Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01845

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the degradation of the endocannabinoid neurotransmitter 2-arachidonoylglycerol, which is implicated in many physiological processes. Beyond the possible utilization of MA

Full text of DOI:10.1021/acs.jmedchem.7b01845

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Article
Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent,
Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors
Mojgan Aghazadeh Tabrizi, Pier Giovanni Baraldi, Stefania Baraldi, Emanuela
Ruggiero, Lucia De Stefano, Flavio Rizzolio, Lorenzo Di Cesare Mannelli, Carla
Ghelardini, Andrea Chicca, Margherita Lapillo, Jürg Gertsch, Clementina Manera, Marco
Macchia, Adriano Martinelli, Carlotta Granchi, Filippo Minutolo, and Tiziano Tuccinardi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01845 • Publication Date (Web): 08 Jan 2018
Downloaded from http://pubs.acs.org on January 8, 2018
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Discovery of 1,5ꢀDiphenylpyrazoleꢀ3ꢀCarboxamide
Derivatives as Potent, Reversible, and Selective
Monoacylglycerol Lipase (MAGL) Inhibitors
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Mojgan Aghazadeh Tabrizi,§ Pier Giovanni Baraldi,§ Stefania Baraldi,§ Emanuela Ruggiero,§
,ϕ
Lucia De Stefano,⊥ Flavio Rizzolio,‡,ϕ Lorenzo Di Cesare Mannelli,ǁ Carla Ghelardini,ǁ Andrea
Chicca,# Margherita Lapillo,†,# Jürg Gertsch,# Clementina Manera,† Marco Macchia,† Adriano
Martinelli,† Carlotta Granchi,† Filippo Minutolo,† and Tiziano Tuccinardi†,ψ,
*
§ Department of Chemical and Pharmaceutical Sciences, University of Ferrara, 44121 Ferrara, Italy
⊥ Graduate School in Chemistry, University of Trieste, 34127 Trieste, Italy
† Department of Pharmacy, University of Pisa, 56126 Pisa, Italy
ϕ
Division of Experimental and Clinical Pharmacology, Department of Molecular Biology and
Translational Research, National Cancer Institute and Center for Molecular Biomedicine, 33081
Aviano (PN), Italy
‡ Department of Molecular Science and Nanosystems, Ca' Foscari Università di Venezia, 30172
VeneziaꢀMestre, Italy
ǁ Department of Neuroscience, Psychology, Drug Research and Child Health, Section of
Pharmacology and Toxicology, University of Firenze, 50139 Firenze, Italy
# Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CHꢀ
3012 Bern, Switzerland
ψ Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College
of Science and Technology, Temple University, 19122 Philadelphia, PA, USA
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ABSTRACT
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Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the
degradation of the endocannabinoid neurotransmitter 2ꢀarachidonoylglycerol, which is implicated in
many physiological processes. Beyond the possible utilization of MAGL inhibitors as antiꢀ
inflammatory, antiꢀnociceptive and antiꢀcancer agents, their application has encountered obstacles
due to the unwanted effects caused by the irreversible inhibition of this enzyme. The possible
application of reversible MAGL inhibitors has only recently been explored, mainly due to the
deficiency of known compounds possessing efficient reversible inhibitory activities. In this work,
we report a new series of reversible MAGL inhibitors. Among them, compound 26 showed to be a
potent MAGL inhibitor (IC50 = 0.51 µM, Ki = 412 nM) with a good selectivity versus fatty acid
amide hydrolase (FAAH), α/βꢀhydrolase domainꢀcontaining 6 (ABHD6) and 12 (ABHD12).
Interestingly, this compound also possesses antiproliferative activities against two different cancer
cell lines and relieves the neuropathic hypersensitivity induced in vivo by oxaliplatin.
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INTRODUCTION
The endocannabinoid system is characterized by a set of neuromodulatory lipids and their receptors,
which are involved in a multiplicity of physiological and pathological conditions. Two different
types of G proteinꢀcoupled receptors (GPCRs) have been discovered and named as central
cannabinoid (CB1) and peripheral cannabinoid (CB2) receptors.1 Endocannabinoid signaling
regulates various phases of mammalian physiological processes, including pain perception, feeding,
emotional state, learning and memory.2ꢀ4 Endocannabinoids are hypothesized to act as retrograde
messengers, where stimulation of the postsynaptic neuron activates the biosynthesis of
endocannabinoids, which are released and transported to activate CB1 receptors expressed mainly in
the CNS on the presynaptic terminal.5 Endocannabinoids are biosynthesized on demand from
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phospholipid precursors at the plasma membrane levels and then released in the extracellular
milieu.6 After activating the receptors, endocannabinoids are transported into the cytoplasm via
facilitated diffusion and degraded by specific enzymes. Several evidence in vitro and in vivo
support the existence of a putative endocannabinoid membrane transporter which mediates the
bidirectional movement of endocannabinoids through the plasma membrane7, 8 in cooperation with
other proteins such as hydrolytic enzymes and intracellular carrier proteins.9, 10 The complex
enzymatic cascades that regulate endocannabinoid production and inactivation include different
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hydrolytic enzymes, such as the fatty acid amide hydrolase (FAAH), Nꢀacylethanolamine
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hydrolyzing acid amidase (NAAA) and monoacylglycerol lipase (MAGL).3,
The selective
diacylglycerol lipases (DAGL) α and β are thought to catalyze the formation of 2ꢀ
arachidonoylglycerol (2ꢀAG), and MAGL degrades this compound into arachidonate and glycerol.
Anandamide (AEA) is a partial CB1 agonist and a weak partial CB2 agonist, whereas 2ꢀAG acts as a
full CB2 agonist.12 The 2ꢀAG levels in the brain are about three orders of magnitude higher than
AEA levels, although the relevance of this difference on their signaling actions is still uncertain,
mainly taking into consideration that their basal extracellular levels, as measured by in vivo
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microdialysis, are within 2ꢀ to 5ꢀfold.13,
In addition to extracellular CB receptors,
endocannabinoids interact with other receptors like the vanilloid receptor 1 (VR1), the transient
potential vanilloid type 1 channels (TRPV1) and other specific GPCRs such as opioid or GPR55
and GPR35 receptors.15 The discovery that the endocannabinoids work together with different
receptor sites has contributed to the identification of this signaling system as a flexible tool to
control diverse functions in the cells and tissues of the organism. Numerous studies suggest the
potential use of selective inhibitors implicated in enzymatic degradation of endocannabinoids that
could provide selective therapeutic chances.16 To reduce the problems connected with CB1 agonists,
an amplification of the actions of AEA and 2ꢀAG by inhibiting their enzymatic degradation has
emerged as a potential strategy to develop the endocannabinoid system for medicinal purposes.
Pharmacological inhibition of FAAH and MAGL was found to decrease pain, inflammation,
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anxiety, and depression in rodent models without the undesired side effects in motility and behavior
observed with direct CB1 agonists.3 First generation MAGL inhibitors include URB602 (compound
1), NꢀArachidonoyl maleimide (NAM, compound 2), and OMDM169 (compound 3).17 Carbamate
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compound 1 (Figure 1) was reported as a MAGL inhibitor with relatively low potency (IC50 = 28
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ꢁM)18 and it was shown to be equally potent against FAAH in vitro.19,
Furthermore, its
administration was shown to attenuate nociception in rodent models of acute, inflammatory, and
neuropathic pain.21, 22 Compound 2 (Figure 1) is an irreversible MAGL inhibitor (IC50 = 140 nM)
that was found to decrease 2ꢀAG hydrolase activity of rat cerebellar membranes.23 NAM is not
selective due to its chemically reactive maleimide functional group that will likely react with many
cysteineꢀcontaining proteins in vivo, thus limiting its use in physiologic studies. Another
carbamateꢀbased derivative 3 (OMDM169, Figure 1) is a MAGL competitive inhibitor (IC50 = 0.89
ꢁM)24 that increased the 2ꢀAG levels at the site of formalinꢀinduced paw inflammation but it also
inhibited pancreatic lipase and DAGLꢀα, because of its structural similarity with tetrahydrolipstatin.
Selective pharmacological tools able to interrupt the in vivo MAGL activity have only become
accessible within the last few years. By now, they have been used to demonstrate the role of this
enzyme in 2ꢀAG signaling termination and the potential translational use of targeting MAGL in the
treatment of nervous system disorders such as pain, anxiety, drug addiction, nausea, and
neuroinflammation.25ꢀ27 Furthermore, MAGL is upregulated in aggressive cancer cells and primary
tumors and its inhibition in aggressive breast, ovarian and melanoma cancer cells impairs cell
migration, invasiveness and tumorigenicity.28 Confirmation of MAGL as the primary brain 2ꢀAG
hydrolase was achieved by the generation of a selective and in vivo active MAGL inhibitor, JZL184
(4, Figure 1). This piperidine carbamate is a potent and selective MAGL inhibitor (IC50 = 8 nM)
that when administered to mice, elevated brain 2ꢀAG levels leading to several cannabinoidꢀrelated
behavioral effects.29 Almost all the reported compounds are characterized by an irreversible MAGL
inhibition mechanism and as reported by Scholsburg et al., the irreversible inhibition of MAGL
produces crossꢀtolerance to CB1 agonists in mice after repeated administrations.30 Furthermore,
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chronic MAGL blockade causes physical dependence, impairs endocannabinoidꢀdependent synaptic
plasticity and desensitizes brain CB1 receptors.30 Considering these drawbacks associated with an
irreversible MAGL inhibition, the development of reversible inhibitors could represent a promising
alternative strategy; however, their use has been investigated only partially so far, mainly because
of the lack of compounds with good MAGL reversible inhibition properties. In 2012 Cisneros et al.
reported a series of oxirane derivatives characterized by a dual MAGL/FAAH reversible inhibition
activity;31 whereas in 2014 HernandezꢀTorres et al. reported the reversible MAGL inhibitor
benzo[d][1,3]dioxolꢀ5ꢀylmethyl 6ꢀ([1,1′ꢀbiphenyl]ꢀ4ꢀyl)ꢀhexanoate 5 (compound c21, Figure 1).
Interestingly, this compound was also tested in vivo and its therapeutic effects were not
accompanied by catalepsy or other motor impairments that have been observed instead after the
administration of irreversible MAGL inhibitors.32 In 2015 Patel et al. developed a series of
Loratadine analogues that showed potent and reversible inhibition of MAGL, accompanied also by
selectivity towards human FAAH. Furthermore, one of these derivatives proved to possess
antagonistic activity towards the Histamine H1 receptor and showed selectivity against cannabinoid
receptors, α/βꢀhydrolase domainꢀcontaining 6 (ABHD6) and α/βꢀhydrolase domainꢀcontaining 12
(ABHD12).33 Finally, very recently Granchi et al. reported a benzoylpiperidine derivative
characterized by a good MAGL inhibitory activity (IC50 = 0.84 µM) and a reversible mechanism of
inhibition.34
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O
NH
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O
N
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N
H
10
O
1, URB602
3, OMDM169
2,
NAM
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O
O
O
O
OH
O
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O2N
N
O
O
5
O
5, c21
4,
JZL-184
Figure 1. Representative MAGL inhibitors.
In the present study, we report the development of a diphenylpyrazole series as a new class of
MAGL reversible inhibitors.
RESULTS AND DISCUSSION
From the analysis of the main available data concerning MAGL reversible inhibition,34ꢀ36 some
pharmacophoric elements seem to be important for inhibitory activity: a) a carbonyl group able to
interact into the oxyanion hole of the enzyme, b) an aromatic portion comprising also an
heterocyclic nucleus, involved in van der Waals interactions in a closed hydrophobic region of the
binding site (red region of Figure S1) and c) a second aromatic portion which interacts in the open
large hydrophobic cavity of MAGL (green region of Figure S1). Following this binding analysis,
we hypothesized that the 1,5ꢀdiphenylpyrazole scaffold, properly substituted with a carbonyl group
and different fragments, could represent the starting point for the identification of new reversible
MAGL inhibitors. Following these indications, we synthesized three different diphenylpyrazole
derivatives (6ꢀ8, Table 1) characterized by different substituents. The compounds were thus tested
for their MAGL inhibition activity together with the CAY10499 derivative,37 which was used as a
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reference compound. As shown in Table 1, the (5ꢀ(4ꢀhydroxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ3ꢀyl)(4ꢀ
(pyrimidinꢀ2ꢀyl)piperazinꢀ1ꢀyl)methanone (8) showed an appreciable activity with an IC50 value of
6.8 µM, whereas the other two compounds were inactive.
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Table 1. MAGL inhibitory activities of diphenylpyrazole derivatives.
compd
Structure
IC50 (µM)
>> 200
6
>> 200
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6.8 ± 0.4
0.14 ± 0.02
CAY10499
As shown in Figure 2, the docking results for this compound highlight the presence of two Hꢀbonds
between the carbonyl oxygen of the compound and the nitrogen backbone of A51 and M123 in the
oxyanion hole. The piperazinylpyrimidine fragment is inserted into the closed hydrophobic region
of the binding site and shows lipophilic interactions with I179, L184, V270 and a πꢀπ interaction
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with Y194. The Nꢀphenyl ring shows lipophilic interactions with L148, A151, L213 and L241,
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whereas the 5ꢀphenol ring shows lipophilic interactions with L205 and a weak Hꢀbond with S176.
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Figure 2. Docking of compound 8 into MAGL.
Using this compound as a starting hit, a first round of structural investigation has been carried out
by maintaining the central pyrazole core fixed and varying: 1) the substituent present on the
nitrogen atom (phenyl or methyl group) of the pyrazole ring (R2, Table 2); 2) the portion linked to
the carbonyl group in position 3 of the pyrazole ring (R3, Table 2) and 3) the position of the
hydroxyl group of the phenolic ring in position 5 (R1, Table 2). As shown in Table 2, the
substitution of the 1ꢀphenyl ring with a methyl group determined an important decrease of activity
(compound 9) compared to 8, as well as the shift of the pꢀhydroxy group to the meta position
(compound 10). The replacement of the pyrimidine with a phenyl ring determined a slight increase
of activity (11, IC50 = 4.7 µM), but when a benzyl group was inserted in place of the phenyl the
activity was about twoꢀfold lower (14, IC50 = 12 µM). When pyrimidine was replaced with a methyl
group (12) or the entire piperazinylpyrimidine fragment with a morpholine ring (13) a tenꢀfold
decrease of activity was observed. Conversely, the best result was achieved by the replacement of
the piperazinylpyrimidine with the 4ꢀbenzylpiperidine, which led to a 3.5 fold increase of the
activity (15, IC50 = 2.0 µM). Finally, the replacement of the 4ꢀbenzylpiperidine with larger
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dimension groups such as the 1ꢀbenzylpiperidinꢀ4ꢀamine of compound 16 and the 2ꢀ(4ꢀ(pyrimidinꢀ
2ꢀyl)piperazinꢀ1ꢀyl)ethanamine of compound 17 led to a completely loss of activity.
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Table 2. MAGL inhibitory activities of 8 derivatives.
O
R3
N
R1
N
R2
compd
8
R1
R2
R3
IC50 (µM)
4ꢀOH
6.8 ± 0.4
4ꢀOH
3ꢀOH
ꢀCH3
132 ± 20
48 ± 2
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10
4ꢀOH
4ꢀOH
4ꢀOH
4.7 ± 0.1
71 ± 3
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61 ± 15
4ꢀOH
4ꢀOH
4ꢀOH
12 ± 1
2.0 ± 0.1
>> 200
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4ꢀOH
>> 200
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As shown in Figure 3, with respect to the piperazinylpirimidine of derivative 8, the 4ꢀ
benzylpiperidine of 15 is inserted deeper inside the closed hydrophobic region of the binding site
and shows stronger lipophilic interactions with L184, V270 and the πꢀπ interaction with Y194. This
different disposition determines also a slight different orientation of the 5ꢀ(4ꢀhydroxyphenyl)ꢀ1ꢀ
phenylꢀ1Hꢀpyrazole fragment with the formation of an Hꢀbond between the hydroxyl group and the
oxygen backbone of P178.
Figure 3. Docking of compound 15 (green) into MAGL. Compound 8 (cyan) is displayed as a
reference compound.
In order to verify the role of the pꢀhydroxyl in the binding process, this group was removed or
replaced with a chlorine atom. As shown in Table 3, in both cases (compounds 19 and 18), the
compounds showed a decrease of MAGL inhibitory activity (IC50 value of 13 and 11 µM,
respectively), supporting the possible attractive interaction of the pꢀhydroxy group with the enzyme,
as suggested by modeling studies. The comparison between the disposition of compounds 8 and 15
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suggests that the phenyl ring of 15 bound to the nitrogen atom in position 1 of the pyrazole ring
appears to be shifted towards the central region of the open binding cavity and could have weaker
lipophilic interactions with L148, A151 and L241, when compared to the phenyl ring of analogue 8.
On these bases, the phenyl ring of 15 was substituted with different groups, such as the pꢀmethoxy
(20), pꢀchloro (21) and pꢀmethyl (22) and with different aromatic portions such as the benzyl (23),
phenylethyl (24) and phenylpropyl (25) fragments. The enzymatic assays revealed that the
introduction of the benzyl group such as in compound 23 determined the highest increase of
inhibitory activity (IC50 = 0.81 µM, Table 3). Then, in order to maximize the lipophilic interactions
of this fragment, methyl groups in para and meta position of the benzyl moiety were introduced
(26, 27 and 28). Finally, a naphtylmethyl group (29) was inserted in the place of the benzyl group.
As shown in Table 3, the metaꢀmethyl substitution of the benzyl ring determined a further slight
improvement of the MAGL inhibitory activity, with a resulting IC50 of 0.51 µM (compound 26).
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Table 3. MAGL inhibitory activities of 4ꢀbenzylpiperidines (compound 15 derivatives).
O
N
R1
N
N
R2
compd
18
R1
R2
IC50 (µM)
Cl
11 ± 3
H
13 ± 1
19
20
21
OH
OH
3.2 ± 0.1
1.4 ± 0.2
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OH
1.5 ± 0.1
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OH
OH
OH
0.81 ± 0.02
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4.3 ± 0.4
1.6 ± 0.1
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OH
OH
0.51 ± 0.03
1.1 ± 0.1
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OH
OH
1.4 ± 0.1
1.6 ± 0.3
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29
As shown in Figure 4, the binding disposition of compound 26 is very similar to that observed for
15, maintaining the lipophilic interactions of the 4ꢀbenzylpiperidine with L184, V270 and the πꢀπ
interaction with Y194, the interaction of the carbonyl group in the oxyanion hole and the Hꢀbond
between the pꢀhydroxyl group and the oxygen backbone of P178. Differently from 15, the mꢀ
methylbenzyl fragment points towards the surface of the binding site and shows stronger lipophilic
interactions with in particular A151 and L241.
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Figure 4. Docking of compound 26 (orange) into MAGL. Compound 8 (cyan) is displayed as a
reference compound.
With the aim of evaluating the reversible or irreversible mechanism of inhibition, the effects of
dilution and preincubation on the inhibitory ability of compound 26 were evaluated. In the dilution
experiments, in case of an irreversible inhibition, the potency should not decrease after dilution.
Differently, in case of a reversible inhibition, the potency level should be substantially reduced after
dilution.38 In our experiment, the inhibition produced by preincubation with a 20 µM concentration
of 26 was measured after a 40X dilution and compared to the potency observed by a 20 µM and a
0.5 µM of compound 26. As shown in Figure 5A, 26 showed a reversible inhibition mechanism, as
the inhibition produced by 0.5 µM of the compound was similar to that of 40X dilution, and was
different to that produced by the compound at a concentration of 20 µM. As a second test, the
activity of 26 was assayed at different preincubation times of the compound with MAGL. In this
assay, the compound is incubated with the enzyme 30 and 60 minutes before the addition of the
substrate and the observed IC50 is then compared with that obtained without a preincubation of the
compound with the enzyme. An irreversible inhibitor will show a higher potency with a higher
incubation time whereas a reversible inhibitor will show a constant inhibition potency independent
from the incubation time. As shown in Figure 5B, this assay confirmed the reversible property of
26, as it did not show any significant increase in inhibition potency with different incubation times.
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Figure 5. Compound 26ꢀMAGL inhibition analysis. A) Dilution assay: the first two columns
indicate the inhibition percentage of compound 26 at a concentration of 20 µM and 0.5 µM. The
third column indicates the inhibition percentage of compound 26 after dilution (final concentration
= 0.5 µM). B) IC50 (µM) values of 26 at different preincubation times with MAGL (0 min, 30 min
and 60 min).
Once confirmed the reversible mechanism of compound 26, its inhibition mode was then
determined by evaluating MichaelisꢀMenten kinetics with various inhibitor concentrations. The
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dataset was plotted as substrate concentration versus MAGL activity and evaluated by using the
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mixedꢀmodel inhibition fit of GraphPad Prism 5.0, thus including competitive, uncompetitive, and
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noncompetitive inhibition terms. This analysis produces the Vmax, Km, Ki and the α value, a
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parameter consisting of a positive number (α is always greater than zero) that can be used as an
indicator of the mechanism of inhibition. In fact, when α is smaller than one, then the mixed model
becomes nearly identical to an uncompetitive model; when it is equal to one, then the mixedꢀmodel
is identical to a noncompetitive inhibition; finally, when α has a very large value, then the mixedꢀ
model becomes identical to a competitive inhibition. As shown in Figure 6, the MichaelisꢀMentenꢀ
type curve resulted in Km values of 0.21 ± 0.07 mM, Vmax value of 42 ± 1 µmol•(minꢀ1mgꢀ1), a Ki
value for 26 of 412 ± 19 nM and an α value greater than 100000, thus supporting a competitive
behavior for the selected compound.
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Figure 6. Inhibition of the activity of MAGL and competitive nature of compound 26.
Selectivity analysis. With the aim of evaluating the selectivity of compound 26 versus the other
major endocannabinoid degrading enzymes, its activities against FAAH, ABHD6 and ABHD12
were tested. As shown in Table 4, compound 26 did not show any significant inhibition of FAAH
and ABHD12 at the concentration of 10
µM (7% and 32% of inhibition for FAAH and ABHD12,
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respectively), while ABHD6 was actually inhibited with an IC50 value of 7.1 ± 2.3 µM. Thus
compound 26 behaves as MAGL inhibitor showing good selectivity over FAAH, ABHD6 and
ABHD12 (see the Experimental section for details). Finally, since the diarylpyrazoles are a wellꢀ
known class of cannabinoid receptor ligands,39 the possible affinities of compound 26 for the
cannabinoid receptors CB1 and CB2 were also evaluated. As reported in Table 4 this compound did
not show a significant binding to any of the two cannabinoid receptor subtypes.
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Table 4. Biological activity of compound 26 on the major components of the endocannabinoid
system. Effects on FAAH, ABHD6, ABHD12, CB1 and CB2 receptors are expressed as IC50 value
(mean ± SD, µM). In bracket the % enzymatic inhibition (FAAH and ABHD12) or receptor binding
(CB1 and CB2 receptors) at 10 ꢁM is reported.
compd
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FAAH
ABHD6
ABHD12
CB1 binding CB2 binding
> 10 (22%) > 10 (35%)
> 10 (7%)
7.1 ± 2.3
> 10 (32%)
Antiproliferative assays. Compound 26 was further tested in in vitro experiments to evaluate its
antiproliferative potency against cancer cells, together with compound CAY10499, which was used
as the reference compound. As suggested by literature data, MAGL is an optimal candidate target
for ovarian cancer.28 Western blot analysis has recently highlighted that MAGL is overexpressed in
OVCAR3 and CAOV3 compared to OVSAHO and COV318 cell lines.34 These cell lines were
selected since they represent more closely highꢀgrade serous ovarian cancer (HGꢀSOC),40 a fatal
tumor. At the molecular level, all cell lines are mutated in the TP53 gene, BRCA2 gene (OVSAHO)
or the cell cycle pathway (OVSAHO, COV318, OVCAR3), which are common events in HGꢀSOC.
Therefore, the antiproliferative activity of the two compounds was tested against these four cell
lines, together with the noncancerous human fibroblast lung cells (MRC5). As shown in Table 5,
compound 26 caused a good inhibition of cell viability, with IC50 values of 12 and 11 ꢁM in the
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OVCAR3 and CAOV3 cell lines, respectively, whereas it proved to be about 7ꢀfold less potent
against ovarian cancer cells that do not overexpress MAGL, such as OVSAHO and COV318 cells.
Furthermore, 26 proved to be inactive also against noncancerous human fibroblast lung cells
(MRC5, IC50 > 100 ꢁM). The covalent reference inhibitor CAY10499 showed inhibition of cell
viability in all the four ovarian cancer cell lines, with IC50 values ranging from 23 to 77 ꢁM without
any significant discrimination between the two MAGLꢀoverexpressing cell lines OVCAR3 and
CAOV3 and the other two lines, OVSAHO and COV318.
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Table 5. Cell growth inhibitory activities (IC50) of compounds 24 and CAY10499.
IC50 values (µM)
OVSAHO
90 ± 6
OVCAR3
12 ± 2
COV318
78 ± 6
CAOV3
11 ± 1
MRC5
> 100
> 100
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23 ± 2
43 ± 6
59 ± 4
77 ± 7
CAY10499
Antinociceptive effects of 26 in animal model of neuropathic pain. The effect of 26 was
evaluated in a mouse model of nociceptive behavior caused by the chemotherapeutic agent,
oxaliplatin (cold plate test). The pain threshold measurements of oxaliplatinꢀtreated animals are
shown in Figure 7. The cold plate test allows to evaluate the response to a thermal non noxious
stimulus defined as allodyniaꢀlike. On day 15, oxaliplatin decreased the licking latency to 10.0 ± 0.5
s in comparison to control mice (17.6 ± 0.4 s). A single treatment p.o. with 26 relieved pain doseꢀ
dependently. The lower dose of 10 mg/kg induced a significant effect 30 min after the
administration. At 30 mg/kg the compound reduced oxaliplatinꢀinduced neuropathic pain 30 and 45
min after treatment and in particular, after 30 min it increased pain threshold to a value similar to
that shown by control animals (vehicle + vehicle).
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Figure 7. Effect of 26 on oxaliplatinꢀinduced neuropathic pain. Oxaliplatin (2.4 mg/kg) was
dissolved in 5% glucose solution and administered i.p. on days 1ꢀ2, 5ꢀ9, 12ꢀ14. On day 15, 26 (10
and 30 mg/kg) was suspended in carboxymethylcellulose (CMC) and p.o. administered. Painꢀ
related behavior (i.e. lifting and licking of the hind paw) was observed by the cold plate test and the
time (s) of the first sign was recorded. **P<0.01 vs Vehicle + Vehicle treated animals; ^P<0.05 and
^^P<0.01 vs oxaliplatin + Vehicle group. Each value represents the mean of 10 mice performed in 2
different experimental sets.
In order to test the effects of the compound after repeated treatments, 26 (30 mg/kg, p.o.) was coꢀ
administered with oxaliplatin from day 1 to day 14 following the same protocol. Pain threshold was
evaluated by cold plate test on days 7 and 15, 24 h after the last administration. As shown in Figure
8 (upper panel), oxaliplatin induced a significant hypersensitivity both the first and the second
week. Compound 26 significantly reduced cold allodynia on day 7 as well as on day 15. The effect
progressively increased overtime. On day 15, 24 h after the last administration, mice treated with 26
showed a complete pain relief. These results highlight: i) the long lasting efficacy of the compound
after repeated treatments, ii) the increasing activity during treatment, suggesting protective
properties against oxaliplatin neurotoxicity, iii) the complete lack of tolerance development.
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Figure 8. Antiꢀneuropathic pain activity of 26 after subchronic treatment. Oxaliplatin (2.4 mg/kg)
was dissolved in 5% glucose solution and administered i.p. on days 1ꢀ2, 5ꢀ9, 12ꢀ14. 26 (30 mg/kg)
was suspended in carboxymethylcellulose (CMC) and administered p.o. using the same protocol as
that for oxaliplatin alone. The cold plate test was performed 24 h after the last administration on
days 7 and 15 observing the painꢀrelated behavior (i.e. lifting and licking of the hind paw).
**P<0.01 vs vehicle + vehicle treated animals; ^P<0.05 and ^^P<0.01 vs oxaliplatin + vehicle
group. Each value represents the mean of 10 mice performed in 2 different experimental sets.
We also quantified by LCꢀMS/MS the amount of 2ꢀAG, AEA, arachidonic acid, prostaglandinꢀE2
(PGE2) and prostaglandinꢀD2 (PGD2) in brain and spinal cord after 15 days of treatment. Animals
treated with oxaliplatin and compound 26 did not show any significant difference in 2ꢀAG levels as
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compared to oxaliplatinꢀtreated mice (see Figure S2). Previous studies reported that covalent
MAGL inhibitors exert significant antiꢀallodynic effects in different mouse models of neuropathic
pain.41ꢀ44 However, in these animal models MAGL inhibition exerted opposite effects on the
modulation of 2ꢀAG concentrations in brain, spinal cord and dorsal root ganglia (DRGs). For
example, Kinsey at al. reported a marked increase of 2ꢀAG levels in brain and spinal cord of mice
treated with a single injection of JZL184 (16 mg/kg, ip) 14 days after chronic constriction injury
(CCI) surgery.41 On the contrary, Khasabova et al. reported no significant change in 2ꢀAG levels in
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brain and spinal cord after 7 days of treatment with cisplatin and JZL184 (10 µg, subcutaneous
injection).45 In the same study, JZL184 was able to recover the cisplatinꢀinduced reduction of 2ꢀAG
level in DRGs. Wilkerson et al. showed that suprathreshold dose of MJN110 (0.1432 mg/kg, ip)
could only marginally increase 2ꢀAG level in the spinal cord of mice subjected to CCI surgery
while not affecting 2ꢀAG level in the brain.44 Our data showed that compound 26 did not increase 2ꢀ
AG levels in brain and spinal cord of mice treated for 15 days, despite inducing significant antiꢀ
allodynic effects. This may be due to a mild and specific increase of 2ꢀAG concentrations in
specific brain or spine regions. Alternatively, compound 26 may exhibit only a negligible
penetration or activity in the central nervous system. Previous studies showed that the antiꢀallodynic
effect of FAAH and MAGL inhibitors is associated with the modulation of AEA and 2ꢀAG levels in
DRGs.45, 46 Thus, we may speculate that the antiꢀallodynic effect induced by compound 26 could be
associated with the modulation of 2ꢀAG levels in the peripheral nervous system (i.e. DRGs). In
agreement with this hypothesis, it was shown that the disposition of cisplatin is limited to peripheral
tissues and DRGs were the most vulnerable neural structure.47 However, we cannot exclude the
possibility that the in vivo effects showed by compound 26 may also be dependent by alternative
mechanisms beyond MAGL inhibition.
Chemistry. The diphenylpyrazole compounds 6ꢀ29 were prepared as shown in Scheme 1. Claisen
condensation of commercially available acetophenones 28bꢀd or 4′ꢀbenzyloxyacetophenone 28a48
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(for the preparation of compounds 6 and 7) with diethyl oxalate afforded the corresponding
diketoesters 29aꢀd49 in quantitative yields. Following cyclization with hydrazines gave both the
corresponding 1,5ꢀdisubstitutedꢀpyrazoleꢀ3ꢀcarboxylic acid esters and the low yield isomer 1,3ꢀ
diphenylꢀ5ꢀcarboxylates, which were easily separated by column chromatography on silica gel.50
Esters 30aꢀp were converted to the corresponding carboxylic acids 31aꢀp by treatment with
aqueous sodium hydroxide. The pyrazoleꢀ3ꢀcarboxylic acids were converted to the corresponding
carboxamide derivatives 6ꢀ29 via coupling reaction with the appropriate amines.
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Scheme 1a
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aReagents and conditions: (i) Diethyl oxalate, NaH, THF, reflux, 1 h; (ii) appropriate hydrazine,
absolute EtOH, 80 °C, 5 h; (iii) NaOH aq (10%), ethanol, reflux, 60 °C, 4 h; (iv) appropriate amine,
EDCI, HOBt, DMF, rt, 16 h.
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CONCLUSIONS
In the present work we have reported a new class of MAGL reversible inhibitors. Starting from (5ꢀ
(4ꢀhydroxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ3ꢀyl)(4ꢀ(pyrimidinꢀ2ꢀyl)piperazinꢀ1ꢀyl)methanone (8) a
structural optimization has been performed leading to the identification of compound 26, which
displayed a high MAGL inhibition activity with a Ki of 412 nM. This reversible inhibitor was then
tested in cellꢀbased assays where it showed promising cell growth inhibitory activities in an
antiproliferative assay in the OVCARꢀ3 and CAOV3 cell lines that overexpress MAGL.
Furthermore, oral administration of 26 dose dependently reversed the lowering of the threshold to
cold stimuli (cold plate test) induced by oxaliplatin, indicating its efficacy in the treatment of
neuropathic pain. The efficacy increased after repeated administration suggesting a possible
protective effect in the absence of tolerance development, even if evidences about the in vivo actual
inhibition of MAGL after administration of the compound are needed. Taken together these results
suggest that these new compounds could open new perspectives for developing novel reversible
MAGL inhibitors.
EXPERIMENTAL SECTION
Pan Assay Interference Compounds (PAINS) analysis. In order to exclude the possibility that the
herein reported class of compounds could act as artifacts or promiscuous bioactive molecules, a
PAINS analysis for the most active compound (26) was carried out.51
PAINS substructural features screening. As reported by Baell and Holloway there is a number of
substructural features which could help to identify compounds that appear as PAINS in many
biochemical high throughput screens.52 The corresponding filters have been included in the Filterꢀ
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itTM software and compound 26 was thus filtered by using this program. The result highlighted that
this compound did not possess any of the substructural features shared by the most common
PAINS.
9
Interference analysis. Some PAINS are associated with color that could interfere photometrically
with the assay. In order to avoid this possibility for each compound concentration a blank analysis
was carried out, and the final absorbance values were obtained subtracting the absorbance
determined by the presence of all the components except the enzyme in the same conditions.
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Aggregation analysis. In order to exclude possible aggregation behaviors, all the enzymatic assays
were carried out in the presence of bovine serum albumin (BSA) 0.1 mg/ml.
Thiol reactive analysis. In some cases, PAINS properties could be caused by a covalent interaction
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between a ligand and cysteines of multiple proteins. In order to investigate this possibility, the
inhibition activity of 26 against MAGL was tested in the presence of the thiolꢀcontaining agent 1,4ꢀ
dithioꢀDLꢀthreitol (DTT).54 This assay suggested that the inhibition potency of 26 was not affected
by the presence of DTT, as it showed an IC50 = 0.50 ± 0.04 µM when assayed with 100 µM DTT,
thus excluding the interaction of this compound with the cysteine residues of the MAGL enzyme.
Selectivity testing. As reported by Dahlin and Walters55 a selectivity analysis is another test that
would contribute to highlight most PAINS. As reported in the text we measured the activity of
compound 26 against FAAH, ABHD6 and ABHD12 resulting in a good MAGL selectivity.
Orthogonal assay. The application of a different readout method for measuring the activity against
the target is another step that contribute to elucidate the PAINS properties. For this reason the
activity of 26 against MAGL was also tested by using an HPLC/UV assay.56 An aliquot of stock
solutions of MAGL was diluted 1:125 with Tris buffer (pH = 7.2; 10 mM, containing 1.0 mM
EDTA) to obtain the working solutions of MAGL at 1.6 ng/µl. The stock and working solutions
were stored at
−
20
°
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containing 1.0 mM EDTA), 5 µl of working solution MAGL (8 ng), 5 µl inhibitor (or solvent as
control) and 5 µl of a solution of 4ꢀnitrophenylacetate (4ꢀNPA) substrate in absolute ethanol (4.25
mM) were added. Samples were incubated for 10 min, then the enzymatic reaction was stopped by
cooling in an ice bath for 10 min and 20 µl of the reaction mixture were taken and analyzed by
HPLC. Shimadzu Prominence HPLC system was used to quantify 4ꢀnitrophenol formed after
enzymatic hydrolysis of 4ꢀNPA by using a diodeꢀarray detector at operation wavelength of 310 nm.
Separation of compounds was carried out on a Kinetex EVO C18 column (5 ꢁm, 150 mm × 4.6
mm, Phenomenex, Inc.). The mobile phase, delivered at a flow rate of 1.0 ml/min and consisted of
methanol and ammonium acetate buffer (pH 4.0; 10 mM) (53:47, v/v). The sample injection volume
was 20 µl. Data were monitored and analyzed using chromatography Software LabSolutions Lite
(Shimadzu). By using this assay method, compound 26 showed an IC50 against MAGL of 0.39 ±
0.05 µM, thus confirming the inhibition activity measured by the colorimetric assay.
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Docking Calculations. The crystal structure of human MAGL (pdb code 3PE635) was minimized
using Amber14 software57 and ff14SB force field at 300 K. The TIP3P explicit solvent model for
water, was used with a 10 Å water cap. In order to neutralize the system, sodium ions were added.
Two minimization steps of were performed; in the first stage, the protein was kept fixed with a
position restraint of 500 kcal/mol Å2 minimizing the positions of the water molecules. In the second
stage, the entire system was minimized through 5000 steps of steepest descent followed by
conjugate gradient (CG) until a convergence of 0.05 kcal/Å•mol. The ligands were built using
Maestro58 and were minimized by means of Macromodel59 in a water environment using the CG
method using the MMFFs force field. Docking analysis was performed by means of the
AUTODOCK 4.0 program;60 Autodock Tools61 was used in order to identify the torsion angles in
the ligands, add the solvent model and assign the Kollman atomic charges to the protein. The ligand
charge was calculated using the Gasteiger method. The ZYH reference inhibitor was used in order
to identify the binding site; a grid of 82, 40, and 30 points in the x, y, and z directions was
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constructed centered on the center of the mass of this compound. A grid spacing of 0.375 Å and a
distanceꢀdependent function of the dielectric constant were used for the energetic map calculations.
Using the Lamarckian Genetic Algorithm, the docked compounds were subjected to 100 runs of the
Autodock search, using 500000 steps of energy evaluation and the default values of the other
parameters. Cluster analysis was performed on the results using an RMS tolerance of 2.0 Å.
MAGL inhibition assay. Human recombinant MAGL, CAY10499 and 4ꢀNPA substrate were from
Cayman Chemical. The enzymatic reaction was carried out at room temperature (final volume of
200 ꢁL in 10 mM Tris buffer, pH 7.2, containing 1 mM EDTA and 0.1 mg/ml BSA). A total of 150
ꢁL of 4ꢀNPA 133.3 ꢁM (final concentration = 100 ꢁM) was added to 10 ꢁL of DMSO containing
the appropriate amount of compound and the reaction was initiated by the addition of 40 ꢁL of
MAGL (11 ng/well). The final concentration of the analyzed compounds ranged for CAY10499
from 10 to 0.00001 ꢁM and for the other compounds from 200 to 0.0128 ꢁM. After the reaction had
proceeded for 30 min, absorbance values were then measured by using a Victor X3 Microplates
Reader (PerkinElmer®) at 405 nm.37 The assay was generated in 96ꢀwell microtiter plates. Two
reactions were also run: one reaction containing no compounds and the second one containing
neither inhibitor nor enzyme. IC50 values were derived from experimental data using the Sigmoidal
dose−response fitting of GraphPad Prism software. To remove possible false positive results, for
each compound concentration a blank analysis was carried out, and the final absorbance results
were obtained detracting the absorbance produced by the presence of all the components except
MAGL in the same conditions. In the enzyme kinetics experiments, compound was tested in the
presence of scalar concentrations of 4ꢀNPA. It was added in scalar amounts (concentration range =
1–8 ꢁM) to a reaction mixture containing Tris buffer and scalar concentrations of 4ꢀNPA (15–1400
ꢁM). Finally, MAGL solution was added (11 ng/well). The MAGL activity was measured by
recording the increase in 4ꢀnitrophenol absorbance using the Victor X3 Microplates Reader
(PerkinElmer®). The experimental data were analyzed by nonꢀlinear regression analysis with
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GraphPad Prism software, using a second order polynomial regression analysis, and by applying the
mixedꢀmodel inhibition fit.
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FAAH inhibition assay. Human recombinant FAAH and AMC arachidonoylamide substrate were
from Cayman Chemical. The FAAH reaction was conducted at room temperature (final volume of
200 ꢁL in 125 mM Tris buffer, pH 9.0, containing 1 mM EDTA and 0.1 mg/ml BSA). A total of
150 ꢁL of AMC arachidonoylamide 13.3 ꢁM (final concentration = 10 ꢁM) was added to 10 ꢁL of
DMSO containing the appropriate amount of compound. The reaction was initiated by the addition
of 40 ꢁL of FAAH (0.9 µg/well) in such a way that the assay was linear over 30 min. The final
concentration of the analyzed compounds ranged for from 200 to 0.0128 µM. After the reaction had
proceeded for 30 min, fluorescence values were then measured by using a Victor X3 PerkinElmer
instrument at an excitation wavelength of 340 nm and an emission of 460 nm. As for MAGL assay,
two reactions were also run: one reaction containing no compounds and the second one containing
neither inhibitor nor enzyme. IC50 values were derived from experimental data using the Sigmoidal
dose−response fitting of GraphPad Prism software. To remove possible false positive results, for
each compound concentration a blank analysis was carried out, and the final fluorescence results
were obtained detracting the fluorescence produced by the presence of all the components except
FAAH in the same conditions. Furthermore, compound 26 was also tested for FAAH inhibition
using mouse brain homogenate as previously reported.8
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CB1 and CB2 receptor binding assays. Receptor binding experiments were performed with
membrane preparations as previously reported.62 Briefly, clean membranes expressing hCB1 or
hCB2 were reꢀsuspended in binding buffer (50 mM TrisꢀHCl, 2.5 mM EDTA, 5 mM MgCl2, 0.5%1
fatty acidꢀfree bovine serum albumin (BSA), pH 7.4) and incubated with vehicle or compounds and
0.5 nM of [3H]CP55,940 for 90 min at 30 °C. Nonꢀspecific binding was determined in the presence
of 10 ꢁM of WIN55,512. After incubation, membranes were filtered through a preꢀsoaked 96ꢀwell
microplate bonded with GF/B filters under vacuum and washed twelve times with 150 ꢁL of iceꢀ
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cold binding buffer. The radioactivity was measured and the results expressed as [3H]CP55,940
binding.
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ABHD6 and ABHD12 inhibition assay. ABHDs activity assays were performed as previously
described.8 The hABHD6 and hABHD12 activity was determined using cell homogenates from
hABHD6 and hABHD12 stably transfected HEK293 cells. Compound was preꢀincubated with 40
ꢁg of cell homogenate for 30 min at 37 °C in assay buffer (Tris 1 mM, EDTA 10 mM plus 0.1%
BSA, pH= 7.6). DMSO was used as vehicle control and WWL70 10 ꢁM or THL 20 ꢁM as positive
controls. Then, 10 ꢁM of 2ꢀOG was added and incubated for 5 min at 37 ºC. The reaction was
stopped by the addition of 400 ꢁL of iceꢀcold CHCl3:MeOH (1:1). The samples were vortexed and
centrifuged (16000 x g, 10 min, 4 ºC). Aliquots (200 ꢁL) of the aqueous phase were assayed for
tritium content by liquid scintillation spectroscopy. Blank values were recovered from tubes
containing no enzyme. Basal 2ꢀOG hydrolysis occurring in nonꢀtransfected HEK293 cells was
subtracted.
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Cell viability assay. OVSAHO, OVCAR3, COV318, CAOV3 and MRC5 (from ATCC) were
maintained at 37 °C in a humidified atmosphere containing 5% CO2 accordingly to the supplier.
Normal (1.5 × 104) and tumor (5 × 102) cells were plated in 96ꢀwell culture plates. The day after
seeding, vehicle or compounds were added at different concentrations to the medium. Compounds
were added to the cell culture at a concentration ranging from 100 to 0.01 µM. Cell viability was
measured after 96 h according to the supplier (Promega, G7571) with a Tecan F200 instrument.
IC50 values were calculated from logistical dose response curves. Averages were obtained from
three independent experiments, and error bars are standard deviations (n = 3).
Animals. Male CDꢀ1 albino mice (Envigo, Varese, Italy) weighing approximately 22–25 g at the
beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro
Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their
arrival. Ten mice were housed per cage (size 26 × 41 cm); animals were fed a standard laboratory
diet and tap water ad libitum, and kept at 23 ± 1 °C with a 12 h light/dark cycle, light at 7 a.m. All
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animal manipulations were carried out according to the European Community guidelines for animal
care (DL 116/92), application of the European Communities Council Directive of 24 November
1986 (86/609/EEC). The ethical policy of the University of Florence complies with the Guide for
the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication
No. 85ꢀ23, revised 1996; University of Florence assurance number: A5278ꢀ01). Formal approval to
conduct the experiments described was obtained from the Animal Subjects Review Board of the
University of Florence. Experiments involving animals have been reported according to ARRIVE
guidelines. All efforts were made to minimize animal suffering and to reduce the number of animals
used.
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Oxaliplatinꢀinduced neuropathic pain model. Mice treated with oxaliplatin (2.4 mg/kg) were
administered i.p. on days 1ꢀ2, 5ꢀ9, 12ꢀ14 (10 i.p. injections)63, 64 oxaliplatin was dissolved in 5%
glucose solution. Control animals received an equivalent volume of vehicle. Behavioral tests were
performed on day 15.
Cold plate test. The animals were placed in a stainless steel box (12 cm × 20 cm × 10 cm) with a
cold plate as floor. The temperature of the cold plate was kept constant at 4 °C ± 1 °C. Painꢀrelated
behavior (licking of the hind paw) was observed and the time (seconds) of the first sign was
recorded. The cutꢀoff time of the latency of paw lifting or licking was set at 60 seconds.65
LCꢀMS/MS quantification of endocannabinoids and other metabolites. The amount of 2ꢀAG,
AEA, arachidonic acid (AA), prostaglandinꢀE2 (PGE2) and prostaglandinꢀD2 (PGD2) were
measured by LCꢀMS/MS in brain and spinal cord of mice treated for treated for 14 days with
oxaliplatin (2.4 mg/kg, i.p.) and compound 26 (30 mg/kg, p.o.) or vehicle. At day 15, animals were
sacrificed by decapitation 1 h after the last dose and brain and spinal cord were collected, quickly
washed in PBS and snapꢀfrozen. Tissue extraction and LCꢀMS/MS conditions were performed as
previously described.8
Statistical analysis. Behavioral measurements were performed on 10 mice for each treatment
carried out in 2 different experimental sets. Results were expressed as mean ± S.E.M. The analysis
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of variance of behavioral data was performed by one way ANOVA, a Bonferroni's significant
difference procedure was used as postꢀhoc comparison. P values of less than 0.05 or 0.01 were
considered significant. Investigators were blind to all experimental procedures. Data were analyzed
using the “Origin 9” software (OriginLab, Northampton, USA).
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General Procedures and Materials. H NMR data were recorded with a Varian VXR 200
spectrometer or a Varian Mercury Plus 400 spectrometers. Chemical shifts (δ) are reported in parts
per million, and J values are given in Hertz. Positive ion electrospray ionization (ESI) mass spectra
were recorded on a doubleꢀfocusing Finnigan MAT 95 instrument with BE geometry. Melting
points (mp) were determined on a BuchiꢀTottoli apparatus and are uncorrected. Chromatography
was performed on Merck 230–400 mesh silica gel and a mixture of ethyl acetate and petroleum
ether in different ratios as mobile phase The purity of tested compounds was determined by
combustion elemental analyses in the Microanalytical Laboratory of the Chemistry Department of
the University of Ferrara with a Yanagimoto MTꢀ5 CHN recorder elemental analyser. All tested
compounds yielded data consistent with a purity of at least 95% as compared with the theoretical
values. TLC was carried out using glass plates coated with silica gel 60 F254 by Merck, and
compounds were visualized by UV lamp (254 nm light source). Organic solutions were dried over
anhydrous Na2SO4. Solvents and reagents were purchased from Aldrich (SigmaꢀAldrich) or Alfa
Aesar (Johnson Matthey Company) and were used without further purification. HPLC analysis: all
target compounds (i.e., assessed in biological assays) were ≥95% pure by HPLC, confirmed via UV
detection (λ = 254 nm). Analytical reversedꢀphase HPLC was conducted using a Kinetex EVO C18
column (5 ꢁm, 150 mm × 4.6 mm, Phenomenex, Inc.). Compounds were dissolved in acetonitrile or
methanol, and injected through a 20 ꢁl loop. Eluent A was water and eluent B was CH3CN; after 5
min at 25% B, a gradient was formed from 25% to 75% of B in 5 min and held at 75% of B for 10
min; flow rate was 1 mL/min. For compound 9, a different gradient profile of mobile phase was
used: after 5 min at 10% B, a gradient was formed from 10% to 25% of B in 5 min and then from
25% to 75% of B in the following 2.5 min and held at 75% of B for 7.5 min; flow rate was 1
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