Pyrazole-Based Acid Ceramidase Inhibitors: Design, Synthesis, and Structure-Activity Relationships

Article abstract of DOI:10.1055/s-0035-1561456

Acid ceramidase (AC) is a lysosomal cysteine amidase responsible for the cleavage of ceramide into sphingosine, which is then phosphorylated to sphingosine 1-phosphate. AC regulates the intracellular levels of ceramide and sphingosine, and AC inhibition may be useful in the treatment of disorders, such as cancer, in which ceramide-mediated signaling may be dysfunctional. Despite their potential experimental and therapeutic value, the number of available small-molecule inhibitors of AC activity remains limited. In the present study is described the discovery of a class of potent pyrazole carboxamide-based AC inhibitors, which were identified using the atomic property field (APF) approach and developed through systematic SAR investigations and in vitro pharmacological characterization. The best compound of this series inhibits AC with nanomolar potency and causes ceramide accumulation and sphingosine depletion in intact G361 proliferative melanoma cells. By expanding the current armamentarium of AC inhibitors, these results should facilitate future efforts to unravel the biology of AC and the therapeutic potential of its inhibition.

Full text of DOI:10.1055/s-0035-1561456

SYNTHESIS
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©
Georg Thieme Verlag Stuttgart · New York
2016, 48, A–R
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feature
Synthesis
E. Diamanti et al.
Feature
Pyrazole-Based Acid Ceramidase Inhibitors: Design, Synthesis,
and Structure–Activity Relationships
Eleonora Diamantia
Giovanni Bottegonia
Luca Goldonia
Natalia Realinia
Chiara Pagliucaa
Fabio Bertozzia
Daniele Piomelli*a,b
Daniela Pizzirania
a
Drug Discovery and Development, Fondazione Istituto
Italiano di Tecnologia, via Morego 30, 16163 Genova,
Italy
Departments of Anatomy and Neurobiology, Pharma-
b
cology and Biological Chemistry, University of California,
Irvine, CA 92697, USA
daniele.piomelli@iit.it
Dedicated to Professor Stuart L. Schreiber on the occasion of his 60th birthday
Received: 24.02.2016
Accepted after revision: 18.04.2016
Published online: 09.06.2016
The sphingolipids are ubiquitous lipid constituents of
eukaryotic cell membranes.1 In addition to this essential
DOI: 10.1055/s-0035-1561456; Art ID: ss-2016-z0135-fa
structural function, sphingolipids are also thought to play
multiple signaling roles and to be involved in the patho-
Abstract Acid ceramidase (AC) is a lysosomal cysteine amidase re-
sponsible for the cleavage of ceramide into sphingosine, which is then
phosphorylated to sphingosine 1-phosphate. AC regulates the intracel-
lular levels of ceramide and sphingosine, and AC inhibition may be use-
ful in the treatment of disorders, such as cancer, in which ceramide-
mediated signaling may be dysfunctional. Despite their potential exper-
imental and therapeutic value, the number of available small-molecule
inhibitors of AC activity remains limited. In the present study is de-
scribed the discovery of a class of potent pyrazole carboxamide-based
AC inhibitors, which were identified using the atomic property field
2
3
4
genesis of cancer, inflammation, and neuropathic pain. In
particular, two key sphingolipids – ceramides and sphin-
gosine 1-phosphate (S1P) – have been shown to regulate
cell fate in opposite manner: ceramides by mediating cellu-
5
6
lar senescence and apoptosis, and S1P by promoting cell
survival and proliferation.7
The ceramides are a highly heterogeneous family of N-
8
acylated sphingosines with long-chain fatty acids. They
(
APF) approach and developed through systematic SAR investigations
hold a central role in sphingolipid metabolism and are the
metabolic precursors of S1P; conversion of one into the
other has been proposed to serve as a ‘rheostat’ in the regu-
and in vitro pharmacological characterization. The best compound of
this series inhibits AC with nanomolar potency and causes ceramide ac-
cumulation and sphingosine depletion in intact G361 proliferative mel-
anoma cells. By expanding the current armamentarium of AC inhibi-
tors, these results should facilitate future efforts to unravel the biology
of AC and the therapeutic potential of its inhibition.
9
lation of cell fate. The lysosomal lipid amidase, acid cera-
midase (AC, encoded by the ASAH-1 gene) is a key element
of this rheostat. It catalyzes the hydrolysis of ceramides into
fatty acid and sphingosine, which is then converted into
S1P by sphingosine kinase (SK) (Scheme 1).10
Key words acid ceramidase, ceramides, atomic property field (APF),
regioisomers, structure–activity relationship (SAR), melanoma
O
NH2
NH2
O
HN
14
AC
SK
HO
O
HO
P
HO
12
1
2
1
2
CerS
SPPase
OH
OH
OH
OH
ceramide
sphingosine
sphingosine-1-phosphate
S1P)
(
Scheme 1 A simplified overview of the sphingolipid rheostat. Representative ceramide species (d18:1/16:0). AC: acid ceramidase; CerS: ceramide
synthase; SK: sphingosine kinase; SPPase: sphingosine phosphate phosphatase.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

B
Synthesis
E. Diamanti et al.
Feature
Biographical Sketches
(
Authors shown clockwise from top left)
organic chemistry from Florence University,
where she worked on the synthesis and de-
velopment of new molecules for targeted
tumor diagnosis and therapy. She has most
recently completed a series of post-doc as-
signments leading drug development proj-
ects for international research institutions,
including the Italian Institute of Technology.
Her scientific investigation has ranged from
hit identification, hit-to-lead, and lead opti-
mization programs to the preclinical devel-
opment of new molecular entities. She has
co-authored 16 publications and holds 4
patent applications. Dr. Pagliuca is currently
working as a Scientist at a global player of
the chemical industry.
tute in La Jolla, California (USA), and then he
joined the Istituto Italiano di Tecnologia
(Genova, Italy) first as a post-doc and later
as a team leader. He is the author of over 40
publications and co-inventor in several pat-
ents. In 2015, he was awarded the DCF prize
for medicinal chemistry by the medicinal
chemistry division of the Italian chemical so-
ciety.
Daniela Pizzirani is Scientist in the Medici-
nal Chemistry Lead Optimization Team at
Chiesi Farmaceutici. After receiving her un-
dergraduate degree in Pharmaceutical
Chemistry and Technology, she performed
her graduate studies in medicinal chemistry
at the University of Bologna. Following com-
pletion of her doctoral studies, she joined
the group of Professor Stuart L. Schreiber at
Harvard University as a postdoctoral associ-
ate in 2007. In 2010, she moved to the Ital-
ian Institute of Technology (IIT), working as
Medicinal Chemist in the group of Professor
Daniele Piomelli.
Dr. Luca Goldoni received his Ph.D. in drug
discovery from the University of Genova
(
Italy) in 2016, focused on metabolomics by
NMR spectroscopy, under the guidance of
Professor Piomelli. He gained 12 years of ex-
perience as an NMR technician in several
chemical and pharmaceutical companies. In
2011 he joined the Italian Institute of Tech-
nology (IIT) as NMR senior specialist where
he is currently responsible for the structure
elucidation of new chemical entities, and
their purity evaluation. From 2013 his re-
search interest also included metabolomics
by NMR spectroscopy.
Daniele Piomelli is the holder of Louise
Turner Arnold Chair in Neurosciences and
Professor of Anatomy and Neurobiology,
Pharmacology and Biological Chemistry at
the University of California, Irvine. In addi-
tion, he is the founding director of the unit
of drug discovery and development (D3) at
the Italian Institute of Technology in Genoa,
Italy.
Fabio Bertozzi received his Ph.D. in organ-
ic chemistry from the University of Pisa, Italy
in 2002. He then joined ACADIA Pharmaceu-
ticals (Denmark) for a post-doctoral fellow-
ship aiming at the development of novel
methodologies for the synthesis of drug-like
structures. After more than five years at
Acadia Pharmaceuticals as medicinal chem-
ist working on several projects at the hit
confirmation/lead optimization stage in the
field of CNS related diseases, Dr. Bertozzi
joined the Istituto Italiano di Tecnologia
Dr. Natalia Realini got her Master’s de-
gree in biology applied to medical science
and her Ph.D. in medical pharmacology, tox-
icology and chemotherapy. In 2010, she
joined Professor Piomelli’s group at the Uni-
versity of California, Irvine and she’s current-
ly a senior postdoctoral associate at the
Italian Institute of Technology, Genova, Italy.
Her research interests include sphingolipid
biology, mainly focused on the biological
role of acid ceramidase in health and diseas-
es.
Eleonora Diamanti received her Master’s
degree in medicinal chemistry at the Univer-
sity of Camerino in 2010. She joined in 2012
Professor Piomelli’s group at the Italian Insti-
tute of Technology (IIT, Genova, Italy) for her
Ph.D. During her doctoral course she spent
(
Genova, Italy) in late 2009 to coordinate
the medicinal chemistry efforts on different
drug discovery projects.
1
0 months at the University of Bristol under
the supervision of Professor V. K. Aggarwal.
She is currently a post doc at the IIT.
Giovanni Bottegoni received his Ph.D. in
pharmaceutical sciences from the University
of Bologna, Italy in 2006. For two years, he
was post-doc at The Scripps Research Insti-
Dr. Chiara Pagliuca earned her Ph.D. in
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

C
Synthesis
E. Diamanti et al.
Feature
In the literature
OH
OH
OH
OH
7
NH
HN
HN
O2N
7
12
12
O
O
O
OEA (1)
D-e-MAPP (2)
B-13 (3)
H
OH
OH
N
O
O
OH
1
4
R1
HN
HN
Br
1
2
O
O
O
1
DP24a (4)
SABRAC (5)
Ceranib-1 (6): R = Me
1
Ceranib-2 (7): R = H
Our previous work
H
O
F
N
O
O
O
H
F
N
N
N
N
H
O
O
carmofur (8)
ARN14974 (9)
Figure 1 Structures of representative AC inhibitors reported in the literature (top) and, specifically, those previously disclosed by our group (bottom)
Since inhibition of AC increases the cellular levels of cer-
amides and concomitantly decreases those of sphingosine
and S1P, it represents a potential strategy to address disor-
ders associated with abnormal levels of these bioactive lip-
ids – including certain types of cancer and inflammation.
A relatively small number of AC inhibitors have been re-
Previous work in our laboratory has identified two new
classes of potent small-molecule inhibitors of AC (Figure 1).
The first includes substituted 2,4-dioxopyrimidine-N1-car-
boxamides such as carmofur (8), which inhibits AC activity
with nanomolar potency.18
Members of this class act synergistically with standard
anti-neoplastic drugs to stop proliferation of various types
of cancer cells, suggesting a potential clinical use as chemo-
sensitizers.18 Recently, we have shown that N3-carbamoyl
uracil derivatives may be useful in normalizing ceramide
levels and concomitantly sensitize proliferative melanoma
cells to the cytotoxic actions of various anti-tumoral agents.
These results are suggestive of the potential utility of AC
blockade as chemosensitizing strategy in proliferative mel-
11
ported in the scientific and patent literature. Ceramide
12
analogues such as oleoylethanolamide (OEA, 1, K ≈ 500 μM),
i
D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-e-
13
MAPP, 2, IC50 >500 μM), and B-13 (3, IC50 = 10 μM), are not
drug-like and display low inhibitory potencies (Figure 1).
Structural modifications of these molecules have led to
the discovery of significantly more potent compounds, such
1
4
as DP24a (4) (IC = 1.3 μM on human recombinant AC)
50
15
19
and SABRAC (5), which inhibit AC activity in intact cells
and cell lysates with nanomolar potency.
anoma. Another class of AC inhibitors identified by our
laboratory includes benzoxazolone-carboxamide deriva-
tives, which display improved chemical and metabolic sta-
Among the AC inhibitors that are not structurally relat-
ed to ceramide, Ceranib-1 and 2 (6,7)1 show micromolar
potency towards intracellular ceramidases, inhibit cell pro-
liferation and induce cell death in certain types of cancer,
alone or in combination with paclitaxel. In addition, cysta-
tin SA (encoded in humans by the CST2 gene) is a physiolog-
6
20
bility, compared to carmofur and its congeners. Focused
structure-activity relationship (SAR) studies around the
benzoxazolone-carboxamide scaffold have led to the iden-
tification of 6-(4-fluorophenyl)-2-oxo-N-(4-phenylbutyl)-
1,3-benzoxazole-3-carboxamide (ARN14974, 9, IC50 = 0.079
μM) as a highly potent systemically active inhibitor of intra-
cellular AC activity.20 Expanded SAR investigations con-
firmed the potential for benzoxazolone-carboxamide deriv-
atives to provide stable and systemically active inhibitors of
ical inhibitor of AC (K ≈ 5 μM), which binds the enzymes
i
and creates an allosteric interference with the active site
17
domains.
21
AC.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

D
Synthesis
E. Diamanti et al.
Feature
Figure 2 Atomic property field (APF)-based screening and selection of five molecular scaffolds
Despite these advances, the number of molecular scaf-
folds that have demonstrated utility as AC inhibitors re-
mains very limited. Yet, broadening chemical diversity in
this class of molecules is essential not only to expand our
ability to study the roles of ceramide and S1P in living ani-
mals, but also to support possible preclinical and clinical
development efforts.
pyrazole derivatives 23a–26a (Table 2, vide infra), and se-
lected this chemical class for further expansion. Our initial
structure-activity relationship (SAR) studies were focused
on compounds 23a and 24a (Scheme 2), investigating the
effects of various substituents at positions 3 and 5 of the
pyrazole ring as well as the role of the acyl function and
side chain length. When two isomeric pyrazole N-carbox-
amides were obtained, the regiochemistry-based SARs
were also evaluated. Finally, the potent inhibitor 24n was
selected for in vitro pharmacology studies and its ability
was tested to interfere with AC activity in intact G361 cells,
a human melanoma cell line characterized by a proliferative
phenotype.
To identify novel molecular scaffolds for AC inhibition,
in the present study, we utilized the atomic property field
22
(
APF) method using carmofur (8) as a template. Five dis-
tinct heterocyclic scaffolds 10–13 and 14a were selected,
which were functionalized as N-carboxamides to yield a
small set of 12 compounds: 15–22, 23a–24a, 25a–26a (Fig-
ure 2).
Chemistry
The ability of these molecules to inhibit AC activity was
examined using lysates of HEK293 cells that overexpressed
human AC (h-AC). We obtained promising results with the
In analogy to hit compounds 8 and 9, we coupled the se-
lected heterocyclic scaffolds 10–13 and 14a with hexyl and
phenylbutyl isocyanate to obtain the corresponding N-car-
Scheme 2 Illustration of the strategy for structure–activity relationship (SAR) study
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

E
Synthesis
E. Diamanti et al.
Feature
boxamide derivatives 15–22 and 23a–26a, in good yields
Scheme S1, Supporting Information and Table 2). The acy-
lation of asymmetrically substituted pyrazolol 14a takes
place competitively at both nitrogen atoms of the pyrazole
ring giving a mixture of two regioisomers 23a–24a and
For the asymmetrically substituted pyrazoles 14c–o, a
mixture of two regioisomers 23 and 24 was obtained in all
cases, with the exception of 3-phenyl-, 3-trifluoromethyl-
and 3-nitro-1H-pyrazoles 14g,i,j, which reacted forming
only one isomer 23g,i,j (Table 1). The ratio between the two
N-acyl compounds 23 and 24 is found to be affected by a
combination of electronic and steric factors of the substitu-
ents in position 3 and 5 of the pyrazole ring.23 Indeed, in the
presence of an electron-donating group, such as methyl,
both pyrazole nitrogen atoms undergo acylation giving a
mixture of the two possible regioisomers, in which the pre-
dominant one is the less sterically hindered compound,
namely 23d and 24d (Table 1). In contrast, the effect of an
electron-withdrawing group, such as trifluoromethyl, de-
creases the reactivity of the neighboring nitrogen atom
leading to the formation of only one regioisomer, which
also corresponds to the less bulky derivative, namely 23i
(Table 1). In the cases of pyrazoles having substituents com-
parable in size but with different electronic properties,
such as methyl and trifluoromethyl groups, both nitrogen
atoms participate in the acylation reaction, which also oc-
curs at the nitrogen atom neighboring the electron-
withdrawing moiety, as for 23n and 24n (Table 1).
(
25a–26a, in overall good yield and in a ratio of 8:92 and
7:93, respectively. The regioisomers were separated by col-
umn chromatography, and the structures assigned by spec-
troscopic analysis. No traces of O-acylated products were
found.
Focusing on the pyrazole scaffold, the three N-acyl de-
rivatives 27–29 were first synthesized by coupling com-
mercial unsubstituted pyrazole 14b to octanoyl chloride,
hexyl chloroformate, and hexyl isocyanate, respectively
(Scheme 3).
Next, the syntheses of a series of unsubstituted and dif-
ferently substituted pyrazole N-carboxamide derivatives
are outlined in Scheme 3 and Table 1. A small set of com-
pounds 30–35, bearing N-carboxamide alkyl chains of dif-
ferent length, was prepared as shown in Scheme 3. The un-
substituted pyrazole 14b was coupled to various alkyl iso-
cyanates in pyridine at room temperature, in the presence
of a catalytic amount of DMAP, to give derivatives 30–33.
For compounds 34 and 35, 14b was first activated with tri-
phosgene followed by in situ quenching with n-decylamine
and N-methylhexylamine, respectively (Scheme 3). Then,
we focused on the synthesis of mono- and disubstituted
pyrazole N-carboxamides. Table 1 shows the reaction con-
ditions adopted for the coupling of a series of substituted
pyrazoles 14c–t with hexyl isocyanate to afford compounds
Unambiguous assignment of the regiochemistry of
asymmetrically substituted pyrazole N-carboxamides was
1
13
achieved by NMR analysis. A set of 1D ( H NMR, C NMR)
1
1
1
13
1
13
and 2D experiments ( H– H COSY, H– C HSQC, H– C
1
15
HMBC and H– N HMBC) on purified regioisomers provid-
ed robust evidence for structural elucidation (Figure 3).
We observed that acylation of one nitrogen atom of the
1
23c–o, the corresponding regioisomers 24c–f, 24h, 24k–o,
pyrazole system implies (i) a marked H-downfield shift of
and derivatives 36–40 as single isomers, in moderate to
good yields.
the adjacent substituent due to anisotropic effect exerted
by the carbonyl group of the urea moiety,24 (ii) a N-high-
15
O
N
N
Cl
O
O
Et3N, CH2Cl2, 12 h
O
2
7 (66%)
N
N
H
N
N
O
Et3N, CH2Cl2, 12 h
O
Cl
1
4b
O
28 (57%)
R3
C
triphosgene, Et3N
N
N
CH2Cl2, 0 °C to r.t., 1 h
3
4
N
R3
then R R N, 12 h
DMAP (cat.), pyridine, 12 h
N
H
O
3
2
3
9: R = n-hexyl (81%)
N
3
0: R = n-pentyl (60%)
N
3
R3
31: R = n-heptyl (92%)
3
4
3
N
R4
34: R = n-decyl, R = H (84%)
32: R = n-octyl (48%)
O
3
4
3
35: R = n-hexyl, R
= Me (88%)
33: R = n-nonyl (80%)
Scheme 3 Synthesis of unsubstituted pyrazole N-acyl derivatives 27–35
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

F
Synthesis
E. Diamanti et al.
Feature
Table 1 Synthesis of Mono- and Disubstituted Pyrazole-N-carboxamides 23c–o, 24c–f, 24h, 24k–o, 36–40
R2
R2
R2
N
O
C
H
N
N
N
R1
N
R1
+
N
H
R1
N
N
DMAP (cat.), pyridine, 12 h
O
N
H
O
23c–o: R1 ≠ R2
24c–f,h,k–o: R1 ≠ R2
14c–t
3
6–40: R1 = R2
Entry
14
14c
R1
R2
OH
Products (yield)a
Ratio (23/24)b
1
2
3
4
5
6
7
8
9
H
23c (25%)
23d (65%)
23e (11%)
23f (75%)
23g (54%)
23h (54%)
23i (82%)
23j (62%)
23k (4%)
23l (6%)
24c (14%)
24d (14%)
24e (50%)
24f (2%)
–
67:33
66:34
14:86
92:8
–
14d
14e
14f
H
Me
NH
H
2
H
OMe
Ph
14g
14h
14i
H
H
Br
24hc
92:8
–
H
CF
NO
NH
3
–
14j
H
2
–
–
14k
14l
Me
Me
Me
Me
Me
Me
Et
2
24k (89%)
24l (2%)
24m (8%)
24n (6%)
24oc
6:94
70:30
70:30
92:8
93:7
10
11
12
13
14
15
16
17
18
OMe
Br
14m
14n
14o
14p
14q
14r
14s
14t
23m (55%)
23n (66%)
23o (75%)
36 (74%)
37 (81%)
38 (83%)
39 (71%)
40 (quant)
CF
3
Ph
Me
Et
n-Pr
i-Pr
t-Bu
n-Pr
i-Pr
t-Bu
a
b
c
Yields refer to purified products.
Ratio between regioisomers was determined by H NMR of the crude mixture.
Not isolated.
1
field shift of the acylated nitrogen atom (≈ 220 ppm), while
the non-acylated nitrogen resonates at lower field (≈ 290
ppm), and (iii) a C-downfield shift of the carbon atom
distal to the urea moiety (Figure 3). Cross-correlation of
these evidences, consistently found throughout regioiso-
mer series 23 and 24, allowed building a robust spectro-
scopic method for structural elucidation of these com-
pounds. In those cases when one regioisomer is formed,
structural assignment was based on the comparison be-
tween spectroscopic data collected for the single isomer
and the corresponding non-acylated pyrazole (see the Sup-
porting Information for complete structural assignment of
representative substituted pyrazole-N-carboxamides).
Pyrazoles that were not commercially available were
prepared as shown in Schemes 4 and 5. 3-Methoxy-1H-pyr-
azole (14f) and 3-methoxy-5-methyl-1H-pyrazole (14l)
were synthesized through a three-step sequence starting
from the corresponding hydroxyl pyrazoles 14c and 14a, re-
spectively (Scheme 4).
25
13
Figure 3 Spectroscopic evidence for structure assignment of regioiso-
mers 23 and 24
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

G
Synthesis
E. Diamanti et al.
Feature
usually included in APF calculations was not taken into con-
sideration. To select the compounds to prioritize, we arbi-
trarily set a threshold for the pseudoenergy value at –1200
score units. This value, which is two-thirds of the 1800
score units assigned to compound 8 into its own APF field,
represented a good compromise between prioritizing a lim-
ited number of compounds and extending the selection be-
yond those derivatives just trivially resembling 8 without
introducing any element of true novelty. Eventually 2317
compounds proceeded to medicinal-chemistry-guided vi-
sual inspection. Five distinct molecular scaffolds were se-
lected based on (i) structural diversity, (ii) ease of prepara-
tion, and (iii) efficient access to structural analogues for SAR
purposes (Figure 2). Scaffolds 10–13 and 14a, displaying
monocyclic and bicyclic as well as heteroaliphatic and het-
eroaromatic systems, were functionalized as N-carboxam-
ide derivatives 15–22, 23a–26a and screened against h-AC
OH 1) Ac O
O
O
2
pyridine
1
0 M NaOH
9
5 °C, 2 h
R1
N
R1
N
R1
N
N
N
N
2) K2CO3, MeI
H
THF/MeOH (1:1)
1 h
H
2
8
-butanone
0 °C, 2 h
O
1
4c: R1 = H
4a: R1 = Me
1
1
1
43: R = H (90%)
44: R = Me (98%)
14f: R = H (61%)
14l: R = Me (56%)
1
1
Scheme 4 Synthesis of substituted pyrazoles 14f and l4l
These compounds were first reacted with 1 equivalent
of acetic anhydride to afford N1-acetyl pyrazoles 41 and 42
as single isomers. Methylation with iodomethane under ba-
sic conditions proceeded smoothly to give compounds 43
and 44, which upon cleavage of the acetyl group delivered
the desired methoxypyrazoles 14f and 14l in overall good
yield. Finally, a Knorr-type reaction was adopted to synthe-
size pyrazoles 14o, 14q, and 14r. Cyclocondensation of
the proper 1,3-dicarbonyl compounds 46–48 with hydra-
zine led to the desired pyrazoles in good to excellent yields
26
29
in a fluorescence-based assay (Table 2).
Compounds 15–20 having a heterocyclic scaffold such
as hydantoin, oxazolidinone, and piperazinone did not sig-
nificantly inhibit AC at 10 μM. Similarly, no inhibition was
seen for compounds 21 and 22, obtained from the heteroar-
omatic bicycle dihydrobenzoimidazolone. By contrast, N-
carboxamides of 3-hydroxy-5-methylpyrazole 23a and 24a
and 25a and 26a showed a marked degree of inhibitory ac-
tivity. These compounds showed median inhibitory con-
centration (IC ) values in the low micromolar range, and an
(
Scheme 5).
R2
N
O
O
NH2NH2•H2O
R1
R1
R2
N
EtOH, 0 °C to r.t.
H
1
5 min
50
2
14o: R = Me; R = Ph (85%)
14q: R = R = Et (98%)
14r: R = R = n-Pr (36%)
1
2
interesting difference in activity between regioisomers
e.g., 23a, IC = 3.137 μM vs 24a, IC50 = 0.455 μM, Table 5,
4
4
4
6: R1 = Me; R = Ph
1
2
1
2
7: R = R = Et
(
50
30
1
2
1
2
8: R = R = n-Pr
vide infra). Based on these results, we selected 23a and
24a as starting points for our SAR analysis.
Scheme 5 Synthesis of 3,5-disubstituted pyrazoles 14o, 14q and 14r.
,3-Diketone 48 was obtained in situ from the corresponding commer-
cially available ketone and acid chloride.
1
The pyrazole-N-carboxamides were tested for their
ability to inhibit the hydrolysis of N- [(1S,2R)-2-hydroxy-1-
(hydroxymethyl)-4-(2-oxochromen-7-yl)oxybutyl]dodec-
Results and Discussion
The primary objective of the present study was to dis-
anamide by recombinant h-AC in a fluorescence-based as-
say. IC50 values are reported in Tables 3–5.
cover novel chemical classes of small-molecule inhibitors of
lysosomal AC activity. We utilized the computer-assisted
APF method – starting from carmofur (8) as a template – to
identify novel scaffolds that, while structurally different,
match the pharmacophore fingerprint of 8. From ZINC,27 we
retrieved a library of over 10 million rule-of-five compliant
compounds (Figure 2). To guarantee synthetic accessibili-
ty, the selected dataset encompassed only commercially
available derivatives. First, the library was pre-filtered by
means of a simple two-dimensional query, retrieving only
those molecules bearing a ureidic group matching the one
displayed by 8. A total of 26,651 compounds were selected,
and assigned a pseudoenergy score that expressed their fit
into the APF fields generated using 8 as template (see Com-
putational Methods). This score expressed the best possible
overlap of the following six properties: hydrogen bond do-
nor propensity, hydrogen bond acceptor propensity, sp2
carbon atom hybridization, lipophilicity, size, and electro-
negativity. Since 8 is a neutral molecule, the charge field
We first investigated the role of acyl function and side
chain. This study was conducted on the unsubstituted pyra-
zole ring to better appreciate possible changes in potency
while avoiding influences due to substituents on the het-
erocycle and regioisomer formation. The unsubstituted pyr-
azole-N-carboxamide 29, bearing a six-carbon alkyl chain,
was found to be moderately potent (IC50 = 0.481 μM, Table 3).
The corresponding amide 27 and carbamate 28 deriva-
tives showed no inhibitory activity. Similarly inactive was
the N-Me derivative 35, where the 1-carboxamide NH moi-
ety of 29 was substituted with a methyl group (Table 3).
Having assessed that the 1-carboxamide NH is manda-
tory for activity, we investigated the influence of side-chain
length by preparing a small set of derivatives 30–34 with
different number of carbon atoms in the side chain. A de-
crease in chain length, as in N-pentylpyrazole-1-carboxam-
ide (30) (IC50 = 1.39 μM), was associated with a decrease in
inhibitory potency. Conversely, increasing the length of the
chain from six to nine methylene units led to progressively
28
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

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E. Diamanti et al.
Feature
Table 2 Screening of Compounds 15–22, 23a–26a on Human AC
Compd
Structure
% Inh at 10 μM
Compd
Structure
% Inh at 10 μM
O
O
O
O
N
N
N
H
N
N
N
H
1
1
5
6
HN
61
21
no inhibition
no inhibition
N
H
O
O
O
O
N
H
11
22
N
H
HN
O
N
H
O
OH
O
O
N
N
1
1
1
2
7
8
9
0
6
11
37
16
23a
24a
25a
26a
96
98
94
92
N
N
O
H
N
H
O
OH
O
O
H
N
N
N
H
N
O
O
N
O
OH
N
O
N
N
N
H
N
HN
N
O
H
OH
O
H
N
O
N
H
N
N
HN
O
higher potency, with the N-nonyl derivative 33 showing the
best IC50 value (0.197 μM) within this set of compounds.
Further extension of the chain length to ten methylene
units had an opposite effect (34, IC50 = 0.993 μM).
Next, we focused on substitutions of the pyrazole ring.
Considering that the initial hit compounds, 23a and 24a,
bear a hydroxyl and a methyl group at positions 3 and 5, re-
spectively, we started our analysis by removing one group
at the time to obtain the monosubstituted 3- and 5-hy-
droxy 23c and 24c and 3- and 5-methyl derivatives 23d and
rivatives 23e and 24e, and 23f and 24f were synthesized.
Interestingly, we did not observe significant difference in
potency between regioisomers 23e and 24e, which inhibit-
ed AC with an IC50 of 0.216 μM and 0.522 μM, respectively.
Although regioisomer 23e was more potent than 24e, the
difference in IC50 was only two-fold. Conversely, methyla-
tion of the hydroxyl group caused a substantial drop in po-
tency, as the 3-methoxy derivative 23f only partially inhib-
ited AC at 10 μM; its isomer 24f showed a limited chemical
stability and was not tested. Replacement of the hydroxyl
group with a bulkier, but planar moiety, such as a phenyl,
led to the single isomer 23g, which inhibited AC with mod-
erate potency (IC50 = 0.766 μM).
24d (Table 4).
The 3-substituted compounds 23c and 23d showed an
inhibitory potency in the micromolar range; the corre-
sponding regioisomers 24c and 24d turned out to be 6- to
Continuing our study on the effect of monosubstitution
of the pyrazole, we turned to electron-withdrawing groups,
as shown with compounds 23h–23i, obtained as single iso-
mers (Table 4). The presence of bromine at position 3 led to
a compound which was equipotent to 23c (23h, IC50 = 2.49
12-fold more potent than 23c and 23d, displaying IC50 val-
ues of 0.196 μM and 0.169 μM, respectively, and 2-fold
more potent than hit compound 24a (IC50 = 0.455 μM). To
further explore the effect of an electron-donating group on
the pyrazole ring, the amino and methoxy-substituted de-
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I
Synthesis
E. Diamanti et al.
Feature
Table 3 Inhibitory Potencies (IC50) of Compounds 27–35 on Human ACa
24d were found to be slightly more potent than the pyrazo-
lol carboxamides 23c and 24c, and with the objective of re-
moving an additional chemical handle suitable for acyla-
tion, we maintained the methyl substituent and replaced
the hydroxyl group with those electron-donating and elec-
tron-withdrawing moieties studied in the monosubstituted
series of compounds. We observed that disubstitution of
the pyrazole ring generally enhances inhibitory potency
against AC and is associated to significant differences in ac-
tivity between the two isomeric series of compounds. The
combination of methyl group with both electron-donating
and electron-withdrawing moieties was generally well tol-
erated, delivering compounds with submicromolar poten-
cies, with the exception of derivative 23l, which showed a
4-fold drop in potency with respect to hit 23a, confirming
the detrimental effect of a methoxy group on this scaffold
for AC inhibition. Of particular interest were the results ob-
tained with the 3-methyl-5-bromo- and 3-methyl-5-triflu-
oromethyl-substituted derivatives 24m and 24n, which
showed IC50 values of 0.011 μM and 0.014 μM, respectively,
representing the most potent compounds within this series
Compd Structure
IC50 (μM) ± S.E.M
N
N
2
2
2
7
8
9
no inhibition
O
N
N
N
N
N
N
no inhibition
0.481 ± 0.029
1.390 ± 0.280
0.224 ± 0.054
0.208 ± 0.049
O
O
N
N
H
O
3
3
3
0
1
2
N
N
H
O
(Table 5).
Finally, the activity of a small set of symmetric 3,5-
disubstituted pyrazole derivatives 36–40 bearing progres-
sively bulkier alkylic substituents was investigated (Table
6).
All the compounds showed double-digit nanomolar po-
tency toward AC indicating that steric hindrance is general-
ly well tolerated, with the exception of the less potent 3,5-
dipropyl derivative 38 (IC50 = 0.294 μM). The highest activi-
ty within this small series was observed with compound 40
bearing a tert-butyl group at positions 3 and 5 (IC50 = 0.011
μM).
At the outset, we set as goal of our project the diversifi-
cation of the available arsenal of small-molecule inhibitors
of AC that can be used as tools to investigate the biology of
this enzyme and, possibly, as starting points for therapeutic
candidates. Having elucidated the main SAR features of this
new chemical class, our efforts were focused on its pharma-
cological characterization. The highly potent inhibitor 24n
was selected for this purpose and its stability was assessed
in buffer and mouse plasma (Table 7).
Compound 24n displayed adequate chemical stability
both at acidic and neutral pH, as well as in cell culture me-
dia. Conversely, the compound had very limited stability in
mouse plasma (half-life, t1/2 = 9 min), making it a promising
candidate for ‘soft drug’ applications (e.g., topical skin or
lung delivery).
N
N
H
O
N
N
H
O
N
3
3
3
4
5
N
0.197 ± 0.032
0.993 ± 0.264
no inhibition
N
H
O
N
N
N
N
H
O
N
3
N
O
a
IC50 values are reported as mean values of two or more determinations.
Accordingly, the compound was tested in the prolifera-
tive melanoma cell line G361. We selected this line based
on previous work, which had shown that AC regulation of
ceramide signaling is important in melanoma cell viabili-
ty. The cells were incubated in the presence of 24n (0.1–
20 μM) and AC inhibition and sphingolipid levels were mea-
sured after various incubation times (Figure 4).
μM), while a trifluoromethyl group turned out to be well
tolerated, as derivative 23i showed an IC50 value of 0.110
μM, being the most potent compound of this set.
As a continuation of the SAR study, we investigated the
effect of disubstitution at position 3 and 5 of the pyrazole
ring (Table 5). As methyl-substituted derivatives 23d and
19
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Synthesis
E. Diamanti et al.
Feature
Table 4 Inhibitory Potencies (IC50) of Compounds 23c–i and 24c–f on Human ACa
Compd
Structure
IC50 (μM) ± S.E.M
Compd
Structure
IC50 (μM) ± S.E.M
OH
OH
H
N
N
N
2
2
2
3c
3d
3e
2.461 ± 1.256
24c
0.196 ± 0.099
N
N
N
N
H
O
O
H
N
N
N
1.100 ± 0.270
0.216 ± 0.010
24d
24e
0.169 ± 0.046
0.522 ± 0.087
N
N
H
O
O
NH2
NH2
H
N
N
N
N
N
N
N
H
O
O
O
O
O
H
N
N
N
2
2
2
3f
32% at 10 μM
24f
unstable
N
N
H
O
3g
0.766 ± 0.326
N
N
N
H
O
Br
N
N
3h
2.492 ± 2.108
0.110 ± 0.025
N
H
O
CF3
N
N
2
3i
N
H
O
a
IC50 values are reported as mean values of two or more determinations.
The compound caused a rapid concentration-dependent
This level of efficacy compares well with those reported for
other classes of AC inhibitors.19 As expected, AC inhibition
was accompanied by accumulation of various species of ce-
ramides: specifically, we observed a significant increase of
those (dihydro)ceramides, which are preferred substrates
for AC (namely, d18:0/16:0 and d18:1/16:0) and a concen-
inhibition of AC activity in these cells. Notably, 24n inhibit-
ed cellular AC activity at concentration as low as 0.1 μM,
and the inhibition was already appreciable after 15 minutes
of incubation and persisted for 6 hours, with a partial re-
covery of enzyme activity after 24 hours (Figure 4, A and B).
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K
Synthesis
E. Diamanti et al.
Feature
tration-dependent decrease in sphingosine (Figure 4, C). At
the highest concentration of 24n tested (20 μM), we ob-
served a time-dependent accumulation of ceramide, which
reached a maximum after 6 hours, and a rapid decrease in
sphingosine levels, which was already detectable after 15
minutes (Figure 4, D). Increases in sphingomyelins and glu-
cosylceramide were also observed after 15 min (Figure S4
in the Supporting Information). The results demonstrate
that 24n is able to effectively inhibit AC in a complex cellu-
lar environment.
Conclusions
In the present study, we utilized the APF-based comput-
er-assisted approach to identify novel scaffolds that may
serve as starting points for the design of small-molecule in-
hibitors of AC activity. Using carmofur (8) as a template, we
probed the ZINC library and retrieved compounds that,
while structurally different, match the pharmacophore fin-
gerprint of 8. Medicinal chemistry-guided visual inspection
of a set of computationally selected compounds identified
five distinct scaffolds that, when appropriately functional-
ized, revealed substituted pyrazole carboxamides as prom-
ising novel AC inhibitors. A series of differently substituted
Table 5 Inhibitory Potencies (IC50) of Compounds 23a, 23k–o, 24a, 24k–n, and 36–40 on Human ACa
Compd
Structure
OH
IC50 (μM) ± S.E.M
Compd
Structure
IC50 (μM) ± S.E.M
OH
H
N
N
2
2
2
2
2
3a
3k
3l
3.137 ± 0.585
24a
0.455 ± 0.112
N
N
N
N
H
O
O
NH2
N
NH2
H
N
0.489 ± 0.173
14.11 ± 0.30
0.264 ± 0.072
0.108 ± 0.020
24k
0.218 ± 0.065
N
N
N
N
H
O
O
O
N
O
H
N
24l
unstable
N
N
N
N
N
N
H
O
O
O
Br
N
Br
H
N
3m
24m
24n
0.010 ± 0.001
0.014 ± 0.010
N
N
N
H
O
CF3
N
CF3
H
N
3n
N
N
N
H
O
O
2
3o
0.317 ± 0.065
N
N
N
H
O
a
IC50 values are reported as mean values of two or more determinations.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

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Synthesis
E. Diamanti et al.
Feature
Table 6 Inhibitory Potencies (IC50) of Compounds 36–40 on Human ACa
Compd
Structure
IC50 (μM) ± S.E.M
Compd
Structure
IC50 (μM) ± S.E.M
N
N
3
3
6
7
0.074 ± 0.037
39
0.043 ± 0.003
N
O
N
N
H
N
H
O
N
0.062 ± 0.006
40
N
0.011 ± 0.001
N
O
N
N
H
N
H
O
3
8
0.294 ± 0.046
N
N
N
O
H
a
IC50 values are reported as mean values of two or more determinations.
Figure 4 Effects of compound 24n in proliferative melanoma cells G361. Concentration dependence of the effect on AC activity (A) and sphingolipid
levels (C). Time-course of the effect of 24n (20 μM) on AC activity (B) and sphingolipid levels (D). Values are reported as the mean ± S.E.M of 3–6
determinations. Independent experiments yielded similar results. * p <0.05, p <0.01, p <0.001 vs vehicle; one-way ANOVA followed by Dunnett’s test.
**
***
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

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E. Diamanti et al.
Feature
Table 7 Stability of Compound 24n by LC-MS Analysis
Compound
Buffer stability (pH 4.5)a t1/2 (min) Buffer stability (pH 7.4)b t1/2 (min) Cell media stabilityc t1/2 (min)
m-Plasma stabilityd t1/2 (min)
2
4n
>360
66
>360
9
a
b
c
NaCl (150 mM), NaH
2
PO (100 mM), trisodium citrate (100 mM), NP40 (1%), DTT (3 mM).
4
PBS (phosphate buffered saline).
DMEM (Dulbecco’s Modified Eagle’s Medium)
Mouse plasma, 37 °C.
d
derivatives were synthesized and an NMR-based model was
built to unambiguously assign the regiochemistry of N-acy-
lation on the pyrazole ring. A systematic SAR study around
the new scaffold highlighted structural features that are im-
portant for AC inhibition. We observed that an unsubstitut-
ed nitrogen atom in the carboxamide moiety is mandatory
for activity, and assessed the best length for the alkylic side
chain as nine methylene units. The electronic nature of sub-
stituents on the pyrazole ring does not significantly impact
the inhibitory potency toward AC, as both electron-donat-
ing and electron-withdrawing moieties are tolerated, with
the exception of the methoxy group that leads to inactive or
unstable compounds 23f–24f, 23l–24l. Conversely, the re-
giochemistry of acylation turned out to be critical, as a 2- to
was used. NMR experiments were run on a Bruker Avance III 400 sys-
1
13
tem (400.13 MHz for H, and 100.62 MHz for C), equipped with a
BBI probe and Z-gradients. Spectra were acquired at 300 K, using
DMSO-d or CDCl as solvents. UPLC-MS analyses were run on a Wa-
6
3
ters ACQUITY UPLC-MS system consisting of an SQD (single quadru-
pole detector) mass spectrometer equipped with an Electrospray Ion-
ization interface and a Photodiode Array Detector. PDA range was
210–400 nm. Analyses were performed on an ACQUITY UPLC HSS T3
C
18 column (50 × 2.1 mmID, particle size 1.8 μm) with a VanGuard
HSS T3 C18 precolumn (5 × 2.1 mmID, particle size 1.8 μm). Mobile
phase was either 10 mM NH OAc in H O at pH 5 adjusted with AcOH
4
2
(
A) and 10 mM NH OAc in MeCN/H O (95:5) at pH 5 (B). Electrospray
4
2
ionization in positive and negative mode was applied. All final com-
pounds, 15–22, 23a, 23c–23o, 24a, 24c–24f, 24h, 24l–24p, 25a, 26a,
and 27–40 showed ≥95% purity by NMR and UPLC-MS analysis.
1
0-fold difference in potency was generally observed be-
Pyrazole-N-carboxamides 23a, 23c–23e, 23h, 23i, 23k, 23m–23n,
24a, 24c–24e, 24h, 24k, 24m, 24n, 29, 33, 36, 37, and 40; General
Procedure (Tables 1 and 3–5, Scheme 3)
tween the two series of regioisomers 23 and 24. Finally, one
of the most potent compounds identified, 24n, was selected
for further characterization. Stability analysis of this deriva-
tive suggested its potential use for ‘soft drug’ applications
and, thus, it was tested on proliferative melanoma cells
G361. The compound 24n effectively engages AC in these
cells leading to the expected variations in the sphingolipid
profile. This novel chemical class further expands and com-
plements the available armamentarium of chemical tools to
study the biology of AC and may offer promising candidates
for the development of topical treatments for cancers and
other hyperproliferative disorders of the skin.
The properly substituted pyrazole (1.0 equiv) was dissolved in anhyd
pyridine. DMAP (0.1 equiv) was added, and the reaction mixture was
stirred under N2 for 30 min. The appropriate isocyanate (1.1–1.5
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 mix-
ture cyclohexane/EtOAc.
N-Hexyl-3-hydroxy-5-methylpyrazole-1-carboxamide (23a) and
N-Hexyl-5-hydroxy-3-methylpyrazole-1-carboxamide (24a)
The reaction was carried out following the general procedure previ-
ously described, dissolving 14a (0.30 g, 3.06 mmol) in anhyd pyridine
(10 mL) and employing hexyl isocyanate (0.49 mL, 3.36 mmol). The
two regioisomers 23a–24a were obtained in a ratio of 8:92, deter-
mined by H NMR of the crude. The mixture was separated by silica
gel column chromatography to afford 23a (cyclohexane/EtOAc, 80:20)
and 24a (cyclohexane/EtOAc, 50:50). 23a (0.02 g, 4%) as a white pow-
All the commercially available reagents and solvents were used as
purchased from vendors without further purification. Anhydrous sol-
vents (pyridine, CH Cl , DMSO) were purchased from Sigma-Aldrich.
1
2
2
Automated column chromatography purifications were done using a
der; mp 59.5 °C.
®
Teledyne ISCO apparatus (CombiFlash Rf) with prepacked silica gel
1
H NMR (400 MHz, CDCl ): δ = 0.89 (t, J = 6.7 Hz, 3 H), 1.23–1.43 (m, 6
columns of different sizes (from 4 g up to 40 g). Mixtures of increasing
polarity of cyclohexane and EtOAc were used as eluents. Purifications
by preparative HPLC-MS were run on a Waters Autopurification sys-
tem consisting of a 3100 Single Quadrupole Mass Spectrometer
equipped with an Electrospray Ionization interface and a 2998 Photo-
diode Array Detector. HPLC system included a 2747 Sample Manager,
3
H), 1.53–1.65 (m, 2 H), 2.55 (s, 3 H), 3.33 (q, J = 6.8 Hz, 2 H), 5.62 (s, 1
H), 4.96 (br s, 1 H), 6.54 (t, J = 5.7 Hz, 1 H).
13
C NMR (101 MHz, CDCl ): δ = 14.1, 14.7, 22.7, 26.6, 29.7, 31.6, 40.5,
3
97.2, 146.2, 151.1, 161.4.
+
+
MS (ESI): m/z = 226 [M + H] , 248 [M + Na] .
MS (ESI): m/z = 224 [M – H]–.
2545 Binary Gradient Module, System Fluidic Organizer and 515
HPLC Pump. PDA range was 210–400 nm. Purifications were per-
TM
formed on a XBridge Prep C OBD column (100 × 19 mmID, particle
18
TM
24a
size 5 ꢀm) with a XBridge Prep C18 (10 × 19 mmID, particle size 5
μm) guard cartridge. Mobile phase was 10 mM NH OAc in MeCN/H O
4
2
Yield: 0.51 g (75%); white powder; mp 108 °C.
(95:5) at pH 5. Electrospray ionization in positive and negative mode
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

N
Synthesis
E. Diamanti et al.
Feature
1
13
H NMR (101 MHz, CDCl ): δ = 11.39 (s, 1 H), 8.85–9.42 (m, 1 H), 5.15
C NMR (101 MHz, CDCl ): δ = 152.0, 150.0, 129.3, 108.6, 40.4, 31.6,
3
3
(s, 1 H), 3.22–3.39 (m, 2 H), 2.21 (s, 3 H), 1.48–1.66 (m, 2 H), 1.18–
29.8, 26.6, 22.7, 14.1, 13.8.
1
.38 (m, 6 H), 0.75–0.90 (m, 3 H).
+
+
–
MS (ESI): m/z = 210 [M + H] , 232 [M + Na] , 208 [M – H] .
13
C NMR (101 MHz, CDCl ): δ = 164.2, 150.0, 149.9, 92.6, 39.9, 31.4,
3
2
9.6, 26.6, 12.2, 22.5, 14.0.
2
4d
+
+
MS (ESI): m/z = 226 [M + H] , 248 [M + Na] .
MS (ESI): m/z = 224 [M – H]–.
Yield: 0.02 g (14%); colorless oil.
1
H NMR (400 MHz, CDCl ): δ = 7.41 (d, J = 1.5 Hz, 1 H), 7.32 (m, 1 H),
3
6.08 (m, 1 H), 3.36 (td, J = 6.0, 7.2 Hz, 2 H), 2.60 (s, 3 H), 1.53–1.70 (m,
N-Hexyl-3-hydroxypyrazole-1-carboxamide (23c) and N-Hexyl-5-
2 H), 1.24–1.43 (m, 6 H), 0.87 (t, J = 7.0 Hz, 3 H).
hydroxypyrazole-1-carboxamide (24c)
13
C NMR (101 MHz, CDCl ): δ = 151.4, 143.0, 140.4, 109.2, 40.3, 31.6,
3
The reaction was carried out following the general procedure de-
29.7, 26.7, 22.7, 14.2, 14.1.
scribed above, dissolving 14c (0.30 g, 3.56 mmol) in anhyd pyridine
+
+
MS (ESI): m/z = 210 [M + H] , 228 [M + NH ] .
4
(17.8 mL) and employing hexyl isocyanate (0.57 mL, 3.92 mmol). The
MS (ESI): m/z = 208 [M – H]–.
two regioisomers 23c and 24c were obtained in a ratio of 67:33, de-
1
termined by H NMR of the crude. The mixture was separated by
3
-Amino-N-hexylpyrazole-1-carboxamide (23e) and 5-Amino-N-
hexylpyrazole-1-carboxamide (24e)
HPLC-MS.
The reaction was carried out following the general procedure de-
scribed above, dissolving 14e (0.20 g, 2.41 mmol) in anhyd pyridine
2
3c
Yield: 0.19 g (25%); light yellow powder; mp 88 °C.
(12 mL) and employing hexyl isocyanate (0.39 mL, 2.65 mmol). The
1
H NMR (400 MHz, CDCl ): δ = 8.04 (d, J = 2.9 Hz, 1 H), 6.45 (t, J = 5.4
3
two regioisomers 23e and 24e were obtained in a ratio of 14:86, de-
Hz, 1 H), 5.88 (d, J = 2.9 Hz, 1 H), 3.38 (td, J = 5.9, 7.1 Hz, 2 H), 1.52–
1
termined by H NMR of the crude. The mixture was separated by sili-
1.70 (m, 2 H), 1.21–1.48 (m, 6 H), 0.89 (t, J = 6.7 Hz, 3 H).
ca gel column chromatography to afford 23e (cyclohexane/EtOAc,
60:40) and 24e (cyclohexane/EtOAc, 70:30).
13
C NMR (101 MHz, CDCl ): δ = 163.1, 149.7, 131.4, 96.5, 40.7, 31.6,
3
29.6, 26.5, 22.7, 14.1.
+
+
+
MS (ESI): m/z = 212 [M + H] , 234 [M + Na] , 250 [M + K] .
23e
MS (ESI): m/z = 210 [M – H]–.
Yield: 0.05 g (11%); yellow oil.
1
H NMR (400 MHz, CDCl ): δ = 7.95 (d, J = 2.8 Hz, 1 H), 6.67–6.91 (m, 1
3
24c
H), 5.77 (d, J = 2.7 Hz, 1 H), 3.45–3.67 (br s, 2 H), 3.34 (td, J = 5.9, 7.1
Hz, 2 H), 1.51–1.66 (m, 2 H), 1.25–1.39 (m, 6 H), 0.87 (t, J = 6.4 Hz, 3
H).
Yield: 0.11 g (14%); white powder; mp 85 °C.
1
H NMR (101 MHz, CDCl ): δ (major rotamer) = 0.89 (t, J = 6.7 Hz, 3
3
13
C NMR (101 MHz, CDCl ): δ = 156.2, 150.1, 130.2, 98.1, 40.3, 31.6,
H), 1.27–1.44 (m, 6 H), 1.53–1.69 (m, 2 H), 3.38 (q, J = 7.0 Hz, 2 H),
3
29.8, 26.6, 22.6, 14.1.
5.50 (app s, 1 H), 7.34 (app s, 1 H), 9.03 (m, 1 H), 10.38 (s, 1 H); δ (mi-
+
+
nor rotamer) = 0.89 (t, J = 6.7 Hz, 3 H), 1.27–1.44 (m, 6 H), 1.53–1.69
MS (ESI): m/z = 211 [M + H] , 233 [M + Na] .
(
(
1
m, 2 H), 3.38 (q, J = 7.0 Hz, 2 H), 5.44 (app s, 1 H), 7.06 (m, 1 H), 7.48
app s, 1 H) ; 1:0.7 ratio of two rotamers.
2
4e
3
C NMR (101 MHz, CDCl ); two rotamers: δ (major rotamer) =
Yield: 0.25 g (50%); colorless oil.
3
1
56.79, 149.86, 142.30, 88.09, 39.97, 31.53, 29.61, 26.58, 22.66, 14.13;
δ (minor rotamer) = 163.86, 149.88, 137.49, 94.76, 40.10, 31.53,
9.61, 26.58, 22.66, 14.13.
1
H NMR (400 MHz, CDCl ): δ = 7.26 (d, J = 1.1 Hz, 1 H), 7.07–7.23 (m, 1
3
H), 5.46–5.78 (br s, 2 H), 5.38 (d, J = 1.8 Hz, 1 H), 3.35 (td, J = 6.0, 7.1
Hz, 2 H), 1.52–1.68 (m, 2 H), 1.25–1.44 (m, 6 H), 0.89 (t, J = 6.6 Hz, 3
H).
13
2
+
+
+
MS (ESI): m/z = 212 [M + H] , 234 [M + Na] , 250 [M + K] .
MS (ESI): m/z = 210 [M – H]–.
C NMR (101 MHz, CDCl ): δ = 153.0, 149.6, 141.2, 89.0, 40.0, 31.6,
3
29.7, 26.6, 22.7, 14.1.
N-Hexyl-3-methylpyrazole-1-carboxamide (23d) and N-Hexyl-5-
methylpyrazole-1-carboxamide (24d)
+
+
MS (ESI): m/z = 211 [M + H] , 233 [M + Na] .
The reaction was carried out following the general procedure de-
scribed above, dissolving 14d (0.05 mL, 0.61 mmol) in anhyd pyridine
3
-Bromo-N-hexylpyrazole-1-carboxamide (23h) and 5-Bromo-N-
hexylpyrazole-1-carboxamide (24h)
(6.1 mL) and employing hexyl isocyanate (0.13 mL, 0.91 mmol). The
The reaction was carried out following the general procedure de-
scribed above, dissolving 14h (0.15 g, 1.02 mmol) in anhyd pyridine
two regioisomers 23d and 24d were obtained in a ratio of 66:34, de-
1
termined by H NMR of the crude. The mixture was separated by sili-
(5.0 mL) and employing hexyl isocyanate (0.22 mL, 1.53 mmol). The
ca gel column chromatography to afford 23d (cyclohexane/EtOAc,
two regioisomers 23h and 24h were obtained in a ratio of 92:8, deter-
mined by H NMR of the crude. The mixture was separated by silica
92:8) and 24d (cyclohexane/EtOAc, 95:5).
1
gel column chromatography to afford 23h (cyclohexane/EtOAc,
23d
99.5:0.5). Compound 24h was not isolated.
Yield: 0.08 g 65%); colorless oil.
1
H NMR (400 MHz, CDCl ): δ = 8.09 (d, J = 2.6 Hz, 1 H), 7.07 (m, 1 H),
23h
3
6
1
.16 (d, J = 2.6 Hz, 1 H), 3.38 (td, J = 6.0, 7.2 Hz, 2 H), 2.28 (s, 3 H),
.51–1.69 (m, 2 H), 1.24–1.43 (m, 6 H), 0.88 (t, J = 6.0 Hz, 3 H).
Yield: 0.10 g (54%); colorless oil.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

O
Synthesis
E. Diamanti et al.
Feature
1
H NMR (400 MHz, CDCl ): δ = 0.88 (t, J = 6.9 Hz, 3 H), 1.23–1.42 (m, 6
The two regioisomers 23m and 24m were obtained in a ratio of
70:30, determined by H NMR of the crude. The mixture was separat-
3
1
H), 1.55–1.67 (m, 2 H), 3.39 (td, J = 6.0, 7.2 Hz, 2 H), 6.40 (d, J = 2.7 Hz,
1
H), 6.84–7.12 (m, 1 H), 8.11 (d, J = 2.7 Hz, 1 H).
ed by silica gel column chromatography to afford 23m (cyclohex-
ane/EtOAc, 99.5:0.5) and 24m (cyclohexane/EtOAc, 90:10).
13
C NMR (101 MHz, CDCl ): δ = 148.7, 130.5, 129.8, 111.6, 40.7, 31.4,
3
2
9.6, 26.6, 22.6, 14.1.
MS (ESI): m/z = 272 and 274 [M – H]–.
23m
Yield: 0.09 g (55%); colorless oil.
1H NMR (400 MHz, CDCl
(
): δ = 7.03–7.22 (m, 1 H), 6.13 (s, 1 H), 3.34
td, J = 6.0, 7.2 Hz, 2 H), 2.59 (s, 3 H), 1.53–1.67 (m, 2 H), 1.23–1.44 (m,
N-Hexyl-3-(trifluoromethyl)pyrazole-1-carboxamide (23i)
3
The reaction was carried out following the general procedure de-
scribed above, dissolving 14i (0.15 g, 1.10 mmol) in anhyd pyridine
6
H), 0.89 (t, J = 6.7 Hz, 3 H).
13
(5.5 mL) and employing hexyl isocyanate (0.24 mL, 1.65 mmol). The
C NMR (101 MHz, CDCl ): δ = 150.3, 145.2, 128.8, 112.2, 40.5, 31.6,
3
crude was purified by silica gel column chromatography (cyclohex-
29.7, 26.7, 22.7, 14.2, 14.1.
ane/EtOAc, 90:10) to afford 23i (0.24 g, 82%) as a colorless oil.
MS (ESI): m/z = 288 and 290 [M + H]+.
1
H NMR (400 MHz, CDCl ) δ = 8.28 (d, J = 2.6 Hz, 1 H), 7.04–7.19 (m, 1
3
H), 6.64 (d, J = 2.7 Hz, 1 H), 3.43 (td, J = 6.0, 7.2 Hz, 2 H), 1.59–1.71 (m,
2
4m
2
H), 1.25–1.36 (m, 6 H), 0.90 (t, J = 6.9 Hz, 3 H).
Yield: 0.012 g (8%); colorless oil.
13
C NMR (101 MHz, CDCl ): δ = 148.7, 144.9, 130.3, (q, J = 38.9 Hz),
1
3
H NMR (400 MHz, CDCl ): δ = 7.26 (s, 1 H), 7.14–7.25 (m, 1 H), 3.35
3
120.6 (q, J = 269.7 Hz), 106.3 (q, J = 2.39 Hz), 50.8, 31.5, 29.6, 26.6,
(td, J = 6.0, 7.2 Hz, 2 H), 2.24 (s, 3 H), 1.55–1.68 (m, 2 H), 1.26–1.43 (m,
22.6, 14.1.
6
13
H), 0.89 (t, J = 6.5 Hz, 3 H).
+
+
MS (ESI): m/z = 264 [M + H] , 286 [M + Na] .
C NMR (101 MHz, CDCl ): δ = 151.7, 149.8, 113.8, 113.1, 40.5, 31.6,
3
2
9.7, 26.7, 22.7, 14.2, 14.0.
3
5
-Amino-N-hexyl-5-methylpyrazole-1-carboxamide (23k) and
-Amino-N-hexyl-3-methylpyrazole-1-carboxamide (24k)
MS (ESI): m/z = 288 and 290 [M + H]+.
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14k (0.10 g, 1.03 mmol) in anhyd pyri-
dine (5.1 mL) and employing hexyl isocyanate (0.165 mL, 1.13 mmol).
The two regioisomers 23k and 24k were obtained in a ratio of 8:94,
determined by H NMR of the crude. The mixture was separated by
silica gel column chromatography to afford 23k (cyclohexane/EtOAc,
N-Hexyl-5-methyl-3-(trifluoromethyl)pyrazole-1-carboxamide
(23n) and N-Hexyl-3-methyl-5-(trifluoromethyl)pyrazole-1-car-
boxamide (24n)
1
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14n (0.30 g, 2.00 mmol) in anhyd pyri-
dine (10 mL) and employing hexyl isocyanate (0.44 mL, 3.00 mmol).
The two regioisomers 23n and 24n were obtained in a ratio of 92:8,
75:25) and 24k (cyclohexane/EtOAc, 85:15).
1
determined by H NMR of the crude. The mixture was separated by
23k
silica gel column chromatography to afford 23n (cyclohexane/EtOAc,
Yield: 0.009 g (4%); brown oil.
96:4) and 24n (cyclohexane/EtOAc, 94:6).
1
H NMR (400 MHz, CDCl ): δ = 6.84–7.08 (m, 1 H), 5.53 (s, 1 H), 3.31
3
(
q, J = 7.0 Hz, 2 H), 3.09–3.45 (br s, 2 H), 2.51 (s, 3 H), 1.46–1.74 (m, 2
2
3n
H), 1.26–1.46 (m, 6 H), 0.88 (t, J = 6.7 Hz, 3 H).
1
Yield: 0.37 g, 67%); colorless oil.
3
C NMR (101 MHz, CDCl ): δ = 154.2, 151.6, 144.7, 99.1, 40.1, 31.6,
1
3
H NMR (400 MHz, CDCl ): δ = 7.18–7.29 (m, 1 H), 6.35 (s, 1 H), 3.38
3
2
9.9, 26.7, 22.7, 14.4, 14.2.
(td, J = 6.0, 7.3 Hz, 2 H), 2.64 (s, 3 H), 1.52–1.75 (m, 2 H), 1.23–1.48 (m,
+
+
+
MS (ESI): m/z = 226 [M + H] , 248 [M + Na] , 264 [M + K] .
MS (ESI): m/z = 224 [M – H]–.
6 H), 0.89 (t, J = 7.3 Hz, 3 H).
13
C NMR (101 MHz, CDCl ): δ = 150.4, 144.9, 142.9, 120.8 (q, J = 269.6
3
Hz), 106.9, 40.6, 31.6, 29.6, 26.6, 22.7, 14.2, 14.1.
24k
+
+
+
MS (ESI): m/z = 226 [M + H] , 248 [M + Na] , 264 [M + K] .
Yield: 0.21 g (89%); white powder; mp 63.5 °C.
MS (ESI): m/z = 224 [M – H]–.
1
H NMR (400 MHz, CDCl ): δ = 7.05 (m, 1 H), 5.39 (br s, 2 H), 5.20 (s, 1
3
H), 3.13–3.45 (m, 2 H), 2.12 (s, 3 H), 1.49–1.72 (m, 2 H), 1.16–1.45 (m,
2
4n
6
H), 0.88 (t, J = 7.0 Hz, 3 H).
Yield: 0.03 g (6%); colorless oil.
13
C NMR (101 MHz, CDCl ): δ = 153.1, 151.4, 150.0, 89.3, 39.9, 31.6,
1
3
H NMR (400 MHz, CDCl ): δ = 7.15 (s, 1 H), 6.62 (s, 1 H), 3.42 (td, J =
3
29.8, 26.7, 22.7, 14.1.
6.1, 7.3 Hz, 2 H), 2.32 (s, 3 H), 1.55–1.72 (m, 2 H), 1.24–1.46 (m, 6 H),
+
+
MS (ESI): m/z = 225 [M + H] , 247 [M + Na] .
0.91 (t, J = 6.30 Hz, 3 H).
13
C NMR (101 MHz, CDCl ): δ = 149.5, 148.2, 133.8, 119.5 (q, J = 268.7
3
3
-Bromo-N-hexyl-5-methylpyrazole-1-carboxamide (23m) and 5-
Hz), 112.3, 40.5, 31.4, 29.5, 26.5, 22.5, 14.0, 13.4.
Bromo-N-hexyl-3-methylpyrazole-1-carboxamide (24m)
+
+
MS (ESI): m/z = 225 [M + H] , 247 [M + Na] .
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14m (0.14 g, 0.85 mmol) in anhyd pyri-
dine (2.6 mL) and employing hexyl isocyanate (0.18 mL, 1.27 mmol).
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

P
Synthesis
E. Diamanti et al.
Feature
N-Hexylpyrazole-1-carboxamide (29)
3,5-Di-tert-butyl-N-hexyl-pyrazole-1-carboxamide (40)
The reaction was carried out following the general procedure de-
scribed above, dissolving 14b (0.12 g, 0.52 mmol) in anhyd pyridine
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14t (0.05 g, 0.27 mmol) in anhyd pyridine
(2.7 mL) and employing hexyl isocyanate (0.06 mL, 0.40 mmol). The
crude was purified by silica gel column chromatography (cyclohex-
ane, 100%) to afford 40 (0.08 g, ~100%) as a colorless oil.
1
(0.5 mL) and employing hexyl isocyanate (0.08 mL, 0.57 mmol). The
crude was purified by silica gel column chromatography (cyclohex-
ane/EtOAc, 90:10) to afford 29 (0.23 g, 81%) as a colorless oil.
1
H NMR (400 MHz, CDCl ): δ = 8.22 (dd, J = 2.7, 0.8 Hz, 1 H), 7.59 (dd,
H NMR (400 MHz, CDCl ): δ = 0.89 (t, J = 6.8 Hz, 3 H), 1.27 (s, 9 H),
3
3
J = 1.6, 0.8 Hz, 1 H), 7.15 (s, 1 H), 6.38 (dd, J = 2.7, 1.6 Hz, 1 H), 3.41 (td,
J = 7.2, 6.0 Hz, 2 H), 1.58–1.69 (m, 2 H), 1.21–1.47 (m, 6 H), 0.82–1.00
1.30–1.42 (m, 6 H), 1.45 (s, 9 H), 1.55–1.68 (m, 2 H), 3.35 (td, J = 6.0,
7.3 Hz, 2 H), 6.02 (s, 1 H), 7.52 (s, 1 H).
(
1
m, 3 H).
13
C NMR (101 MHz, CDCl ): δ = 161.3, 156.4, 151.4, 104.7, 40.6, 33.1,
3
3
C NMR (101 MHz, CDCl ): δ = 149.8, 142.0, 128.7, 108.3, 40.5, 31.6,
32.3, 31.6, 30.5, 30.1, 29.9, 29.6, 26.8, 22.7, 14.1.
3
2
9.8, 26.6, 22.7, 14.1.
+
+
MS (ESI): m/z = 308 [M + H] , 228 [M + Na] .
MS (ESI): m/z = 194 [M – H]–.
MS (ESI): m/z = 306 [M – H]–.
N-Nonylpyrazole-1-carboxamide (33)
Computational Methods
The reaction was carried out following the general procedure de-
scribed above, dissolving 14b (0.07 g, 1.03 mmol) in anhyd pyridine
APF Field Generation
(1.0 mL) and employing nonyl isocyanate (0.30 mL, 1.54 mmol). The
First, compound 8 was generated in three-dimensional structure
crude was purified by silica gel column chromatography (cyclohex-
31
within ICM (ICM3.7, Molsoft LLC, San Diego, CA, USA). Cartesian co-
ordinates were translated into internal coordinates and MMFF32 force
field atom types and charges were assigned. The starting conforma-
tion of compound 8 was obtained assigning an elongated orientation
to the acyl side chain. Then, continuous three-dimensional pharma-
cophoric grid potentials were calculated accounting for relevant
physico-chemical features of 8. According to the original implementa-
tion reported by Totrov, seven APF potentials were selceted: hydrogen
bond donor and acceptor propensity, lipophilicity, size, charge, hy-
bridization, and electronegativity. These grids encompassed the tem-
plate molecule plus a 5Å margin.
ane/EtOAc, 95:5) to afford 33 (0.19 g, 80%) as a white powder; mp 54
°
C.
1
H NMR (400 MHz, CDCl ): δ = 8.22 (d, J = 2.6 Hz, 1 H), 7.59 (d, J = 1.5
3
Hz, 1 H), 7.15 (s, 1 H), 6.38 (dd, J = 1.6, 2.7 Hz, 1 H), 3.41 (td, J = 6.0, 7.2
Hz, 2 H), 1.56–1.69 (m, 2 H), 1.19–1.44 (m, 12 H), 0.87 (t, J = 6.8 Hz, 3
H).
13
C NMR (101 MHz, CDCl ): δ = 150.2, 142.4, 129.0, 108.6, 40.8, 32.3,
3
30.1, 29.9, 29.7, 27.3, 23.1, 14.6.
MS (ESI) m/z = 238 [M + H]+.
MS (ESI): m/z = 236 [M – H]–.
APF Screening
N-Hexyl-3,5-dimethylpyrazole-1-carboxamide (36)
We retrieved a library of over ten million compounds from ZINC (‘In-
Stock’, ‘Drug-like’ subset as of March 2011). In ICM, this molecular
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14p (0.10 g, 0.10 mmol) in anhyd pyri-
dine (1.0 mL) and employing hexyl isocyanate (0.09 mL, 0.15 mmol).
The crude was purified by silica gel column chromatography (cyclo-
hexane/EtOAc, 99:1) to afford 36 (0.16 g, 74%) as a colorless oil.
1
27
dataset was prescreened by means of a 2D substructure query to re-
trieve all those compounds bearing a ureidic group having one nitro-
gen atom included in a ring system and the other as unsubstituted NH
group. Selected compounds were automatically assigned three-di-
mensional internal coordinates, bond orders, tautomeric forms, ste-
reochemistry, hydrogen atoms, protonation states, MMFF force field
atom types, and charges by the ICM converting procedure. Then, the
conformation of each compound was globally optimized using the bi-
ased probability Monte Carlo method within the precalculated APF
H NMR (400 MHz, CDCl ): δ = 7.19–7.25 (m, 1 H), 5.88 (s, 1 H), 3.34
3
(td, J = 6.0, 7.2 Hz, 2 H), 2.55 (s, 3 H), 2.19 (s, 3 H), 1.55–1.65 (m, 2 H),
1.27–1.42 (m, 6 H), 0.88 (t, J = 6.8 Hz, 3 H).
13
C NMR (101 MHz, CDCl ): δ = 151.5, 150.0, 143.6, 109.7, 40.2, 31.6,
3
29.8, 26.7, 22.7, 14.1, 13.7.
33
field. Torsional variables describing flexible rings were sampled ex-
plicitly. The intramolecular energy contribution was also considered
to avoid unrealistically strained solutions. To provide a simulation of
suitable length, the thoroughness parameter was set equal to the pre-
viously validated value of 1. Based on the fit of its optimized confor-
mation into the APF field, each compound was assigned a pseudo-en-
ergy value and the library was ranked accordingly.
MS (ESI): m/z = 224 [M + H]+.
3,5-Diethyl-N-hexylpyrazole-1-carboxamide (37)
The reaction was carried out following the general procedure de-
scribed as above, dissolving 14q (0.10 g, 0.80 mmol) in anhyd pyri-
dine (1.0 mL) and employing hexyl isocyanate (0.18 mL, 1.21 mmol).
The crude was purified by silica gel column chromatography (cyclo-
hexane/EtOAc, 90:10) to afford 37 (0.16 g, 81%) as a colorless oil.
1
Pharmacology
H NMR (400 MHz, CDCl ): δ = 7.20–7.26 (m, 1 H), 5.96 (s, 1 H), 3.34
Preparation of Enzyme-Enriched Lysate: Hek293 cells stably express-
3
19
(
td, J = 6.0, 7.2 Hz, 2 H), 3.03 (q, J = 0.9, 7.4 Hz, 2 H), 2.57 (q, J = 7.6 Hz,
H), 1.53–1.67 (m, 2 H), 1.28–1.45 (m, 6 H), 1.24 (dt, J = 7.5, 9.2 Hz, 6
H), 0.88 (t, J = 6.9 Hz, 3 H).
ing human AC (Hek293-hAC) were suspended in 20 mM Tris·HCl
(pH 7.5) with 0.32 M sucrose, sonicated and centrifuged at 800 g for
30 min at 4 °C. Supernatants were then centrifuged at 12 000 g for 30
min at 4 °C. Pellets were re-suspended in PBS buffer (pH 7.4) and sub-
jected to three freeze-thaw cycles at –80 °C. The suspension was fi-
nally centrifuged at 105 000 g for 1 h at 4 °C and protein concentra-
tion was measured in the supernatant with bicinchoninic acid based
protein assay.
2
13
C NMR (101 MHz, CDCl ): δ = 155.5, 151.4, 149.6, 105.7, 40.2, 31.9,
3
29.7, 26.5, 22.6, 21.6, 21.4, 14.3, 13.0.
MS (ESI): m/z = 252 [M + H]+.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–R

Q
Synthesis
E. Diamanti et al.
Feature
Fluorescence-Based in Vitro Assay
quantified in the multiple reaction monitoring (MRM) mode. The
capillary voltage was set at 3 kV. The cone voltage was set at 25 V. The
source temperature was set to 120 °C. Desolvation gas and cone gas
Compounds were assessed for their ability to inhibit hydrolysis of the
fluorogenic substrate, N-[(1S,2R)-2-hydroxy-1-(hydroxymethyl)-4-
(
N ) flow were set to 800 and 50 L/h, respectively. Desolvation tem-
(2-oxochromen-7-yl)oxybutyl]dodecanamide, which is converted to
2
perature was set to 600 °C. Detection and analysis were controlled by
Waters MassLynx software version 4.1. Calibration curves were pre-
pared for every experiment. Data were normalized for protein con-
centration, measured using the bicinchoninic acid (BCA) assay
umbelliferone by AC in the fluorescence-based in vitro assay, as previ-
20,34
ously described.
Lysosomal lysate, enriched with AC, was pre-
pared from Hek293 cells stably expressing human AC and preincubat-
ed with test compounds and a positive control (diluted 20 × from
DMSO stock solutions at different concentrations) for 10 min. Then,
the fluorogenic substrate (diluted 40 × from EtOH stock solution, final
concentration 5 μM) was added and the mixture was incubated for 3
(Pierce).
Cell Lines
h at 37 °C, stopped with MeOH, and treated with NaIO (fresh solution
4
G361 human melanoma cells were purchased from Sigma Aldrich, Mi-
in 100 mM glycine/NaOH buffer pH 10.6) followed by 2 h incubation
at 37 °C in the dark. Fluorescence intensities were measured (exci-
tation/emission: 355/460 nm) and plotted as a function of compound
concentrations. IC50 values were calculated by nonlinear regression
analysis using GraphPad Prism 5 (GraphPad Software Inc., CA, USA)
applying a standard slope curve fitting. Fluorescence-based assay was
used for in vitro IC50 determinations.
lan, Italy. Cells were cultured at 37 °C and 5% CO in Dulbecco’s Modi-
fied Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 2
mM L-glutamine and antibiotics.
2
Sphingolipid Levels Measurement
Sphingolipids were quantified by LC-MS analysis according to the
36
method previously described.
AC Assay in Total Lysate
AC activity in G361 cells was performed using a previously described
Acknowledgment
LC-MS based method.1
8a,20
Briefly, cells (10 cells) were suspended in
TENTN buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X100,
.25% NP40, 2 mM EDTA, protease inhibitors), subjected to sonication
6
The authors thank Andrea Armirotti and Giuliana Ottonello for ana-
lytical stability data, Abdul Basit for sphingolipid level measurements,
and Silvia Venzano for handling compounds.
0
and centrifuged at 800 g for 15 min at 4 °C. Supernatants were used as
total lysates. Lysates (50–100 μg protein) were incubated in AC buffer
(
100 mM sodium phosphate, 0.1% Nonidet P-40, 150 mM NaCl, 3 mM
DTT, 100 mM sodium citrate, pH 4.5) with 50 μM N-lauroyl ceramide
Nu-Chek Prep, Elysian, MN, USA) as substrate, reactions were carried
Supporting Information
(
on for 1 h at 37 °C. Reactions were stopped by addition of a mixture of
Supporting information for this article is available online at
CHCl /MeOH (2:1, v/v) containing 1 nmol 11-lauroleic acid (NuChek
http://dx.doi.org/10.1055/s-0035-1561456.
S
u
p
p
ortioIgnfmr oaitn
S
u
p
p
o
nrtogI
i
f
rm oaitn
3
Prep). The organic phases were collected, dried under nitrogen and
analyzed by UPLC/MS (Acquity, Waters) in the negative-ion mode
monitoring the reaction product (lauric acid, m/z = 199) using 11-lau-
roleic acid as internal standard. Blank samples without protein ex-
tract or without substrate were used as negative controls.
References
(
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Lipids were extracted according to Bligh and Dyer.35 Samples were
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©
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