Benzoxazolone carboxamides: Potent and systemically active inhibitors of intracellular acid ceramidase

Article abstract of DOI:10.1002/anie.201409042

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences surv

Full text of DOI:10.1002/anie.201409042

Angewandte
Chemie
DOI: 10.1002/anie.201409042
Enzyme Inhibition Hot Paper
Benzoxazolone Carboxamides: Potent and Systemically Active
Inhibitors of Intracellular Acid Ceramidase**
Daniela Pizzirani, Anders Bach, Natalia Realini, Andrea Armirotti, Luisa Mengatto, Inga Bauer,
Stefania Girotto, Chiara Pagliuca, Marco De Vivo, Maria Summa, Alison Ribeiro, and
Daniele Piomelli*
[
4]
Abstract: The ceramides are a family of bioactive lipid-derived
messengers involved in the control of cellular senescence,
inflammation, and apoptosis. Ceramide hydrolysis by acid
ceramidase (AC) stops the biological activity of these sub-
stances and influences survival and function of normal and
neoplastic cells. Because of its central role in the ceramide
metabolism, AC may offer a novel molecular target in
disorders with dysfunctional ceramide-mediated signaling.
Here, a class of benzoxazolone carboxamides is identified as
the first potent and systemically active inhibitors of AC.
Prototype members of this class inhibit AC with low nano-
molar potency by covalent binding to the catalytic cysteine.
Their metabolic stability and high in vivo efficacy suggest that
these compounds may be used as probes to investigate the roles
of ceramide in health and disease, and that this scaffold may
represent a promising starting point for the development of
novel therapeutic agents.
metabolism and have attracted considerable attention
because of their proposed participation in cellular senes-
[
5]
[6]
[7]
cence, inflammation, and apoptosis. Additionally, the
ceramides are metabolic precursors of sphingosine-1-phos-
phate (S1P), a lipid mediator that enhances cell survival and
proliferation by activating selective G protein-coupled recep-
tors. The pharmacology of sphingolipid signaling is still
nascent, yet small-molecule modulators of ceramide produc-
tion and degradation might open new avenues of therapeutic
intervention in pathological conditions, including cancer,
[
8]
[
9]
inflammation, and pain.
AC is a lysosomal cysteine amidase that catalyzes the
hydrolysis of ceramide into sphingosine and fatty acid
(Figure 1). As this reaction is a crucial step in ceramide
T
he sphingolipids are a class of bioactive lipid molecules that
[
1]
serve multiple regulatory functions. They contribute to key
[2]
cellular processes and are involved in the pathogenesis of
Figure 1. A simplified overview of the biosynthetic pathway leading
from ceramide to sphingosine-1-phosphate. AC: acid ceramidase. SK:
sphingosine kinase.
[
3]
inflammation and neuropathic pain. The ceramides, a highly
heterogeneous family of N-acylated sphingosines with long-
chain fatty acids, hold a central position in sphingolipid
[
+]
[+]
[10]
[
*] Dr. D. Pizzirani, Dr. A. Bach, Dr. N. Realini, Dr. A. Armirotti,
degradation and S1P biosynthesis,
AC inhibition can
[
$]
Dr. L. Mengatto, Dr. I. Bauer, Dr. S. Girotto, Dr. C. Pagliuca,
impact the balance between cell proliferation and death,
influencing both growth and survival of normal and neoplastic
cells.
Although several AC inhibitors have been reported, the
majority of these compounds are structural analogues of
ceramide and are limited in their use by low inhibitory
potency and/or lack of systemic activity (Figure 2, and
Table S1 in the Supporting Information).
We previously identified the antineoplastic drug carmofur
(5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1-carboxamide, 8;
Figure 3) as the first nanomolar inhibitor of AC (median
inhibitory concentration, IC , for rat AC = 29 nm).
result prompted us to expand the class of 2,4-dioxopyrimi-
dine-1-carboxamide derivatives and led us to discover the first
single-digit nanomolar inhibitors of AC activity.
tantly, selected compounds in this series sensitize certain
types of cancer cells to the actions of cytotoxic agents, such as
-fluorouracil and taxol, which is suggestive of a potential
clinical use as chemosensitizers. Despite their considerable
potency, the utility of these agents is hindered by low chemical
and metabolic stability. Even the best compounds in this class
have extremely short half-life times in mouse plasma (t1/2 = 1–
Dr. M. De Vivo, Dr. M. Summa, Dr. A. Ribeiro, Prof. D. Piomelli
Drug Discovery and Development, Istituto Italiano di Tecnologia
Via Morego 30, 16163 Genova (Italy)
E-mail: piomelli@uci.edu
Prof. D. Piomelli
Departments of Anatomy and Neurobiology, Pharmacology and
Biological Chemistry, University of California Irvine
Irvine, CA 92697 (USA)
[
11,12]
$
[
] Current address: Janssen Pharmaceutica
Turnhoutseweg 30, 2340 Beerse (Belgium)
+
[
] These authors equally contributed to this work.
[14]
[
**] Dr. A. Bach was supported by a Carlsberg Foundation fellowship.
The authors thank Dr. Oscar Sasso for help with in vivo experiments,
Dr. Elisa Romeo for technical support in molecular biology, and Dr.
Silvia Venzano for handling of compounds.
5
0
This
[15]
Impor-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409042.
ꢀ
2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
5
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
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Figure 4. Kinetic analysis of the AC reaction in the presence of vehicle
DMSO 1%, n=6) or 9a (20 nm or 50 nm, n=6). Vmax (vehi-
cle)=10793 RFU; Vmax (9a, 20 nm)=7808 RFU; Vmax (9a,
0 nm)=3889 RFU. Km (vehicle)=6.8 mm; Km (9a, 20 nm)=6.8 mm; Km
9a, 50 nm)=7.2 mm. RFU=relative fluorescence units. Data are
(
5
(
expressed as mean of two independent experiments.
Figure 2. Structures of a representative ceramide species (d18:1/16:0)
[
13]
and various AC inhibitors (1–7). OEA: oleoylethanolamide.
Figure 3. Initial hits used for the discovery of nanomolar AC inhibitors.
[
14]
3
min), a limitation that extensive structure–stability stud-
[15]
ies have been as yet unable to overcome. Thus, the field still
needs suitable small molecules to foster probe and drug
discovery efforts.
Here, we outline the discovery of the first class of potent
AC inhibitors endowed with pronounced systemic activity.
Elucidation of the mechanism through which these molecules
inhibit AC along with in vivo pharmacokinetic and pharmo-
codynamic studies support the potential of this chemotype for
the development of agents that target AC.
During the screening on human AC of a small library of
compounds generated in our laboratory, we identified 2-oxo-
N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (9a) as
a nanomolar inhibitor of this enzyme (IC50 for human AC =
Figure 5. Compound 9a inhibits AC by covalent modification of the
catalytic Cys-143. Possible covalent adducts formed upon nucleophilic
attack of AC catalytic Cys-143 on 9a (A). Extracted-ion chromatograms
of doubly-charged native peptide from a control incubation with
DMSO (m/z: 483.23, black trace) and the same peptide covalently
modified by 9a (m/z: 570.79, red trace) (B). Tandem mass spectrum
of the covalently modified peptide, confirming peptide sequence
6
4 nm; Figure 3). Kinetic studies using human recombinant
AC showed that 9a causes a concentration-dependent
reduction in maximal catalytic velocity (Vmax) without affect-
ing the Michaelis–Menten constant (K ; Figure 4).
m
This result, which is suggestive of a noncompetitive
mechanism of action, was extended by liquid chromatography
mass spectrometry (LC-MS) experiments, which showed that
(CTSIVAEDK) and mass increase of Cys-143 (+175.10 Da); mass
increase and peptide sequence are consistent with the formation of
9
a covalently binds to the catalytic cysteine, Cys-143, of the
adduct A (+C H NO) (C).
1
1
13
[
16]
enzyme. Because 9a contains two electrophilic carbonyl
groups, it may form distinct adducts with AC. As illustrated in
Figure 5A, the nucleophilic attack of Cys-143 on the carbonyl
group of urea would result in adducts A or B, whereas attack
on the carbonyl group of the carbamate would result in
adducts C or D. Incubation of purified recombinant human
AC with 9a, followed by trypsin digestion and peptide
analysis by LC-MS, showed the presence of a covalent
adduct of 9a with the N-terminal peptide of AC (CTSI-
VAEDK, Figure 5B, red trace). Control incubations of AC
with DMSO showed only the native unmodified peptide
(Figure 5B, black trace). Tandem MS analysis showed the
formation of adduct A and indicated that the mass increase,
corresponding to the carboxamide side chain of 9a, is carried
by the N-terminal cysteine, confirming that this residue is the
target of covalent binding (Figure 5C). We found no evidence
supporting alternative hypotheses and no adducts of 9a with
other AC tryptic peptides. These results indicate that 9a
2
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Chemie
inhibits human AC through S-acylation of catalytic Cys-143,
with the benzoxazolone ring acting as the leaving group.
Encouraged by the potency and drug-likeness of 9a, which
is based on the privileged scaffold of benzoxazolone, we
sought to expand this chemical class and identify structural
features that are critical for AC inhibition. Focused structure–
activity relationship (SAR) studies were first aimed at
exploring the role of the urea moiety of 9a, with a small set
of compounds (9b–e) that were prepared as described in
Scheme 1.
entries 1–5). We reasoned then that we could modulate the
reactivity of the 3-carboxamide by introducing substituents
with different electronic properties on the heterocyclic
scaffold. To test this hypothesis, we prepared compounds
16a and 17a, which bear a bromine and a p-fluorophenyl
group at position 6, respectively. Compound 16a was obtained
by coupling the commercial 6-bromobenzoxazolone 18 to
4-phenylbutyl isocyanate 11, while 17a was accessed by a two-
step synthesis involving a Suzuki–Miyaura reaction to give 19,
followed by N3 acylation (Scheme 2).
[
17]
Scheme 2. Syntheses of substituted benzoxazolone derivatives 16a
and 17a.
The introduction of a bromine in the para position to the
urea moiety, as in compound 16a, led to a two-fold increase in
potency relative to 9a (16a, IC = 33 nm), while replacement
Scheme 1. Syntheses of benzoxazolone derivatives 9a–e.
5
0
of bromine with p-fluorophenyl had no significant effect (17a,
IC = 79 nm; Table 1, entries 6–7).
Replacement of the carbonylic oxygen atom of the urea
5
0
moiety of 9a with a sulfur atom gave compound 9b. This
We expected that modulating the reactivity of these
derivative showed limited chemical stability in [D ]DMSO,
compounds would impact their stability. Therefore, we tested
derivatives 9a, 16a, and 17a for their stability in buffers at
acidic and neutral pH values, as well as in mouse plasma
(Table 2). All compounds showed adequate stability under
6
and was not studied further. The role of the 3-carboxamide
NH group was then investigated by preparing the N-methyl
derivative 9c and the corresponding carbamate 9d. The two
compounds were efficiently accessed by activating the
benzoxazolone 10 with triphosgene, followed by in situ treat-
ment with the appropriate commercial amine 13 or alcohol 14,
respectively. Both compounds 9c and 9d had no inhibitory
activity on AC. Loss of activity was also observed when the
urea moiety was replaced with an amide, as in compound 9e.
These results demonstrate that the 3-carboxamide NH moiety
is a key structural feature for AC inhibition (Table 1,
Table 2: Stability of compounds 9a and 16a–17a by LC-MS analysis.
Entry
Compound
Buffer
Buffer
stability
(pH 7.4)
m-Plasma
stability
[
a]
[b]
[c]
stability
pH 4.5)
(
t1/2 [min]
t1/2 [min]
t1/2 [min]
1
2
3
9a
16a
17a
126Æ3
294Æ30
>360
45
24
>300
60
8
>120
Table 1: Inhibitory potencies (IC ) of compound 9a, analogues 9b–9e,
and 16a-17a on human AC.
5
0
[
a] NaCl (150 mm), NaH PO (100 mm), trisodium citrate (100 mm),
2 4
NP40 (1%), DTT (3 mm). [b] PBS. [c] Mouse plasma, 378C.
standard assay conditions (pH 4.5). When tested in phos-
phate-buffered saline (PBS, pH 7.4), the introduction of
bromine in the para position to the urea moiety, as in
compound 16a, led to a decrease of stability compared to 9a
1
[a]
Entry
Compound
R
X
Y
IC50 [nm] ÆS.E.M
64Æ7
(16a, t1/2 = 24 min), whereas replacement of bromine with a
1
2
3
4
5
6
7
9a
9b
9c
9d
9e
16a
17a
H
H
H
H
H
Br
O
S
NH
NH
NCH3
O
CH2
NH
NH
p-fluorophenyl group promoted stability (17a, t1/2 > 300 min).
As expected, an electron-withdrawing group increased the
electrophilicity of the carbonyl group of urea and made the
resulting benzoxazolone a better leaving group upon nucle-
ophilic attack, accounting for the lower stability observed in
neutral buffer. Interestingly, the highly conjugated system
resulting from the introduction of the phenyl ring, as in
not stable
no inhibition
no inhibition
no inhibition
31Æ9
O
O
O
O
O
p-F-Phe
79Æ31
[
a] IC50 values are expressed as means of at least three determinations.
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Communications
compound 17a, stabilizes the benzoxazolone 3-carboxamide
scaffold and, at the same time, appears to be well tolerated in
terms of AC inhibitory potency. Stability experiments in
mouse plasma showed that 17a has a substantially longer
plasma half-life than does 9a (17a, t1/2 > 120 min) and is
considerably more stable than the corresponding bromine
derivative 16a (Table 2).
Furthermore, metabolic stability studies in mouse liver
microsomes showed that 89% of 17a was recovered after an
incubation time of one hour. Lastly, compound 17a was tested
for off-target effects on a set of enzymes that includes
proteases (aspartic, cysteine, and serine), lipoxygenases,
cyclooxygenases, group IV phospholipase (sPLA ), and mon-
2
oacylglycerol lipase. The compound showed no significant
activity toward these targets, with the exception of a weak
inhibitory effect on the aspartic protease cathepsin D (67%
inhibition at 10 mm; Table S2, Supporting Information).
The favorable profile of 17a prompted us to test its ability
to inhibit AC in intact cells. Human colon adenocarcinoma
SW403 cells and mouse macrophage-like Raw 264.7 cells
were incubated in the presence of 17a (0.1–20 mm). AC
activity and sphingolipid levels were measured after various
incubation times. The compound inhibited cellular AC
activity with an IC50 of 825 nm in SW403 and 400 nm in Raw
Figure 7. Time-course of the effects of 17a (20 mm) in SW403 (A, C)
and Raw 264.7 cells (B, D) on AC activity (A, B) and sphingolipid
levels (C, D). Values are expressed as means ÆS.E.M of at least three
determinations. Experiments were repeated twice with similar results.
2
64.7 cells (Figure 6A,B). Consistent with these results,
Figure 8. In vivo profile of 17a. Plasma pharmacokinetic profile of 17a
À1
À1
after i.p. (10 mgkg ) and i.v. (1 mgkg ) administration in mice (A).
Identification of 19 as primary in vivo metabolite of 17a: super-
imposed MRM traces of a standard sample of 17a (retention time
3
.91 min, 1 mm calibrator, red trace) and a sample collected 1 h after
À1
i.p. administration of 17a in mice (10 mgkg ; black trace) (B). The
peak at 1.4 min corresponds to the primary metabolite of 17a (19,
Figure 6. Effects of compound 17a in SW403 (A, C) and Raw 264.7
cells (B, D), after a 3 h incubation. Concentration dependence of the
effects on AC activity (A, B) and sphingolipid levels (C, D). Values are
expressed as means ÆS.E.M of at least three determinations. Experi-
ments were repeated twice with similar results.
2
27 Da molecular mass, m/z: 228 detected in ESI mode). Effects of
À1
17a (10 mgkg , 3 h) on AC activity in mouse tissues (C) and
sphingolipid levels in lungs (D). Values are expressed as means
ÆS.E.M (n=6).
incubation with 17a resulted in an increase in the levels of
ceramide (d18:1/16:0) and a corresponding decrease in the
levels of sphingosine. The levels of dihydroceramide (d18:0/
Pharmacokinetic analyses showed that 17a quickly enters
the bloodstream after a single intraperitoneal (i.p. 10 mgkg )
À1
administration in mice (Figure 8A), reaching a maximal
[1b]
À1
1
6:0), which is cleaved by AC to sphinganine, were also
plasma concentration, Cmax, of 1767.9 ngmL and displaying
increased (Figure 6C,D).
a half-life time of 458 min in circulation. Relevant pharma-
cokinetic parameters are reported in Table S3 (Supporting
Information). The primary in vivo metabolite of 17a, the
hydrolysis product 19 (Figure 8B), did not inhibit AC in vitro
at 10 mmmm.
The effects of 17a persisted for 6 h, with a partial recovery
of enzyme activity and consequent decrease in sphingolipid
levels observed after 24 h (Figure 7). The results indicate that
1
7a inhibits AC in a complex cellular environment, leading to
À1
the intended biochemical response, that is, increased ceram-
ide and decreased sphingosine levels.
Injection of 17a in mice (10 mgkg , i.p.) caused a sub-
stantial reduction in AC activity in multiple organs, including
4
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brain, liver, heart, lungs, and kidney (Figure 8C). Highest
levels of baseline AC activity and AC inhibition were found in
lung tissue, which was selected for further analyses. As
expected, we observed a significant increase in ceramide
species that are preferred substrates for AC (namely, d18:1/
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with longer fatty acyl chains, which are not preferred
substrates for AC, were not affected (Table S4, Supporting
Information). Compound 17a appeared to be well tolerated
at the dosage tested. These findings show that 17a inhibits AC
in live animals, resulting in the predicted alteration of the
balance between ceramide and sphingosine.
In conclusion, we identified the first class of potent and
systemically active inhibitors of intracellular AC activity.
These compounds act as covalent inhibitors of AC. Focused
SAR studies showed that an unsubstituted nitrogen atom in
the 3-carboxamide moiety is mandatory for activity, and
highlighted that the introduction of a p-fluorophenyl group on
the benzoxazolone ring balanced potency and stability (both
chemical and metabolic), as shown with compound 17a.
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inhibit AC in various cell lines as well as in vivo. Thus, our
results identify compound 17a as the first potent chemical
probe that may be used to investigate the physiopathological
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novel therapeutic agents. Further expansion of this chemical
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Keywords: acid ceramidase · cancer · ceramide ·
enzyme inhibition · sphingosine-1-phosphate
.
inhibitory activity data for compounds 1–7 and assay methods.
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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Angewandte
Communications
Communications
Enzyme Inhibition
A key regulator of the sphingolipid
metabolism is acid ceramidase (AC),
which offers a molecular target in disor-
ders such as cancer and inflammation, in
which sphingolipid signaling might be
dysfunctional. The shown compound is
metabolically stable, inhibits AC activity
both in vitro and in vivo, and can thus be
used as a potent chemical probe to
investigate the roles of ceramides in
physiology and pathology.
D. Pizzirani, A. Bach, N. Realini,
A. Armirotti, L. Mengatto, I. Bauer,
S. Girotto, C. Pagliuca, M. De Vivo,
M. Summa, A. Ribeiro,
D. Piomelli*
&&&& — &&&&
Benzoxazolone Carboxamides: Potent
and Systemically Active Inhibitors of
Intracellular Acid Ceramidase
6
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ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!