.84
(t, J = 6.8 Hz, 3H), 1.20−1.35 (m, 6H), 1.48−1.58 (m, 2H), 2.58 (s,
3H), 3.31 (td, J = 5.7, 7.0 Hz, 2H), 4.21 (s, 2H), 7.19−7.32 (m, 5H),
7.65 (s, 1H), 8.85 (s, 1H), 9.11 (t, J = 5.7 Hz, 1H). 13C NMR (101
MHz, CDCl3) δ 13.99, 22.50, 26.52, 29.19, 31.39, 38.99, 41.23, 57.44,
122.21, 127.46, 128.11, 128.36, 128.88, 137.04, 150.22, 150.42, 160.32.
MS (ESI) m/z: 359 [M − H]+. MS (ESI) m/z: 357 [M − H]−.
N-Hexyl-5-morpholino-2,4-dioxopyrimidine-1-carboxamide
(4j). The reaction was carried out following method A, dissolving 5j
(0.06 g, 0.32 mmol) in dry pyridine (3 mL) and employing DMAP
(0.07 g, 0.35 mmol) and hexyl isocyanate (0.05 mL, 0.35 mmol). The
crude product was purified by preparative HPLC−MS to afford
41.63, 100.15, 138.41, 148.91, 150.70, 158.08. MS (ESI) m/z: 317 [M
− H]−.
5-Fluoro-N-hexyl-3-methyl-2,4-dioxopyrimidine-1-carboxa-
mide (8a). The reaction was carried out following method A,
dissolving 12a (0.04 g, 0.31 mmol) in dry pyridine (1.6 mL) and
employing DMAP (0.04 g, 0.34 mmol) and hexyl isocyanate (0.08 mL,
0.56 mmol). The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc 70:30) to afford compound
1
8a (0.06 g, 71%) as a colorless oil. H NMR (400 MHz, CDCl3) δ
0.90 (t, J = 6.4 Hz, 3H), 1.21−1.44 (m, 6H), 1.56−1.69 (m, 2H),
3.34−3.44 (m, 2H), 3.40 (s, 3H), 8.47 (d, J = 6.6 Hz, 1H), 9.23 (m,
1H). 13C NMR (101 MHz, CDCl3) δ 14.12, 22.65, 26.64, 28.71, 29.28,
31.51, 41.61, 121.11 (d, J = 37.4 Hz), 140.62 (d, J = 238.2 Hz), 149.61,
151.01, 156.69 (d, J = 26.4 Hz). MS (ESI) m/z: 272 [M − H]+.
N-Hexyl-3-methyl-2,4-dioxopyrimidine-1-carboxamide (8b).
The reaction was carried out following method A, dissolving 12b (0.08
g, 0.60 mmol) in dry pyridine (3 mL) and employing DMAP (0.08 g,
0.66 mmol) and hexyl isocyanate (0.16 mL, 1.08 mmol). The crude
was purified by silica gel column chromatography (cyclohexane/
EtOAc 60:40) to afford compound 8b (0.12 g, 79%) as a colorless oil.
1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 6.5 Hz, 3H), 1.26−1.45
(m, 6H), 1.53−1.68 (m, 2H), 3.35 (s, 3H), 3.36−3.42 (m, 2H), 5.93
(d, J = 8.5 Hz, 1H), 8.39 (d, J = 8.5 Hz, 1H), 9.29 (m, 1H). 13C NMR
(101 MHz, CDCl3) δ 14.13, 22.73, 26.68, 28.00, 29.30, 31.53, 41.41,
103.42, 136.70, 150.39, 152.64, 162.09. MS (ESI) m/z: 276 [M −
Na]+.
1
compound 4j (0.06 g, 60%) as a white powder. H NMR (400 MHz,
CDCl3) δ 0.77−0.95 (m, 3H), 1.26−1.43 (m, 6H), 1.51−1.66 (m,
2H), 2.90−3.07 (m, 4H), 3.36 (td, J = 5.7, 7.1 Hz, 2H), 3.73−3.94 (m,
4H), 7.83 (s, 1H), 8.32 (s, 1H), 9.10 (t, J = 5.7 Hz, 1H). 13C NMR
(101 MHz, CDCl3) δ 13.98, 22.50, 26.50, 29.18, 31.37, 41.28, 50.32,
66.54, 122.44, 128.33, 150.06, 150.14, 159.59. MS (ESI) m/z: 347 [M
− Na]+. MS (ESI) m/z: 323 [M − H]−.
N-Hexyl-5-(4-methylpiperazin-1-yl)-2,4-dioxopyrimidine-1-
carboxamide (4k). The reaction was carried out following method A,
dissolving 5k (0.15 g, 0.71 mmol) in dry pyridine (2 mL) and
employing DMAP (0.10 g, 0.79 mmol) and hexyl isocyanate (0.11 mL,
0.79 mmol). The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc 70:30) to afford compound
4k (0.04 g, 17%) as a white powder. 1H NMR (400 MHz, DMSO-d6)
δ 0.83−0.94 (m, 3H), 1.20−1.37 (m, 6H), 1.45−1.55 (m, 2H), 2.27
(br s, 3H), 2.94 (br s, 4H), 3.23−3.36 (m, 6H), 7.55 (s, 1H), 9.22 (t, J
= 5.6 Hz, 1H), 11.79 (br s, 1H). 13C NMR (101 MHz, DMSO-d6) δ
14.35, 22.45, 26.35, 29.14, 31.31, 40.80, 45.88, 49.37, 54.65, 121.17,
128.26, 150.72, 150.86, 160.61. MS (ESI) m/z: 338 [M − H]+. MS
(ESI) m/z: 336 [M − H]−.
N-Hexyl-2,4-dioxo-5-phenylpyrimidine-1-carboxamide (4l).
The reaction was carried out following method A, dissolving 5l
(0.05 g, 0.26 mmol) in dry pyridine (2.6 mL) and employing DMAP
(0.04 g, 0.28 mmol) and hexyl isocyanate (0.07 mL, 0.47 mmol). The
crude was purified by silica gel column chromatography (cyclohexane/
EtOAc 70:30) to afford compound 4l (0.04 g, 49%) as a white powder.
1H NMR (400 MHz, DMSO-d6) δ 0.87 (t, J = 6.5 Hz, 3H), 1.16−1.37
(m, 6H), 1.43−1.61 (m, 2H), 3.23−3.32 (m, 2H), 7.30−7.46 (m, 3H),
7.48−7.57 (m, 2H), 8.28 (s, 1H), 9.19 (t, J = 5.6 Hz, 1H), 11.94 (s,
1H). 13C NMR (101 MHz, CDCl3) δ 14.14, 22.67, 26.66, 29.31, 31.53,
41.50, 77.16, 116.87, 128.53, 128.76, 131.49, 136.05, 149.92, 151.03,
161.31. MS (ESI) m/z: 333 [M − NH4]+. MS (ESI) m/z: 314 [M −
H]−.
3-Ethyl-5-fluoro-N-hexyl-2,4-dioxopyrimidine-1-carboxa-
mide (9a). The reaction was carried out following method A,
dissolving 13a (0.04 g, 0.30 mmol) in dry pyridine (1.3 mL) and
employing DMAP (0.04 g, 0.33 mmol) and hexyl isocyanate (0.07 mL,
0.50 mmol). The crude was purified by silica gel column
chromatography (petroleum ether/EtOAc 85:15) to afford compound
1
9a (0.02 g, 25%) as a colorless oil. H NMR (400 MHz, CDCl3) δ
0.90 (t, J = 6.8 Hz, 3H), 1.26 (t, J = 7.1 Hz, 3H), 1.29−1.43 (m, 6H),
1.56−1.68 (m, 2H), 3.28−3.50 (m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 8.45
(d, J = 6.6 Hz, 1H), 9.22−9.29 (m, 1H). 13C NMR (101 MHz,
CDCl3) δ 12.74, 14.14, 22.65, 26.66, 29.29, 31.52, 37.74, 41.61, 121.11
(d, J = 37.4 Hz), 140.72 (d, J = 238.6 Hz), 149.73, 150.69, 156.40 (d, J
= 29.7 Hz). MS (ESI) m/z: 157 [M − CONH(CH2)5CH3]−.
3-(Cyclopropylmethyl)-5-fluoro-N-hexyl-2,4-dioxopyrimi-
dine-1-carboxamide (10a). The reaction was carried out following
method A, dissolving 14a (0.05 g, 0.26 mmol) in dry pyridine (1.4
mL) and employing DMAP (0.04 g, 0.29 mmol) and hexyl isocyanate
(0.07 mL, 0.48 mmol). The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc 85:15) to afford compound 10a
(0.06 g, 74%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.39−
0.45 (m, 2H), 0.47−0.56 (m, 2H), 0.89 (t, J = 6.9 Hz, 3H), 1.20−1.27
(m, 1H), 1.28−1.43 (m, 6H), 1.53−1.70 (m, 2H), 3.39 (td, J = 5.6, 7.2
Hz, 2H), 3.88 (d, J = 7.3 Hz, 2H), 8.46 (d, J = 6.6 Hz, 1H), 9.26 (m,
1H). 13C NMR (101 MHz, CDCl3) δ 4.07, 9.63, 14.12, 22.65, 26.65,
29.28, 31.51, 41.60, 46.96, 121.16 (d, J = 37.5 Hz), 140.76 (d, J =
238.3 Hz), 149.76, 151.11, 156.82 (d, J = 26.5 Hz). MS (ESI) m/z:
312 [M − H]+, 334 [M − Na]+.
N-Hexyl-5-iodo-2,4-dioxopyrimidine-1-carboxamide (4m).
The reaction was carried out following method A, dissolving 5m
(0.10 g, 0.42 mmol) in dry pyridine (2 mL) and employing DMAP
(0.06 g, 0.46 mmol) and hexyl isocyanate (0.07 mL, 0.46 mmol). The
crude was purified by silica gel column chromatography (cyclohexane/
EtOAc 90:10) to afford compound 4m (0.03 g, 19%) as a white
N-Butyl-2,4-dioxopyrimidine-1-carboxamide (20b). The re-
action was carried out following method A, dissolving 5b (0.99 g, 8.75
mmol) in dry pyridine (20 mL) and employing DMAP (1.17 g, 9.62
mmol) and butyl isocyanate (1.08 mL, 9.62 mmol). The crude was
crystallized in refluxing ethanol (20 mL) and filtered at room
temperature to afford compound 20b (1.05 g, 57%) as a white powder.
1H NMR (400 MHz, DMSO-d6) δ 0.89 (t, J = 7.3 Hz, 3H), 1.26−1.38
1
powder. H NMR (400 MHz, CDCl3) δ 0.77−0.95 (m, 3H), 1.22−
1.46 (m, 6H), 1.55−1.66 (m, 2H), 3.41 (td, J = 5.6, 7.1 Hz, 2H), 8.49
(s, 1H), 8.86 (s, 1H), 8.96 (t, J = 5.6 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 13.98, 22.50, 26.46, 29.08, 31.34, 41.42, 134.64, 143.41,
148.71, 150.97, 159.06. MS (ESI) m/z: 366 [M − H]+.
N-Hexyl-5-bromo-2,4-dioxopyrimidine-1-carboxamide (4n).
The reaction was carried out following method A, dissolving 5n (0.30
g, 1.57 mmol) in dry pyridine (7.8 mL) and employing DMAP (0.21 g,
1.72 mmol) and hexyl isocyanate (0.27 mL, 1.88 mmol). The crude
was purified by silica gel column chromatography (cyclohexane/
EtOAc from 70:30 to 100% EtOAc) to afford compound 4n (0.31 g,
62%) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 0.87 (t, J
= 6.5 Hz, 3H), 1.24−1.32 (m, 6H), 1.40−1.61 (m, 2H), 3.26 (td, J =
5.7, 7.0 Hz, 2H), 8.47 (s, 1H), 9.07 (t, J = 5.7 Hz, 1H), 12.20 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 14.12, 22.65, 26.61, 29.23, 31.49,
(m, 2H), 1.46−1.56 (m, 2H), 3.27 (td, J = 5.7, 7.0 Hz, 2H), 5.79 (d, J
= 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 9.11 (t, J = 5.7 Hz, 1H), 11.71
(s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 13.54, 19.39, 30.76, 40.01,
103.47, 138.72, 149.93, 151.47, 162.76. MS (ESI) m/z: 210 [M − H]−.
N-Pentyl-2,4-dioxopyrimidine-1-carboxamide (21b). The re-
action was carried out following method A, dissolving 5b (0.10 g, 0.89
mmol) in dry pyridine (3 mL) and employing DMAP (0.12 g, 0.98
mmol) and pentyl isocyanate (0.13 mL, 0.98 mmol). The crude was
purified by silica gel column chromatography (cyclohexane/EtOAc
1
90:10) to afford compound 21b (0.02 g, 10%) as a white powder. H
I
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NMR (400 MHz, CDCl3) δ 0.94 (t, J = 6.7 Hz, 3H), 1.30−1.41 (m,
4H), 1.60−1.70 (m, 2H), 3.41 (td, J = 5.6, 7.1 Hz, 2H), 5.91 (dd, J =
8.5, 2.0 Hz, 1H), 8.25 (s, 1H), 8.44 (d, J = 8.5 Hz, 1H), 9.06 (br s,
1H). 13C NMR (101 MHz, CDCl3) δ 13.93, 22.27, 28.97, 28.86, 41.26,
103.82, 138.90, 150.80, 150.82, 161.72.
N-Heptyl-2,4-dioxopyrimidine-1-carboxamide (22b). The
reaction was carried out following method A, dissolving 5b (0.37 g,
3.34 mmol) in dry pyridine (11 mL) and employing DMAP (0.45 g,
3.67 mmol) and heptyl isocyanate (0.70 mL, 4.35 mmol). The crude
was triturated in dichloromethane to afford compound 22b (0.58 g,
68%) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 0.86 (t, J
= 6.7 Hz, 3H), 1.25−1.28 (m, 8H), 1.47−1.53 (m, 2H), 3.23−3.29 (m,
2H), 5.79 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 9.11 (t, J = 5.7
Hz, 1H), 11.71 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 13.75,
21.72, 25.93, 28.10, 28.85, 30.98, 40.41, 102.90, 139.00, 150.43,
151.91, 163.19. MS (ESI) m/z: 254 [M − H]+, 271 [M − NH4]+. MS
(ESI) m/z: 252 [M − H]−.
h. The mixture was poured into water (50 mL). The product
precipitated, and it was filtered. The desired carboxamide was then
washed with cyclohexane (3 × 10 mL) and dried under reduced
pressure.
5-Ethyl-N-hexyl-2,4-dioxopyrimidine-1-carboxamide (4f).
The reaction was carried out following method B, dissolving 5-
ethyluracil (0.10 g, 0.71 mmol) in dry DMSO (4 mL) and employing
hexyl isocyanate (0.11 mL, 0.78 mmol). The crude was purified by
silica gel column chromatography (EtOAc/cyclohexane 90:10) to
1
afford compound 4f (0.03 g, 16%) as a white powder. H NMR (400
MHz, CDCl3) δ 0.88 (t, J = 6.3 Hz, 3H), 1.16 (t, J = 7.4 Hz, 3H),
1.21−1.50 (m, 6H), 1.54−1.68 (m, 2H), 2.42 (q, J = 7.4 Hz, 2H), 3.37
(td, J = 5.7, 7.0 Hz, 2H), 8.21 (s, 1H), 9.15 (br s, 1H), 9.54 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 12.67, 14.31, 20.19, 22.56, 26.58,
29.23, 31.43, 41.26, 117.92, 134.02, 150.12, 151.53, 163.31. MS (ESI)
m/z: 266 [M − H]−.
N-Hexyl-5-methoxy-2,4-dioxopyrimidine-1-carboxamide
(4h). The reaction was carried out following method B, dissolving 5-
methoxyuracil (0.05 g, 0.33 mmol) in dry DMSO (1.6 mL) and
employing hexyl isocyanate (0.09 mL, 0.59 mmol). The crude was
triturated with cyclohexane to afford compound 4h (0.05 g, 56%) as a
5-Fluoro-N-octyl-2,4-dioxopyrimidine-1-carboxamide (23a).
The reaction was carried out following method A, dissolving 5a (0.05
g, 0.38 mmol) in dry pyridine (3.8 mL) and employing DMAP (0.05 g,
0.42 mmol) and octyl isocyanate (0.08 mL, 0.46 mmol). The crude
was purified by silica gel column chromatography (cyclohexane/
EtOAc 80:20) to afford compound 23a (0.05 g, 46%) as a white
1
white powder. H NMR (400 MHz, DMSO-d6) δ 0.86 (t, J = 6.9 Hz,
3H), 1.26−1.31 (m, 6H), 1.47−1.54 (m, 2H), 3.27 (td, J = 5.7, 7.0 Hz,
2H), 3.66 (s, 3H), 7.71 (s, 1H), 9.22 (t, J = 5.6 Hz, 1H), 11.94 (s, 1H).
13C NMR (101 MHz, DMSO-d6) δ 13.86, 21.97, 25.87, 28.67, 30.83,
40.35, 56.49, 116.17, 136.52, 150.13, 150.24, 158.78. MS (ESI) m/z:
270 [M − H]+, 287 [M − NH4]+, 308 [M − K]+. MS (ESI) m/z: 268
[M − H]−.
1
powder. H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.1 Hz, 3H),
1.21−1.39 (m, 10H), 1.51−1.67 (m, 2H), 3.39 (td, J = 7.1, 5.5 Hz,
2H), 8.48 (d, J = 6.8 Hz, 1H), 8.93−9.05 (m, 1H), 9.10 (s, 1H). 13C
NMR (101 MHz, CDCl3) δ 14.20, 22.75, 26.94, 29.27, 31.89, 41.63,
123.40 (d, J = 37.3 Hz), 140.89 (d, J = 241.3 Hz), 149.13, 150.03, 156.
156.43. MS (ESI) m/z: 284 [M − H]−.
General Procedure for the Synthesis of 2,4-Dioxopyrimi-
dine-1-carboxamides 4g and 4o24 via Method C (Table 1). The
properly 5-substituted uracil (1.0 equiv) was dissolved in dry pyridine,
and hexyl isocyanate (1.1−1.5 equiv) was added. The reaction mixture
was heated under microwave irradiation at 100 °C for 10 min. The
solvent was evaporated under reduced pressure, and the crude
products were purified by silica gel column chromatography, eluting
with dichloromethane or dichloromethane/acetone mixture.
N-Hexyl-5-(hydroxymethyl)-2,4-dioxopyrimidine-1-carboxa-
mide (4g). The reaction was carried out following method C,
dissolving 5-hydroxymethyluracil (0.07 g, 0.49 mmol) in dry pyridine
(2.5 mL) and employing hexyl isocyanate (0.08 mL, 0.54 mmol).The
crude was purified by silica gel column chromatography (dichloro-
methane/acetone 60:40) to afford compound 4g (0.04 g, 27%) as a
white powder. 1H NMR (400 MHz, CDCl3) δ 0.90 (t, J = 6.9 Hz, 3H),
1.31−1.39 (m, 6H), 1.56−1.64 (m, 2H), 2.40 (t, J = 6.5 Hz, 1H), 3.39
(td, J = 5.6, 7.1, 2H), 4.48 (d, J = 6.6 Hz, 2H), 8.33 (s, 1H), 8.45 (s,
1H), 9.00 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 14.13, 22.66,
26.63, 29.27, 31.51, 41.47, 58.82, 115.26, 136.19, 149.74, 151.09,
162.49. MS (ESI) m/z: 292 [M − Na]+, 308 [M − K]+. MS (ESI) m/
z: 268 [M − H]−.
N-Octyl-2,4-dioxopyrimidine-1-carboxamide (23b). The re-
action was carried out following method A, dissolving 5b (0.08 g, 0.72
mmol) in dry pyridine (7.2 mL) and employing DMAP (0.09 g, 0.78
mmol) and octyl isocyanate (0.23 mL, 1.30 mmol). The crude was
triturated in a mixture of dichloromethane/MeOH/EtOAc to afford
compound 23b (0.14 g, 73%) as a white powder. 1H NMR (400 MHz,
DMSO-d6) δ 0.85 (t, J = 6.7 Hz, 3H), 1.24−1.31 (m, 10H), 1.49−1.52
(m, 2H), 3.23−3.28 (m, 2H), 5.79 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.3
Hz, 1H), 9.11 (t, J = 5.7 Hz, 1H), 11.71 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 13.91, 22.04, 26.19, 28.57, 28.63, 31.18, 40.33, 103.43,
138.73, 149.93, 151.49, 162.72. MS (ESI) m/z: 266 [M − H]−.
N-Octyl-2,4-dioxo-5-(trifluoromethyl)pyrimidine-1-carboxa-
mide (23e). The reaction was carried out following method A,
dissolving 5e (0.10 g, 0.55 mmol) in dry pyridine (5.5 mL) and
employing DMAP (0.08 g, 0.61 mmol) and octyl isocyanate (0.28 mL,
1.64 mmol). The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc from 70:30 to cyclohexane/
EtOAc 60:40) and then washed with n-heptane to afford compound
23e (0.04 g, 22%) as a white powder. 1H NMR (400 MHz, CDCl3) δ
0.88 (d, J = 6.8 Hz, 3H), 1.18−1.44 (m, 10H), 1.53−1.70 (m, 2H),
3.41 (td, J = 7.1, 5.5 Hz, 2H), 8.90−8.97 (m, 3H). 13C NMR (101
MHz, CDCl3) δ 14.20, 22.75, 26.92, 29.20, 29.26, 31.89, 41.80, 121.16
(q, J = 268.7 Hz), 139.73, 148.25, 150.83, 156.86. MS (ESI) m/z: 334
[M − H]−.
N-Nonyl-2,4-dioxopyrimidine-1-carboxamide (24b). The re-
action was carried out following method A, dissolving 5b (0.07 g, 0.62
mmol) in dry pyridine (3.1 mL) and employing DMAP (0.08 g, 0.69
mmol) and nonyl isocyanate (0.16 mL, 1.30 mmol). The crude was
triturated in dichloromethane to afford compound 24b (0.10 g, 57%)
as a white powder. 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.8 Hz,
3H), 1.26−1.32 (m, 12H), 1.57−1.61(m, 2H), 3.38 (td, J = 5.6, 7.2
Hz, 2H), 5.88 (dd, J = 2.0, 8.5 Hz, 1H), 8.37−8.39 (m, 1H), 8.42 (d, J
= 8.5 Hz, 1H), 9.03 (t, J = 5.8 Hz, 1H). 13C NMR (101 MHz, CDCl3)
δ 14.24, 22.80, 26.99, 29.32, 29.35, 29.59, 31.98, 41.44, 103.97, 139.07,
149.78, 151.46, 162.06. MS (ESI) m/z: 282 [MH]+. MS (ESI) m/z:
280 [M − H]−.
Synthesis of N-Hexyl-5-methylamino-2,4-dioxopyrimidine-
1-carboxamide (4p). Compound 4i (0.10 g, 0.28 mmol) was
dissolved in EtOAc (30 mL) and hydrogenated in an H-Cube
apparatus (using 10% Pd/C as catalyst, 1 bar hydrogen pressure, at 40
°C). The reaction mixture was concentrated under reduced pressure.
The crude was purified by silica gel column chromatography
(cyclohexane/EtOAc 85:15) to afford compound 4p (0.02 g, 27%)
1
as a white powder. H NMR (400 MHz, DMSO-d6) δ 0.78−0.99 (m,
3H), 1.17−1.36 (m, 6H), 1.45−1.55 (m, 2H), 2.57 (s, 3H), 3.27 (td, J
= 5.7, 6.9 Hz, 2H), 5.01 (s, 1H), 7.05 (s, 1H), 9.32 (t, J = 5.7 Hz, 1H),
11.87 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 13.85, 21.96, 25.89,
28.71, 29.70, 30.82, 40.21, 106.74, 126.49, 150.01, 150.68, 160.01. MS
(ESI) m/z: 267 [M − H]−.
Synthesis of N-Hexyl-2,4-dioxohexahydropyrimidine-1-car-
boxamide (7). The reaction was carried out following the procedure
described for compound 4p, dissolving 4b (0.03 g, 0.12 mmol) in
THF (7 mL) and using 10% Pd/C as catalyst and 40 bar hydrogen
pressure at 50 °C. The solvent was removed under reduced pressure to
afford compound 7 (0.03 g, quantitative) as a white powder. 1H NMR
(400 MHz, DMSO-d6) δ 0.86 (t, J = 6.6 Hz, 3H), 1.22−1.34 (m, 6H),
1.41−1.51 (m, 2H), 2.57 (t, J = 6.6 Hz, 2H), 3.18 (td, J = 5.6, 6.8 Hz,
General Procedure for the Synthesis of 2,4-Dioxopyrimi-
dine-1-carboxamides 4f and 4h via Method B (Table 1). The
properly 5-substituted uracil (1.0 equiv) was dissolved in dry DMSO,
and hexyl isocyanate (1.1−1.8 equiv) was added. The reaction mixture
was heated to 50 °C and stirred under nitrogen atmosphere for 4−12
J
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Journal of Medicinal Chemistry
Article
2H), 3.87 (t, J = 6.6 Hz, 2H), 8.71 (t, J = 5.3 Hz, 1H), 10.67 (s, 1H).
13C NMR (101 MHz, DMSO-d6) δ 13.84, 21.97, 25.94, 28.94, 30.35,
pyridine (4 mL). To the resulting solution, isobutyl chloroformate
(0.06 mL, 0.47 mmol) was added. The reaction was stirred at room
temperature under nitrogen for 3 h, and then the solvent was removed
under reduced pressure. The crude was purified by silica gel column
chromatography (cyclohexane/EtOAc 90:10) to afford compound 18a
(0.03 g, 22%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.86−
0.92 (m, 3H), 1.01 (d, J = 6.7 Hz, 6H), 1.20−1.42 (m, 6H), 1.55−1.65
(m, 2H), 2.11 (hept, J = 6.7 Hz, 1H), 3.31−3.52 (m, 2H), 4.26 (d, J =
6.7 Hz, 2H), 8.48 (d, J = 6.7 Hz, 1H), 8.83 (t, J = 5.5 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 14.12, 18.88, 22.64, 26.59, 27.81, 29.21,
31.48, 41.78, 76.84, 122.77 (d, J = 37.62 Hz), 140.42 (d, J = 243.18
Hz), 148.09, 148.36 (d, J = 50.64 Hz), 148.64, 154.33. MS (ESI) m/z:
358 [M − H]+, 375 [M − NH4]+, 396 [M − K]+.
30.85, 37.35, 39.85, 152.98, 153.77, 170.75. MS (ESI) m/z: 240 [M −
H]−.
Synthesis of 5-Fluoro-N-hexyl-3-(2-methylpropanoyl)-2,4-
dioxopyrimidine-1-carboxamide (15a). 5-Fluoro-N-hexyl-2,4-di-
oxopyrimidine-1-carboxamide (4a, 0.04 g, 0.15 mmol) was dissolved in
dry dichloromethane (0.8 mL). To the resulting solution, cooled to 0
°C, triethylamine (0.09 mL, 0.62 mmol) was added, followed by
isobutyryl chloride (0.05 mL, 0.50 mmol). The mixture was allowed to
warm to room temperature and stirred under nitrogen atmosphere for
45 min. Water was added and the aqueous phase extracted with
dichloromethane (3 × 15 mL). The combined organic layers were
washed with saturated NaHCO3 solution and brine, dried over
Na2SO4, and the solvent was removed under reduced pressure. The
crude was purified by silica gel column chromatography (cyclohexane/
EtOAc 85:15) to afford compound 15a (0.01 g, 20%) as a colorless oil.
1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.0 Hz, 3H), 1.24−1.40
(m, 6H), 1.31 (d, J = 7.0 Hz, 6H), 1.57−1.64 (m, 2H), 3.05 (hept, J =
7.0 Hz, 1H), 3.38 (td, J = 5.5, 7.2 Hz, 2H), 8.48 (d, J = 6.7 Hz, 1H),
8.86 (t, J = 5.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 14.11, 17.94,
22.63, 26.60, 29.21, 31.48, 40.02, 41.74, 122.78 (d, J = 37.4 Hz),
140.55 (d, J = 243.3 Hz), 148.92, 149.40, 155.64 (d, J = 28.5 Hz),
177.21. MS (ESI) m/z: 256 [M − COC(CH3)2]−.
3-Benzoyl-5-fluoro-N-hexyl-2,4-dioxopyrimidine-1-carboxa-
mide (19a). The reaction was carried out following the procedure
described for compound 18a, dissolving 4a (0.05 g, 0.20 mmol) in dry
pyridine (4 mL) and employing benzoyl chloride (0.03 mL, 0.25
mmol). The crude was purified by silica gel column chromatography
(cyclohexane/EtOAc 90:10) to afford compound 19a (0.03 g, 41%) as
a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H),
1.24−1.39 (m, 6H), 1.49−1.65 (m, 2H), 3.36 (td, J = 5.7, 7.0 Hz, 2H),
7.47−7.61 (m, 2H), 7.65−7.79 (m, 1H), 7.89−8.01 (m, 2H), 8.58 (d, J
= 6.7 Hz, 1H), 8.82 (t, J = 5.7 Hz, 1H). 13C NMR (101 MHz, CDCl3)
δ 13.82, 22.48, 26.55, 29.07, 31.41, 40.32, 41.74, 123.05 (d, J = 37.38
Hz), 130.32, 130.62, 135.89, 140.38 (d, J = 243.88 Hz), 148.65, 149.33
(d, J = 12.50 Hz), 155.46, 165.98. MS (ESI) m/z: 378 [M − Na]+.
Synthesis of N-Hexyl-N-methyl-2,4-dioxopyrimidine-1-car-
boxamide 27b. Sodium salt 29b (3.54 g, 26.38 mmol) was
suspended in dry acetonitrile (50 mL). Triphosgene (2.53 g, 7.91
mmol) was added, and the suspension was stirred at room temperature
for 20 min. N-Methylhexylamine (4.0 mL, 26.4 mmol) was added
dropwise, and the reaction mixture was stirred at room temperature for
12 h. The suspension was filtered, and the filtrate was evaporated
under reduced pressure. The crude was purified by silica gel column
chromatography (EtOAc/cyclohexane 90:10) to afford compound 27b
Synthesis of Methyl 5-Fluoro-3-(hexylcarbamoyl)-2,6-dioxo-
pyrimidine-1-carboxylate (16a). 5-Fluoro-N-hexyl-2,4-dioxopyri-
midine-1-carboxamide (4a, 0.05 g, 0.20 mmol) was dissolved in dry
dichloromethane (4 mL), and pyridine (0.04 mL, 0.43 mmol) was
added, followed by methyl chloroformate (0.02 mL, 0.23 mmol) at 0
°C. The mixture was stirred at room temperature for 18 h, and then
the solvent was removed under reduced pressure. The crude was
purified by silica gel column chromatography (cyclohexane/EtOAc
1
90:10) to afford compound 16a (0.02 g, 32%) as a colorless oil. H
NMR (400 MHz, CDCl3) δ 0.86−0.92 (m, 3H), 1.22−1.43 (m, 6H),
1.55−1.65 (m, 2H), 3.33−3.43 (m, 2H), 4.09 (s, 3H), 8.48 (d, J = 6.7
Hz, 1H), 8.79 (t, J = 5.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
14.33, 22.35, 26.52, 28.98, 31.50, 41.69, 56.85, 122.71 (d, J = 37.5 Hz),
140.17 (d, J = 242.8 Hz), 148.38, 148.46, 148.51, 153.88 (d, J = 29.3
Hz). MS (ESI) m/z: 316 [M − H]+, 333 [M − NH4]+.
1
(0.12 g, 2%) as a white powder. H NMR (400 MHz, DMSO-d6)
showed a 66:34 ratio of two rotamers with the following chemical
shifts: major rotamer, δ 0.87 (t, J = 6.7 Hz, 3H), 1.09−1.37 (m, 6H),
1.45−1.56 (m, 2H), 2.97 (s, 3H), 3.10−3.20 (m, 1H), 3.43−3.53 (m,
1H), 5.69 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 11.49 (t, J =
5.7 Hz, 1H); minor rotamer, δ 0.83 (t, J = 6.7 Hz, 3H), 1.09−1.37 (m,
6H), 1.45−1.56 (m, 2H), 2.87 (s, 3H), 3.23−3.32 (m, 2H), 5.70 (d, J
= 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 11.49 (t, J = 5.7 Hz, 1H). 13C
NMR (101 MHz, DMSO-d6) showed the presence of two rotamers
with the following chemical shifts: major rotamer, δ 13.85, 21.97,
25.49, 26.08, 30.91, 35.62, 48.88, 102.42, 141.49, 148.41, 151.33,
163.34; minor rotamer, δ 13.84, 21.87, 25.41, 27.05, 30.73, 34.32,
50.17, 102.38, 141.59, 148.78, 151.11, 163.27. MS (ESI) m/z: 252 [M
− H]−.
b. Pharmacology. Recombinant Acid Ceramidase Expres-
sion. Rat AC was cloned from a brain cDNA library using primers
based on the sequence obtained from the National Center for
Biotechnology Information (NCBI) database: 5′rAC (5′-GACCATG-
CTGGGCCGTAGT-3′) and 3′rAC (5′-CCAGCCTATACAAG-
GGTCT-3′). The PCR (High Fidelity PCR Master, Roche) product
was subcloned into a pEF6-V5/His vector (Invitrogen) to construct a
mammalian expression vector encoding V5/His-tagged rat AC. HEK
293 cells were transfected with pEF6-rAC-V5/His using Super-Fect
reagent (Qiagen) and screened with G418 (0.3 mg/mL).
Acid Ceramidase (AC) Activity. AC activity was analyzed as
previously described.25 Briefly, cells were suspended in 20 mM Tris-
HCl (pH 7.5) containing 0.32 M sucrose, sonicated, and centrifuged at
800g for 15 min at 4 °C. The supernatants were centrifuged again at
12000g for 30 min at 4 °C. The pellets were suspended in phosphate
buffered saline (PBS) and subjected to two freeze−thaw cycles at −80
°C. The suspensions were centrifuged at 105000g for 1 h at 4 °C. The
supernatants containing recombinant AC were kept at −80 °C until
use. Protein concentration was measured using the bicinchoninic acid
(BCA) assay (Pierce). Recombinant rat AC (50 μg) was preincubated
with inhibitors (final DMSO concentration 1%) in assay buffer (100
Ethyl 5-Fluoro-3-(hexylcarbamoyl)-2,6-dioxopyrimidine-1-
carboxylate (17a). The reaction was carried out following the
procedure described for compound 16a, dissolving 4a (0.05 g, 0.20
mmol) in dry dichloromethane (2.0 mL) and employing pyridine
(0.09 mL, 1.14 mmol) and ethyl chloroformate (0.05 mL, 0.57 mmol).
The crude was purified by silica gel column chromatography
(cyclohexane/EtOAc 80:20) to afford compound 17a (0.03 g, 46%)
1
as a colorless oil. H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.5 Hz,
3H), 1.23−1.41 (m, 6H), 1.44 (t, J = 7.1 Hz, 3H), 1.55−1.65 (m, 2H),
3.38 (td, J = 5.6, 7.1 Hz, 2H), 4.53 (q, J = 7.1 Hz, 2H), 8.48 (d, J = 6.8
Hz, 1H), 8.82 (t, J = 5.7 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
13.87, 14.13, 22.64, 26.60, 29.21, 31.49, 41.78, 67.17, 122.77 (d, J =
37.4 Hz), 140.38 (d, J = 242.7 Hz), 147.92, 148.57, 148.70, 155.16 (d,
J = 31.5 Hz). MS (ESI) m/z: 347 [M − NH4]+.
Methyl 5-Fluoro-3-(octylcarbamoyl)-2,6-dioxopyrimidine-1-
carboxylate (25a). The reaction was carried out following the
procedure described for compound 16a, dissolving 23a (0.20 g, 0.70
mmol) in dry dichloromethane (7.0 mL) and employing pyridine
(0.22 mL, 2.80 mmol) and methyl chloroformate (0.05 mL, 0.57
mmol). The crude was purified by silica gel column chromatography
(cyclohexane/EtOAc 85:15) to afford compound 25a (0.16 g, 66%) as
1
a colorless oil. H NMR (400 MHz, CDCl3) δ 0.74−1.00 (m, 3H),
1.11−1.43 (m, 11H), 1.47−1.69 (m, 2H), 1.55−1.65 (m, 2H), 3.37
(td, J = 5.6, 7.1 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 8.47 (d, J = 6.7 Hz,
1H), 8.78 (t, J = 5.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 14.17,
22.73, 26.88, 29.18, 29.23, 31.87, 41.76, 56.96, 122.85 (d, J = 37.5 Hz),
140.30 (d, J = 242.6 Hz), 148.52, 148.60, 148.65, 154.01 (d, J = 29.3
Hz). MS (ESI) m/z: 344 [M − H]+.
Synthesis of Isobutyl 5-Fluoro-3-(hexylcarbamoyl)-2,6-diox-
opyrimidine-1-carboxylate (18a). 5-Fluoro-N-hexyl-2,4-dioxopyr-
imidine-1-carboxamide (4a, 0.10 g, 0.39 mmol) was dissolved in dry
K
dx.doi.org/10.1021/jm301879g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mM sodium phosphate, 0.1% Nonidet P-40, 150 mM NaCl, 3 mM
DTT, 100 mM sodium citrate, pH 4.5) for 30 min at 37 °C. Reactions
were started by the addition of 100 μM N-lauroylceramide (Nu-Chek
Prep, Elysian, MN) and carried on for 30 min at 37 °C. Reactions were
stopped by addition of a mixture of chloroform/methanol (2:1, vol/
vol) containing 1 nmol of heptadecanoic acid (HDA, NuChek Prep).
The organic phases were collected, dried under nitrogen, and analyzed
by liquid chromatography/mass spectrometry in the negative-ion
mode monitoring the reaction product (lauric acid, m/z = 199) using
HDA as internal standard. Lipids were eluted on an XDB Eclipse C18
column isocratically at 2.2 mL·min−1 for 1 min with a solvent mixture
of 95% methanol and 5% water, both containing 0.25% acetic acid and
5 mM ammonium acetate. The column temperature was 50 °C.
Electrospray ionization was in the negative mode. Capillary voltage
was 4 kV, and fragmentor voltage was 100 V. Nitrogen was used as
drying gas at a flow rate of 13 L·min−1 and at a temperature of 350 °C.
Nebulizer pressure was set at 60 psi. We monitored [M − H]− in the
selected-ion monitoring mode. Calibration curves were generated with
authentic lauric acid (Nu Check Prep).
c. Computational Methods. Electronic structure calculations
were performed with Gaussian 09 software.26 All the calculations
adopted the B3LYP hybrid density functional, and a 6-311+g(d) basis
set was employed.27 All the optimizations were carried out in
polarizable conductor calculation model (CPCM), and the chosen
solvent was water.28 The structures were optimized with tight
convergence parameters and ultrafine grid. Finally, natural bond
orbital (NBO) analysis and second-order perturbation theory analysis
of Fock matrix in NBO basis were carried out to better understand the
effect of the local changes in the electronic structure induced by
different substituents.21 This last part was carried out through the
NBO 3.1 software included in the Gaussian package. All the structures
were prepared using Gaussview 5.0.26 In the alkyl side chain,
methylene units beyond the first were omitted.
ABBREVIATIONS USED
■
AC, acid ceramidase; OEA, oleoylethanolamide; 5-FU, 5-
fluorouracil; DMAP, 4-dimethylaminopyridine; DMSO, di-
methyl sulfoxide; BSA, bis(trimethylsilyl)acetamide; TFA,
trifluoroacetic acid; BOC, tert-butyloxycarbonyl; NBO, natural
bond orbital; PLS, partial least squares; HDA, heptadecanoic
acid
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures, analytical and spectroscop-
ical data of intermediates and final compounds, analytical
stability data, additional computational details on the NBO and
PLS studies, and MTT cell viability assay conditions. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For D.P.: e-mail, piomelli@uci.edu. For T.B.: phone, +39-010-
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71781533; fax, +39-010-71781228; e-mail, tiziano.bandiera@iit.
it.
Author Contributions
‡D.P. and C.P. contributed equally to design and perform
research.
Notes
The authors declare the following competing financial interests.
Two patent applications protecting the class of compounds
disclosed in this paper were filed by the following authors:
Piomelli, D.; Realini, N.; Mor, M.; Pagliuca, C.; Pizzirani, D.;
Scarpelli, R.; Bandiera, T.
ACKNOWLEDGMENTS
■
The authors thank Giuliana Ottonello for analytical stability
data, Sine Mandrup Bertozzi for reverse phase HPLC
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Silvia Venzano for handling of compounds.
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M
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