Sandra Codony1, Carla Calvó-Tusell2, Elena Valverde1, Sílvia Osuna2,3, Christophe Morisseau4, M Isabel Loza5, José Brea5, Concepción Pérez6, María Isabel Rodríguez-Franco6, Javier Pizarro-Delgado7,8,9, Rubén Corpas10,11, Christian Griñán-Ferré12, Mercè Pallàs12, Coral Sanfeliu10,11, Manuel Vázquez-Carrera7,8,9, Bruce D Hammock4, Ferran Feixas2, Santiago Vázquez1. 1. Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Departament de Farmacologia, Toxicologia i Química Terapèutica, Facultat de Farmàcia i Ciències de l'Alimentació, and Institute of Biomedicine (IBUB), Universitat de Barcelona, Av. Joan XXIII, 27-31, Barcelona 08028, Spain. 2. CompBioLab Group, Departament de Química and Institut de Química Computacional i Catàlisi (IQCC), Universitat de Girona, C/ Maria Aurèlia Capmany 69, Girona 17003, Spain. 3. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain. 4. Department of Entomology and Nematology and Comprehensive Cancer Center, University of California Davis, One Shields Avenue, Davis 95616, California, United States. 5. Drug Screening Platform/Biofarma Research Group, CIMUS Research Center. Departamento de Farmacoloxía, Farmacia e Tecnoloxía Farmacéutica, University of Santiago de Compostela (USC), Santiago de Compostela 15782, Spain. 6. Institute of Medicinal Chemistry, Spanish National Research Council (CSIC), C/Juan de la Cierva 3, Madrid 28006, Spain. 7. Pharmacology Section. Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine of the University of Barcelona (IBUB), University of Barcelona, Av. Joan XXIII, 27-31, Barcelona 08028, Spain. 8. Spanish Biomedical Research Center in Diabetes and Associated Metabolic Diseases (CIBERDEM)-Instituto de Salud Carlos III, Madrid 28029, Spain. 9. Pediatric Research Institute-Hospital Sant Joan de Déu, Esplugues de Llobregat 08950, Spain. 10. Institute of Biomedical Research of Barcelona (IIBB), CSIC and IDIBAPS, Barcelona 08036, Spain. 11. CIBER Epidemiology and Public Health (CIBERESP)-Instituto de Salud Carlos III, Madrid 28029, Spain. 12. Pharmacology Section. Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institut de Neurociències, University of Barcelona, Av. Joan XXIII, 27-31, Barcelona 08028, Spain.
Abstract
The pharmacological inhibition of soluble epoxide hydrolase (sEH) is efficient for the treatment of inflammatory and pain-related diseases. Numerous potent sEH inhibitors (sEHIs) present adamantyl or phenyl moieties, such as the clinical candidates AR9281 or EC5026. Herein, in a new series of sEHIs, these hydrophobic moieties have been merged in a benzohomoadamantane scaffold. Most of the new sEHIs have excellent inhibitory activities against sEH. Molecular dynamics simulations suggested that the addition of an aromatic ring into the adamantane scaffold produced conformational rearrangements in the enzyme to stabilize the aromatic ring of the benzohomoadamantane core. A screening cascade permitted us to select a candidate for an in vivo efficacy study in a murine model of cerulein-induced acute pancreatitis. The administration of 22 improved the health status of the animals and reduced pancreatic damage, demonstrating that the benzohomoadamantane unit is a promising scaffold for the design of novel sEHIs.
The pharmacological inhibition of soluble epoxide hydrolase (sEH) is efficient for the treatment of inflammatory and pain-related diseases. Numerous potent sEH inhibitors (sEHIs) present adamantyl or phenyl moieties, such as the clinical candidates AR9281 or EC5026. Herein, in a new series of sEHIs, these hydrophobic moieties have been merged in a benzohomoadamantane scaffold. Most of the new sEHIs have excellent inhibitory activities against sEH. Molecular dynamics simulations suggested that the addition of an aromatic ring into the adamantane scaffold produced conformational rearrangements in the enzyme to stabilize the aromatic ring of the benzohomoadamantane core. A screening cascade permitted us to select a candidate for an in vivo efficacy study in a murine model of cerulein-induced acute pancreatitis. The administration of 22 improved the health status of the animals and reduced pancreatic damage, demonstrating that the benzohomoadamantane unit is a promising scaffold for the design of novel sEHIs.
In mammals, arachidonic acid, a polyunsaturated fatty acid, is
metabolized by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome
P450s (CYPs). The COX and LOX pathways lead mainly to the production
of pro-inflammatory lipid mediators, such as prostaglandins and leukotrienes,
and are pharmaceutically targeted.[1] In
contrast, the CYP pathway produces both pro- and anti-inflammatory
lipid mediators, such as the pro-inflammatory 20-hydroxyeicosatetraenoic
acid or the potent anti-inflammatory epoxyeicosatrienoic acids (EETs).[2] However, the EETs are rapidly metabolized by
the soluble epoxide hydrolase (sEH, EPHX2, EC 3.3.2.3)
into the corresponding dihydroxyeicosatrienoic acids, which are less
biologically active.[3,4] The pharmacological inhibition
of sEH in vivo stabilizes the concentration of EETs,
reducing inflammatory and pain states, suggesting sEH as a pharmacological
target for the treatment of inflammatory diseases.[5,6]X-ray crystallographic studies revealed that sEH
has an L-shaped
active pocket with the catalytic residues situated at the corner.
Although each side of the pocket (10 and 15 Å long) accepts a
variety of functional groups, the entire pocket is essentially hydrophobic.[7] Indeed, a number of very potent sEH inhibitors
(sEHIs) feature lipophilic moieties such as adamantyl or phenyl groups
(Figure ), limiting
their usefulness.[5,6]
Figure 1
Structure of the three sEHIs that have
entered human clinical trials.
Structure of the three sEHIs that have
entered human clinical trials.Nevertheless, several compounds have been tested in human. AR9281,
which was developed by Arête Therapeutics for the treatment
of hypertension in diabetic patients, failed in a phase II clinical
trial largely due to its poor pharmacokinetic properties, likely related
with its adamantyl unit.[8] GSK2256294, developed
for chronic obstructive pulmonary disease by GlaxoSmithKline, has
entered clinical trials for obese smokers and other indications such
as subarachnoidal hemorrhage or diabetic patients with insulin resistance.[9] More recently, taking the AR9281 failure into
account, EicOsis has recently replaced the adamantyl moiety of AR9281
by an aromatic ring for its drug candidate EC5026, which has recently
completed human phase 1a clinical trials for the treatment of neuropathic
pain.[10]Using urea-based sEHIs with
lipophilic units of very different
sizes, we recently found that the pocket of the sEH can accommodate
polycycles of a quite diverse volume and that the replacement of the
adamantane moiety by larger polycyclic rings, such as the diamantane
moiety, may be better than the replacement by smaller ones. Indeed,
urea 6, a diamantane analogue of the well-known sEHI 4, t-AUCB, and 5, t-TUCB, showed to be a subnanomolar inhibitor of the human sEH (hsEH)
(Figure ).[11] Therefore, herein we are testing new sEH that
merge the adamantyl and phenyl groups in a unique polycyclic scaffold.
Figure 2
Structure
and IC50 in hsEH of compounds 4–6.
Structure
and IC50 in hsEH of compounds 4–6.
Results and Discussion
Design and Synthesis of New sEHIs
Taking into account
that both adamantane and aromatic ring moieties
fit very well in the hydrophobic pocket of the sEH and that the replacement
of adamantane by larger polycyclic rings seems to be a promising strategy
to obtain more potent sEHIs, a novel series of compounds bearing the
very versatile benzohomoadamantane scaffold as the hydrophobic moiety
were designed and synthesized. This polycyclic, readily accessible,[12−15] system features a homoadamantane unit fused with an aromatic ring
and permits several chemical derivatizations in its structure (Figure ). Potent sEHIs and
optimum drug-like properties could be achieved by modifying the substituents
in the benzohomoadamantane unit and/or the right-hand side (RHS) of
the molecule (Figure ).
Figure 3
Known adamantyl- and phenyl-based sEHIs 1a, b, 3, 4a, b, 5, and 7a, b, and general structure, I, of the new sEHI reported in this work. (see below). For
simplicity, only IC50 values obtained for the hsEH are
reported.
Known adamantyl- and phenyl-based sEHIs 1a, b, 3, 4a, b, 5, and 7a, b, and general structure, I, of the new sEHI reported in this work. (see below). For
simplicity, only IC50 values obtained for the hsEH are
reported.Thioureas, carbamates and, particularly
ureas are good pharmacophores
for an sEHI.[6] For this reason, thiourea 9, carbamate 11, and urea 13 were
first synthesized in order to explore their relative potency as an
sEHI and to select the more suitable pharmacophore for the novel polycyclic
scaffold (Scheme ).
The three compounds were easily synthesized in low to moderate yields
from known 9-methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine[13] (II, R = CH3, X = CH2) and 4-(trifluoromethyl)phenyl isothiocyanate, 8, p-tolyl chloroformate, 10, and 2,3,4-trifluorophenyl
isocyanate, 12, respectively (Scheme ).
Scheme 1
Synthesis of New Compounds 9, 11, 13, 14, 16–18, and 20–23
Reagents and conditions:
(a)
4-(trifluoromethyl)phenyl isothiocyanate (8), Et3N, DCM, overnight (b) p-tolyl chloroformate
(10), Et3N, DCM, overnight; (c) 2,3,4-trifluorophenyl
isocyanate (12), DCM, overnight; (d) Triphosgene, sat.
NaHCO3, DCM, 30 min; (e) 1-acetyl-4-aminopiperidine (15), DCM, overnight; (f) 4-((trans-4-aminocyclohexyl)oxy)benzoic
acid hydrochloride[20] (19),
Et3N, DMF, overnight. See the Experimental
Section and the Supporting Information for further details.
Synthesis of New Compounds 9, 11, 13, 14, 16–18, and 20–23
Reagents and conditions:
(a)
4-(trifluoromethyl)phenyl isothiocyanate (8), Et3N, DCM, overnight (b) p-tolyl chloroformate
(10), Et3N, DCM, overnight; (c) 2,3,4-trifluorophenyl
isocyanate (12), DCM, overnight; (d) Triphosgene, sat.
NaHCO3, DCM, 30 min; (e) 1-acetyl-4-aminopiperidine (15), DCM, overnight; (f) 4-((trans-4-aminocyclohexyl)oxy)benzoic
acid hydrochloride[20] (19),
Et3N, DMF, overnight. See the Experimental
Section and the Supporting Information for further details.The inhibitory activities
of the three compounds in hsEH were evaluated
using a previously reported sensitive fluorescent-based assay (Table ).[16] While carbamate 11 was
a very weak inhibitor (IC50 = 12.7 μM) and thiourea 9 displayed only a moderate inhibition (IC50 =
138 nM), urea 13 revealed as a very potent hsEHI (IC50 = 1 nM) (Table ). The superior potency of the urea is in agreement with previous
results in other series of sEHIs.[17,18] For this reason,
no further carbamates and thioureas derivatives were envisaged and
the urea group was chosen as the main pharmacophore for the synthesis
of further inhibitors.
Table 1
Inhibition of hsEH
and Microsomal
Stability Values of the New Benzohomoadamantane-Based sEHI
Cpd
hsEHaIC50 (nM)
microsomal stabilityb (h/m/r)
9
138
NDc
11
>10,000
ND
7
7.7
79/77/81
13
1.0
7/0.2/ND
14
20
77/23/33
1, AR9281
8.0
72/100/87
16
3.1
1/0.5/ND
17
941
ND
18
>10,000
ND
4, t-AUCB
0.5
94/92/46
20
0.9
70/10/2
21
28
90/83/ND
Reported IC50 values
are the average of three replicates. The fluorescent assay as performed
here has a standard error between 10 and 20% suggesting that differences
of twofold or greater are significant. Because of the limitations
of the assay, it is difficult to distinguish among potencies <0.5
nM.[16]
Percentage of the remaining compound
after 60 min of incubation with human, mice, and rat microsomes obtained
from Tebu–Xenotech in the presence of NADP at 37 °C.
ND: not determined.
Reported IC50 values
are the average of three replicates. The fluorescent assay as performed
here has a standard error between 10 and 20% suggesting that differences
of twofold or greater are significant. Because of the limitations
of the assay, it is difficult to distinguish among potencies <0.5
nM.[16]Percentage of the remaining compound
after 60 min of incubation with human, mice, and rat microsomes obtained
from Tebu–Xenotech in the presence of NADP at 37 °C.ND: not determined.Having found that this novel scaffold
may successfully replace
the adamantane and/or the phenyl group found in known sEHIs, a series
of benzohomoadamantane derivatives related with the potent sEHI AR9281, t-AUCB, t-TUCB, EC5026, and 7 were synthesized in order to explore their potency and DMPK properties.
Of note, a very recent work has described that the replacement of
a methylene unit of the adamantane moiety by an oxygen atom led to
more soluble compounds while only slightly reducing the inhibitory
activity against the sEH (e.g., 1b, 4b, and 7b in Figure ).[19] In this sense,
an oxygen atom was introduced in the benzohomoadamantane scaffold
in order to explore whether a similar trend was also followed within
this new family of sEHIs (Figure and Scheme ).The synthesis of the new sEHI started from the suitably
substituted
benzohomoadamantane amines of general structure II.[12−15] Thus, the synthesis of urea 14 involved the reaction
of 5-methyl-1,5,6,7-tetrahydro-1,5:3,7-dimethanobenzo[e]oxonin-3(2H)-amine[12] (II, R = CH3, X = O) with 2,3,4-trifluorophenylisocyanate, 12, in dichloromethane (Scheme ). For the obtention of the piperidine derivatives,
we first prepared the isocyanate of the corresponding polycyclic amine II (II, R = CH3, X = CH2;[13]II, R = CH3, X = O;[12]II, R = H, X =
O[12]) by reaction with triphosgene and saturated
the aqueous solution of NaHCO3. Once the desired isocyanate
of general structure III (III, R = CH3, X = CH2; III, R = CH3, X = O; III, R = H, X = O) was obtained, it was reacted
with 1-acetyl-4-aminopiperidine 15 in dichloromethane
to obtain ureas 16–18 in moderate overall yields
(Scheme ). Finally,
the t-AUCB analogues 20–23 were
obtained in low to moderate yields by the reaction, in the presence
of triethylamine, of the corresponding isocyanate, obtained from II (II, R = CH3, X = CH2;[13]II, R = CH3, X = O;[12]II, R = Cl, X
= CH2;[15]II, R
= F, X = CH2[15]) and triphosgene,
in DMF with 4-((trans-4-aminocyclohexyl)oxy)benzoic
acid, 19, prepared as previously reported[20] (Scheme ).
sEH Inhibition and DMPK
Assays
The
potency of the new compounds as inhibitors of the hsEH was tested
using a previously reported sensitive fluorescent-based assay.[16] Gratifyingly, the potency of the new benzohomoadamantane
ureas was in the same range as that of diphosgene their corresponding
adamantane-based analogues (compare 13vs7, 16vs AR9281, and 20vs t-AUCB) (Table ). The comparison of the compounds presenting
a methylene unit in the polycyclic scaffold with their analogues featuring
an oxygen atom (13vs14, 16vs17, and 20vs21, Scheme and Table ) showed that, in all cases, the compound bearing an
oxygen atom was less potent; these results are in line with those
previously found within the adamantane series of sEHIs.[19] Interestingly, the AR9281 analogues 16 and 17 showed the largest difference (300-fold difference, Table ). Also, the replacement
of the methyl group at the R position of the polycyclic scaffold by
a hydrogen atom in the AR9281 analogues produced a dramatic drop of
the inhibitory activity (17vs18, >10-fold decrease in potency, Table ).Considering the metabolism liability
of the adamantane and adamantane-related scaffolds,[21,22] we evaluated the in vitro stability in human, mice,
and rat microsomes of the new ureas bearing the benzohomoadamantane
moiety (Table ). Within
the trifluorophenyl series, the substitution of the adamantane nucleus
by the benzohomoadamantane scaffold showed an important decrease of
the microsomal stability (7vs13, Table ). By contrast, in the corresponding oxa-analogue, 14, the stability seemed to be restored in human, but marginally in
mice and rat microsomes (13vs14, Table ). Moreover, the analogue of AR9281, 16, presented very
high metabolic liability in all three species, as less than 1% of
the compound remains after being incubated with microsomes for 60
min (AR9281 vs16, Table ). Finally, within the t-AUCB series, the replacement of the adamantane moiety
by the benzohomoadamantane scaffold led to similar stability in human
microsomes but to lower stability in mice and rat microsomes. Interestingly,
in the oxa-analogue 21, the stability was also maintained
in mice microsomes (20 and 21vs
t-AUCB, Table ). Although it seems that the ureas presenting the oxa-benzohomoadamantane
moiety were more stable in microsomes, taking into account that all
these derivatives (14, 17, 18, and 21) were considerably less potent, this oxa-polycyclic
scaffold was abandoned and only the ureas featuring the benzohomoadamantane
core were further evaluated.Overall, the t-AUCB family of compounds presented
the most favorable properties in terms of potency and microsomal stability,
and this series was selected for further optimization. As the adamantane
nucleus contributes to the high lipophilicity of the known sEHI that
compromises the solubility of these compounds, we next measured the
solubility of the selected t-AUCB series in a 1%
DMSO: 99% PBS buffer solution. As expected, the solubility decreases
from the adamantane-based t-AUCB to the benzohomoadamantane
analogue 20 (Table ) likely due to the increase of carbon atoms from the
adamantane nucleus (10 atoms) to the new polycyclic scaffold (16 atoms).
Taking this into account, novel substitutions in the R position of
the benzohomoadamantane scaffold were explored toward improving solubility
while maintaining or enhancing both potency and microsomal stability
of 20. Thus, the methyl group of 20 was
replaced by chlorine and fluorine atoms, leading to compounds 22 and 23, respectively. The potency, microsomal
stability, solubility, and permeability of both compounds were assessed
in order to explore their properties (Table ).
Table 2
IC50 in
Human, Murine,
and Rat sEH, Microsomal Stability, Solubility, and Permeability Values
of the t-AUCB Related Compounds
permeability (Caco-2)
sEH IC50 (nM)a
Papp (nm/s)
Cpd
human
murine
rat
microsomal stabilityb (h/m/r)
solubilityc (μM)
A→B
B→A
ERd
LD50e(μM)
IC50hLOX-5f(μM)
IC50hCOX-2g(μM)
4, t-AUCB
0.5
1.7
8.0h
94/92/46
25
1.9
210.3
111
NDi
ND
ND
20
0.9
9.9
0.4
70/10/2
4
10
123.7
12.4
>100
>100
>10
22
0.4
0.4
0.4
89/29/52
13
21.5
46.6
2.1
>100
>100
>10
23
0.5
0.5
0.4
77/36/60
7
0.9
219.1
243.9
>100
>100
>10
Reported
IC50 values
are the average of three replicates. The fluorescent assay as performed
here has a standard error between 10 and 20%, suggesting that differences
of twofold or greater are significant. Because of the limitations
of the assay, it is difficult to distinguish among potencies <0.5
Nm.[16]
Percentage of remaining compound
after 60 min of incubation with human, mice, and rat microsomes obtained
from Tebu–Xenotech in the presence of NADP at 37 °C.
Solubility in a 1% DMSO: 99%
PBS
buffer solution, see the Experimental Section for details.
The efflux
ratio was calculated
as ER = (Papp B → A)/(Papp A → B). See the Experimental Section for further details.
sEHI cytotoxicity tested by propidium
iodide staining after 24 h of incubation in SH-SY5Y cells. See the Experimental Section for further details.
IC50 in human LOX-5 (hLOX-5). See the Experimental Section for further details.
IC50 in human COX-2 (hCOX-2) performed by Eurofins
(catalogue reference 4186).
Taken from ref (24).
ND: Not determined.
Reported
IC50 values
are the average of three replicates. The fluorescent assay as performed
here has a standard error between 10 and 20%, suggesting that differences
of twofold or greater are significant. Because of the limitations
of the assay, it is difficult to distinguish among potencies <0.5
Nm.[16]Percentage of remaining compound
after 60 min of incubation with human, mice, and rat microsomes obtained
from Tebu–Xenotech in the presence of NADP at 37 °C.Solubility in a 1% DMSO: 99%
PBS
buffer solution, see the Experimental Section for details.The efflux
ratio was calculated
as ER = (Papp B → A)/(Papp A → B). See the Experimental Section for further details.sEHI cytotoxicity tested by propidium
iodide staining after 24 h of incubation in SH-SY5Y cells. See the Experimental Section for further details.IC50 in human LOX-5 (hLOX-5). See the Experimental Section for further details.IC50 in human COX-2 (hCOX-2) performed by Eurofins
(catalogue reference 4186).Taken from ref (24).ND: Not determined.Satisfactorily, the evaluation
of the inhibition activity against
the hsEH showed that both compounds 22 and 23 were slightly more potent than 20, with IC50 values in the same range as t-AUCB. Of note, 22 and 23 presented the same potency inhibiting
the human, murine, and rat enzymes, while t-AUCB
was three- and twenty-fold less potent with the murine and the rat
enzymes, respectively (Table ).Regarding metabolic stability, compounds 22 and 23 presented better stabilities than 20 (Table ). Furthermore,
the
experimental solubility values of these new halogenated compounds
were determined. In line with a previous work with adamantane derivatives,[23] the solubility increases when the methyl group
is replaced by a halogen atom (compare 20vs22 and 23, Table ), particularly for the chlorinated compound 22.The Caco-2 cell permeability model was used in order
to evaluate
the permeability of the compounds. Apparent permeability values (Papp)
were determined from the amount permeated through the Caco-2 cell
membranes at both apical-basolateral (A-B) and basolateral-apical
(B-A) direction. Gratifyingly, compounds 20 and 22 presented higher permeability values than t-AUCB, 22 being the one that presented the best profile
(Table ). Moreover,
the cytotoxicities of 20, 22, and 23 were evaluated in SH-SY5Y cells by propidium iodide staining
after 24 h of incubation. None of the compounds showed to be cytotoxic
at the highest concentration tested (100 μM).Finally,
inhibitors 20, 22, and 23 were
tested for selectivity against hCOX-2
and hLOX-5, two enzymes involved in the AA cascade.
Neither 20 nor the halogenated analogues 22 and 23 significantly inhibited these enzymes (see Table ).Next, considering
the best permeability of 20 and 22, both
compounds were selected for CYPs and hERG inhibition
assays. Cytochromes P450 (CYP) inhibition was evaluated using human
recombinant cytochrome P450 enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A, through a fluorescence-detection method. These assays were
of great interest not only for the detection of possible drug–drug
interactions but also in terms of selectivity as EETs are formed by
several cytochrome P450 isoforms, particularly CYP2C19. Satisfactorily,
the tested compounds did not significantly inhibit the evaluated cytochromes.
We considered acceptable IC50 values around 2 μM
in CYP2C19 taking into account that both compounds presented 2000-fold
more potency inhibiting the sEH.Regarding the hERG inhibition
assay, both compounds inhibited the
channel only 4 and 44% at 10 μM, respectively (Table ). Overall, both compounds presented
high potency inhibiting human, murine, and rat enzymes and did not
significantly inhibit either cytochromes or hERG. Considering the
biological profiling of the new sEH inhibitors, compounds 20 and 22 were selected as the candidates for the in vivo studies.
Table 3
Inhibition (Expressed
as % of Inhibition
at 10 μM or IC50) of Recombinant Human Cytochromes
P450 Enzymes and Inhibition of the hERG Channel (Expressed as % of
Inhibition at 10 μM)
cytochrome
inhibitiona
CYP 1A2
CYP 2C9
CYP 2C19
(μM)
CYP 2D6
CYP
3A4b
Cpd
(BFC)
(DBF)
hERG channel inhibition (% at 10 μM)
20
1 ± 2
17 ± 3
1.9
1 ± 1
2 ± 2
14 ± 1
4
22
14 ± 4
31 ± 3
2.2
11 ± 3
1 ± 2
52 ± 4
44
The cytochrome inhibition was tested
at 10 μM. IC50 was calculated for those compounds
that presented >50% of inhibition.
For the study of CYP3A4, two different
substrates were used: benzyloxytrifluoromethylcoumarin (BFC) and dibenzylfluorescein
(DBF).
The cytochrome inhibition was tested
at 10 μM. IC50 was calculated for those compounds
that presented >50% of inhibition.For the study of CYP3A4, two different
substrates were used: benzyloxytrifluoromethylcoumarin (BFC) and dibenzylfluorescein
(DBF).
In silico Study: Molecular
Basis of Benzohomoadamantane-Derived Soluble Epoxide Hydrolase Inhibitors
The incorporation of an aromatic ring into the adamantane scaffold
can potentially impact the orientation and molecular interactions
of benzohomodamantane sEHIs compared to adamantane derivatives. To
unravel how bulky benzohomodamantane ureas are accommodated in the
active site of sEH and to understand the molecular basis of their
inhibitory mechanism, molecular dynamics (MD) simulations were performed
for compounds t-AUCB, 20, 22, and 23. The MD simulations revealed that the addition
of an aromatic ring onto the adamantane scaffold of t-AUCB triggers conformational rearrangements in the active site and
adjacent regions to stabilize the benzohomoadamantane scaffold. These
interactions, together with a network of hydrogen bonds and hydrophobic
interactions with the urea and benzoic acid moieties, are key for
retaining the inhibitors in the active site.First, we explored
the preferred binding mode of the selected sEHI and the flexibility
of the active site of the sEH–inhibitor complex. The L-shaped
active site pocket of sEH consists of three regions: the left-hand
side (LHS) and the RHS pockets and a central narrow channel defined
by catalytic residues Asp335, Tyr383, and Tyr466 that connects the
LHS and RHS hydrophobic cavities (see Figure ).[25] Previously,
we showed that the active site of EHs present high plasticity.[19,26] Available X-ray structures of sEH in complex with adamantyl ureas
indicate that the adamantane scaffold can occupy both LHS and RHS
pockets.[27] In the case of t-AUCB (PDB: 5AM3), the inhibitor is orientated with the benzoic acid group occupying
the RHS, while adamantane sits in the LHS (see Figure ). To corroborate that this is also the preferred
orientation for benzohomoadamantane derivatives, we carried out molecular
docking calculations for compounds 20, 22, and 23 (see the Experimental Section for details). All the binding poses featuring the urea moiety interacting
with Asp335 oriented the benzohomoadamantane scaffold in the LHS and
the benzoic acid in the RHS, as observed for t-AUCB.
The LHS pocket presents enough space to accommodate the bulky benzohomoadamantane
scaffold (see Figure ). To evaluate the stability and molecular interactions of the inhibitor
in the active site of sEH, we carried out three replicas of 250 ns
MD simulations for t-AUCB, 20, 22, and 23, starting from this orientation, that
is the benzohomoadamantane occupying the LHS pocket. All inhibitors
show considerable stability and no sign of unbinding or significant
reorientations are observed along the MD simulation time. To evaluate
the impact of the inhibitors on the active site conformational plasticity,
we monitored the changes on the active site volume along the MD simulations
(see Figures b and S1) using the POcket Volume MEasurer (POVME,
see the Supporting Information for details).[28] As observed previously, in the apo state, the total volume encompassing LHS, RHS, and the central channel
displays wide fluctuations from 70 to 700 Å3 (average
volume 290 ± 133 Å3).[19] The size of the LHS pocket is mainly determined by flexible Ile363,
Ile375, Met469, and Asn472 side chains, while the shape of the RHS
pocket fluctuates with frequent side chain conformational changes
of Leu408, Met419, and Phe497 residues (see Figure S1). When t-AUCB, 20, 22, and 23 compounds are bound in the active site, an
expansion of the active site volume with respect to the average apo value is observed, which becomes stable at around 330–400
Å3 (see Figures b,c and S1). The average
volumes determined for the last 150 ns of each MD simulation are 335
± 33, 396 ± 37, 351 ± 25, and 356 ± 32 Å3 for t-AUCB, 20, 22, and 23, respectively. As expected, benzohomadamantane
inhibitors show wider active sites than t-AUCB,compound 20, with a bulkier methyl group, being the one with the larger
volume. The analysis of the root-mean-square fluctuation (RMSF) from
the MD simulations shows that some active site residues become more
rigid in the presence of inhibitors (see Figure S2). All inhibitors are able
to restrict the conformational plasticity of the active site, indicating
that they are tightly bound (see the population shift toward narrow
distributions of active site volumes in Figure S1).
Figure 4
(a) Representation of the sEH structure (PDB: 5AM3), active site catalytic
residues (nucleophilic Asp335, Tyr383, Tyr466), and t-AUCB inhibitor. The LHS pocket is colored in orange, the RHS pocket
is colored in cyan, and the central channel in purple. (b) Plot of
the fluctuations of the active site volume for the apo state (orange
line, 290 ± 133 Å3), t-AUCB
(purple line, 335 ± 33 Å3), compound 20 (red line, 396 ± 37 Å3), compound 22 (blue line, 351 ± 25 Å3), and compound 23 bound (green line, 356 ± 32 Å3) along
a representative 250 ns MD simulation trajectory. The average volumes
are calculated for the last 150 ns of the MD simulation. (c) Representative
sEH structure with the active site volume obtained from MD simulations
of compound 22.
(a) Representation of the sEH structure (PDB: 5AM3), active site catalytic
residues (nucleophilic Asp335, Tyr383, Tyr466), and t-AUCB inhibitor. The LHS pocket is colored in orange, the RHS pocket
is colored in cyan, and the central channel in purple. (b) Plot of
the fluctuations of the active site volume for the apo state (orange
line, 290 ± 133 Å3), t-AUCB
(purple line, 335 ± 33 Å3), compound 20 (red line, 396 ± 37 Å3), compound 22 (blue line, 351 ± 25 Å3), and compound 23 bound (green line, 356 ± 32 Å3) along
a representative 250 ns MD simulation trajectory. The average volumes
are calculated for the last 150 ns of the MD simulation. (c) Representative
sEH structure with the active site volume obtained from MD simulations
of compound 22.To gain a deeper insight into the molecular basis of the inhibitory
mechanism of benzohomoadamantane ureas, the non-covalent interactions
between the selected inhibitors and the active site residues of sEH
were analyzed with NCIplot on the most visited MD conformations (see Figures and S3).[29] First, we analyzed
the interactions established in the RHS pocket and the central channel
where all inhibitors share a common scaffold: the benzoic acid group.
For t-AUCB, 20, 22, and 23 compounds, the carboxylate unit is stabilized by two hydrogen
bonds with Ser412 and Ser415 that are located at the entrance of the
RHS pocket, the interaction with Ser415 being more stable along the
MD simulations (see Figure b and S3). The aromatic ring of
the benzoic acid is further stabilized in the RHS pocket through CH···π
interactions by the side chains of Trp525 and Phe497. The side chain
of Phe497 transitions from the solvent to the active site to form
a network of stable hydrophobic interactions that includes the benzoic
acid group and the aromatic side chains of residues Trp525, Phe497,
and catalytic Tyr383. Moreover, MD simulations show that four water
molecules permanently occupy the RHS pocket in t-AUCB, 20, 22, and 23 (see Figures S4 and S5). These water molecules establish
a network of interactions with the carboxylate group of the inhibitor
and with residues Leu417, Ser407, Val416, Ser415, Ser412, and Lys495
that provides extra stabilization of the benzoic acid group in the
RHS pocket. The urea moiety establishes hydrogen bonds with three
catalytic residues: Asp335, Tyr383, and Tyr466 located in the central
channel of the active site pocket. MD simulations show that the three
hydrogen bonds remain significantly stable along the whole simulation
time for all inhibitors with no significant differences (see Figure c). t-AUCB and 20 are able to retain a tighter hydrogen bond
(below 3 Å) between the carbonyl of the urea and the OH of Tyr466
than halogenated compounds 22 and 23. These
results indicate that all inhibitors remain stable in the active site
pocket, forming similar interactions consistent with reported IC50 values. Previously, we have shown that less potent inhibitors
displayed fluctuations in the interactions between the urea motif
and the catalytic residues, shifting the ensemble toward longer distances.[19] All inhibitors share a common scaffold on the
RHS of the urea and MD simulations reported a similar behavior in
terms of interactions and conformational dynamics in the RHS and central
channel regions. The network of hydrogen bonds and π–π
stacking interactions is key to retain the inhibitor in the active
site.
Figure 5
(a) Representative structure of 22 bound in the active
site of sEH obtained from the most visited conformations along the
MD simulations. The benzohomoadamantane moiety occupies the LHS pocket
while the benzoic acid group lays in the RHS pocket. The central urea
unit establishes hydrogen bonds with Asp335 (d1), Tyr466 (d2), and Tyr383 (d3). The PDB 5AM3 has been used as the starting point for
all MD simulations. (b) Most relevant molecular interactions in the
RHS. Average distances (in Å) obtained from the last 150 ns of
MD simulations are represented. Hydrogen bonds between the oxygens
of the carboxylate group of 22 and the hydrogen of the
OH group of Ser412 and Ser415 are shown. The π–π
stacking average distances are computed between the most proximal
carbon atoms of each ring. The CH···π and hydrophobic
interaction average distances are computed between the hydrogen atoms
and the centroid of each aromatic ring (c) Histogram plots of the
distance between the carboxylic group of the catalytic Asp335 and
the amide groups of the inhibitor [d1(CγAsp335-NHINH)] and the distance between the carbonyl group of the urea
inhibitor and the OH group of Tyr466 residue [d2(OHTyr466-OINH)] along the MD simulations of t-AUCB (purple), 20 (red), 22 (blue),
and 23 (green). (d) Most relevant molecular interactions
in the LHS. Average distances (in Å) obtained from the last 150
ns of MD simulations are represented. The CH···π
interaction is calculated between the hydrogen of benzohomoadamantane
unit and the centroid of the benzoid ring of Trp336. The NH···π
interaction is monitored between the amide hydrogen of Gln384 and
the center of the aromatic ring of the benzohomoadamantane scaffold.
(a) Representative structure of 22 bound in the active
site of sEH obtained from the most visited conformations along the
MD simulations. The benzohomoadamantane moiety occupies the LHS pocket
while the benzoic acid group lays in the RHS pocket. The central urea
unit establishes hydrogen bonds with Asp335 (d1), Tyr466 (d2), and Tyr383 (d3). The PDB 5AM3 has been used as the starting point for
all MD simulations. (b) Most relevant molecular interactions in the
RHS. Average distances (in Å) obtained from the last 150 ns of
MD simulations are represented. Hydrogen bonds between the oxygens
of the carboxylate group of 22 and the hydrogen of the
OH group of Ser412 and Ser415 are shown. The π–π
stacking average distances are computed between the most proximal
carbon atoms of each ring. The CH···π and hydrophobic
interaction average distances are computed between the hydrogen atoms
and the centroid of each aromatic ring (c) Histogram plots of the
distance between the carboxylic group of the catalytic Asp335 and
the amide groups of the inhibitor [d1(CγAsp335-NHINH)] and the distance between the carbonyl group of the urea
inhibitor and the OH group of Tyr466 residue [d2(OHTyr466-OINH)] along the MD simulations of t-AUCB (purple), 20 (red), 22 (blue),
and 23 (green). (d) Most relevant molecular interactions
in the LHS. Average distances (in Å) obtained from the last 150
ns of MD simulations are represented. The CH···π
interaction is calculated between the hydrogen of benzohomoadamantane
unit and the centroid of the benzoid ring of Trp336. The NH···π
interaction is monitored between the amide hydrogen of Gln384 and
the center of the aromatic ring of the benzohomoadamantane scaffold.Significant differences were observed in the LHS
pocket, where
the benzohomoadamantane scaffold is placed. For t-AUCB, 20, 22, and 23, the
adamantane unit is mostly stabilized by the side chain of Trp336 through
stable CH···π interactions (see Figure d and S3). In all cases, additional hydrophobic interactions with the side
chains of Met339 and Val498 that wrap the adamantane in the LHS pocket
are observed. The incorporation of an aromatic ring into the adamantane
scaffold of t-AUCB induces a series of conformational
rearrangements in the active site, which further stabilize both the
adamantane and aromatic groups. In particular, the benzohomoadamantane
scaffold is reoriented in the beginning of the MD simulations to position
the aromatic ring toward the amide group of the side chain of Gln384
for establishing NH···π interactions that retain
the benzohomoadamantane group fixed in the LHS pocket (see Figure d and S3). This interaction is observed in all MD simulations
in the presence of 20, 22, and 23 inhibitors and, once formed, remains stable along the whole simulation
time. In t-AUCB, the amide group of Gln384 forms
a network of hydrogen bond interactions with the OH group of Tyr383
and the urea moiety that is partially disrupted in the presence of
benzohomoadamantane. Additionally, Phe381 moves away from the LHS
pocket to accommodate the aromatic rings of 20, 22, and 23, establishing frequent π-stacking
interactions. Met468, that in the t-AUCB X-ray structure
is pointing toward the solvent, moves toward the active site to establish
hydrophobic interactions with the benzohomoadamantane moiety. The
hydrophobic nature of the LHS pocket prevents the permanence of water
molecules in this cavity (see Figure S5). However, transient entrance of one or two water molecules is frequently
observed along the MD simulations of 20, 22, and 23 compounds. Finally, the symmetric adamantyl
unit in t-AUCB freely rotates inside the LHS pocket,
while the asymmetry introduced in the benzohomoadamantane scaffold
limits its rotation inside the active site (see Figure S6). The strong and stable hydrophobic and NH···π
interactions significantly reduce the rotation of the benzohomoadamantane
moiety inside the LHS pocket (see Figure S6). This limited flexibility can pose some impediments in the binding
pathway of benzohomoadamante derivatives.A significant conformational
rearrangement is observed for compounds 22 and 23 in the course of the 250 ns of the
MD simulation. The active site loop (493–500 residues) containing
Leu499 is significantly displaced from the reference X-ray structure
(see Figures and
RMSF S2). To explore whether this conformational
change can take place in longer time-scales also for t-AUCB and 20, we performed 500 ns of accelerated MD
(aMD) simulations for all compounds.[30] The
analysis of aMD simulations confirms that this rearrangement occurs
in the presence of all compounds (see Figure S7 and the Supporting Information for aMD details). This
rearrangement includes the motion of the bulky Leu499 side chain that
leaves the active site and the approximation of the carbonyl backbone
of Val498 toward the benzohomoadamante moiety (see Figure b,c). Based on the analysis
of NCIplot, the backbone of Val498 can establish dipole-induced dipole
interactions generated by halogens F and Cl on the benzohomoadamantane
scaffold. The computational mutagenesis of L499A and subsequent MD
simulations on the L499A sEH variant showed that the loop is not perturbed
along the simulation time, indicating that the displacement of the
bulky side-chain of Leu499 can play a key role in accommodating the
benzohomoadamantane scaffold. However, further investigations are
required to explore the contribution of this conformational rearrangement
in the thermodynamics and kinetics of inhibitor binding. Similar conformational
changes in loops located at the vicinity of the active site have been
described in other EH as key for substrate binding.[26] The molecular insight gained from MD simulations paves
the way toward the rational improvement of benzohomoadamantane scaffolds
for enhanced inhibition.
Figure 6
(a) Overlay of two representative structures
of t-AUCB (purple, PDB 5AM3) and 22 (blue) bound in
the active site of sEH. The
image indicates the motion of the loop (493–500) containing
Val498 and Leu499 with a black arrow. The loop is colored in purple
for the X-ray t-AUCB conformation and in blue for
the 22 conformation. The PDB 5AM3 have been used as the starting point
for all MD simulations. (b) X-ray structure (PDB 5AM3), where Leu499 is
pointing toward the adamantane moiety. (c) Most visited active site
conformation with 22 bound, where Leu499 is displaced
from the active site and Val498 carbonyl points toward the benzohomoadamantane
moiety.
(a) Overlay of two representative structures
of t-AUCB (purple, PDB 5AM3) and 22 (blue) bound in
the active site of sEH. The
image indicates the motion of the loop (493–500) containing
Val498 and Leu499 with a black arrow. The loop is colored in purple
for the X-ray t-AUCB conformation and in blue for
the 22 conformation. The PDB 5AM3 have been used as the starting point
for all MD simulations. (b) X-ray structure (PDB 5AM3), where Leu499 is
pointing toward the adamantane moiety. (c) Most visited active site
conformation with 22 bound, where Leu499 is displaced
from the active site and Val498 carbonyl points toward the benzohomoadamantane
moiety.
Pharmacokinetic
Studies of Selected Compounds 20 and 22
The pharmacokinetic characterization
of 20 and 22 was performed in male C57BL/6
mice by intraperitoneal administration of 3 mg/kg of each compound.
As shown in Table , both compounds demonstrated good absorption and elimination characteristics.
Notwithstanding, compound 22 presented a better profile
than 20 considering its larger half-life (5.2 h), higher Cmax (3583 ng/mL) and AUC (23,328.12 h*ng/mL),
and lowest clearance (0.13 L/h/kg). Considering its superior pharmacokinetic
profile and its better potency and solubility (Tables and 4), 22 was selected for conducting the in vivo efficacy
study in the well-known murine model of cerulein-induced acute pancreatitis
(AP).[31−33]
Table 4
Pharmacokinetic Parameters in the
Male C57BL/6 Mouse for Compounds 20 and 22 after 3 mg/kg IP Administrationa
Cpd
dose (mg/Kg)
HL (h)
Tmax (h)
Cmax (ng/mL)
AUClast (h*ng/mL)
AUCINF (h*ng/mL)
Vd (L/Kg)
Cl (L/h/Kg)
20
3
1.17
0.50
1610
2260
2323
2.18
1.29
22
3
5.2
2
3583
22543
23328
0.96
0.13
See the Experimental
Section and Tables S2 and S3 and Figures S8 and S9 in the Supporting Information
See the Experimental
Section and Tables S2 and S3 and Figures S8 and S9 in the Supporting Information
In vivo Efficacy Study
AP is a potentially life-threatening gastrointestinal disease,
and its incidence has been increasing over the last few decades. The
onset of the disease is thought to be triggered by intra-acinar cell
activation of digestive enzymes that results in interstitial edema,
inflammation, and acinar cell death that often leads to a systemic
inflammation response.[31−34] The efficacy of the new sEHI 22 at 0.1 and 0.3 mg/kg
was assessed in the cerulein-induced AP murine model. The experimental
procedure for the in vivo efficacy study followed
already published protocols.[35]First,
the health status of the animals was analyzed by monitoring their
change in body weight along the experimental procedure. After food
replacement (with the last cerulein injection), control animals gained
some weight, and, as expected, it was not observed in animals receiving
cerulein only. In contrast, animals treated with both doses (0.3 and
0.1 mg/kg) of 22 showed an increased body weight although
only the group treated at 0.3 mg/kg reached statistical significance
(p < 0.01 vs Cerulein group)
(Figure ).
Figure 7
Percentage
of body weight change at the end of the study vs t = 0 h. Effect of 12 consecutive administrations of
cerulein (50 μg/kg, IP) and treatment with 22 (single
dose, 0.3 or 0.1 mg/kg, IP) on C57BL/6 male mice body weight. Results
are expressed as mean ± SEM (n = 3–9).
*p < 0.05, **p < 0.01, ****p < 0.0001 vs Cerulein group (ANOVA-one
way).
Percentage
of body weight change at the end of the study vs t = 0 h. Effect of 12 consecutive administrations of
cerulein (50 μg/kg, IP) and treatment with 22 (single
dose, 0.3 or 0.1 mg/kg, IP) on C57BL/6 male mice body weight. Results
are expressed as mean ± SEM (n = 3–9).
*p < 0.05, **p < 0.01, ****p < 0.0001 vs Cerulein group (ANOVA-one
way).In addition, the concentrations
of 22 in plasma and
pancreatic tissue were measured 10 h post-administration. Taking into
account that 22 is a subnanomolar inhibitor of the murine
sEH, we confirmed that the administration of both doses produced enough
plasma levels of compound 22 to inhibit the sEH (175
nM for the dosage of 0.3 mg/kg and 14 nM for the dosage of 0.1 mg/kg).
Moreover, the compound concentration in pancreas was also analyzed
showing 29.9 ng/g for the dosage of 0.3 mg/kg, confirming that 22 was able to reach the pancreatic tissue. However, no amount
of 22 was detected in pancreatic tissues with the 0.1
mg/kg dose.Finally, histologic analysis of pancreas was assessed
in order
to determine if treatment with 22 reduced the severity
of the cerulein-induced pancreatitis. Pathologic changes were studied
on H&E-stained pancreas sections (Figure A).
Figure 8
Results of the histologic analysis of pancreas
from mice treated
with vehicle (control), cerulein, and cerulein plus either 0.1 or
0.3 mg/kg of compound 22. (A) Representative H&E-stained
sections of the pancreas from the in vivo efficacy
study. Arrows indicate inflammatory cells and edema. Bold arrows indicate
intracellular vacuoles. (B–E) Histologic scoring of pancreatic
tissues. (B), edema. (C), inflammatory cells (mononuclear and polymorphonuclear).
(D), parenchymal atrophy. (E), total scoring (pancreatic parenchymal
atrophy, vacuolar degeneration of cells, edema, hemorrhage, mononuclear
inflammatory cells, mononuclear inflammatory cells, polimorfonuclear
inflammatory cells and necrosis). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control. #p < 0.05 vs cerulein.
Results of the histologic analysis of pancreas
from mice treated
with vehicle (control), cerulein, and cerulein plus either 0.1 or
0.3 mg/kg of compound 22. (A) Representative H&E-stained
sections of the pancreas from the in vivo efficacy
study. Arrows indicate inflammatory cells and edema. Bold arrows indicate
intracellular vacuoles. (B–E) Histologic scoring of pancreatic
tissues. (B), edema. (C), inflammatory cells (mononuclear and polymorphonuclear).
(D), parenchymal atrophy. (E), total scoring (pancreatic parenchymal
atrophy, vacuolar degeneration of cells, edema, hemorrhage, mononuclear
inflammatory cells, mononuclear inflammatory cells, polimorfonuclear
inflammatory cells and necrosis). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control. #p < 0.05 vs cerulein.As expected, the cerulein
control group presents pancreatic damage
representative of AP, including edema (Figure B), infiltration of inflammatory cells (Figure C), and parenchymal
atrophy (Figure D).
By contrast, treatment with both doses of 22 ameliorated
cerulein-induced effects. The higher dose (0.3 mg/kg) more efficiently
reversed the pancreatic damage, edema, and neutrophil infiltration
(Figure and Tables
S4–S7 in the Supporting Information).
Conclusions
The sEH has been identified
as a suitable target for several inflammatory
diseases. For this reason, many adamantane-based and aryl-based sEHIs
have been designed and three selected compounds, AR9281, GSK2256294,
and EC5026, have reached human clinical trials. In this work, we found
that the aromatic and adamantane fragment can be merged, leading to
the very versatile benzohomoadamantane scaffold. Therefore, three
series of compounds replacing the adamantane moiety of AR9281, 7,
and t-AUCB by this new polycycle have been synthesized
and biologically evaluated. The in vitro profiling
of these sEHIs (solubility, cytotoxicity, metabolic stability, CYP450s, hLOX-5, hCOX-2 and hERG inhibition) allowed
to select a suitable candidate for an in vivo efficacy
study in a murine model of AP, for which sEHIs showed effectiveness
at ameliorating this condition.[19,35,36] The administration of 22 improved the general health
status of cerulein-induced AP mice and significantly reduced pancreatic
damage. Hence, the benzohomoadamantane moiety emerges as a suitable
hydrophobic scaffold for the design of novel sEHIs. The molecular
insight provided by MD simulations indicated that sEH reshapes the
active site pocket to stabilize the aromatic ring of the benzohomoadamantane
scaffold. Due to the promising results obtained with compound 22, more research around benzohomoadamantane-based sEHIs for
the treatment of inflammatory and pain-related diseases is currently
ongoing.
Experimental Section
Chemical Synthesis
General Methods
Commercially available
reagents and solvents were used without further purification unless
stated otherwise. Preparative normal phase chromatography was performed
on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf
silica gel cartridges. Thin-layer chromatography was performed with
aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1.05554),
and spots were visualized with UV light and 1% aqueous solution of
KMnO4. Melting points were determined in open capillary
tubes with a MFB 595010M Gallenkamp. 400 MHz 1H and 100.6
MHz 13C NMR spectra were recorded on a Varian Mercury 400
or on a Bruker 400 AVANCE III spectrometers. 500 MHz 1H
and 125.7 13C NMR spectra were recorded on a Varian Inova
500 spectrometer. The chemical shifts are reported in ppm (δ
scale) relative to internal tetramethylsilane, and coupling constants
are reported in Hertz (Hz). Assignments given for the NMR spectra
of selected new compounds have been carried out on the basis of DEPT,
COSY 1H/1H (standard procedures), and COSY 1H/13C (gHSQC and gHMBC sequences) experiments.
IR spectra were run on the PerkinElmer Spectrum RX I, PerkinElmer
Spectrum TWO, or Nicolet Avatar 320 FT-IR spectrophotometers. Absorption
values are expressed as wave-numbers (cm–1); only
significant absorption bands are given. High-resolution mass spectrometry
(HRMS) analyses were performed with an LC/MSD TOF Agilent Technologies
spectrometer. The elemental analyses were carried out in a Flash 1112
series Thermofinnigan elemental microanalyzator (A5) to determine
C, H, N, and S. The structure of all new compounds was confirmed by
elemental analysis and/or accurate mass measurement, IR, 1H NMR and 13C NMR. The analytical samples of all the new
compounds, which were subjected to pharmacological evaluation, possessed
purity ≥95% as evidenced by their elemental analyses.
To a solution of 9-fluoro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride
(180 mg, 0.67 mmol) in DCM (3 mL) and saturated aqueous NaHCO3 solution (2 mL), triphosgene (74 mg, 0.25 mmol) was added.
The biphasic mixture was stirred at room temperature for 30 min and
then the two phases were separated and the organic one was washed
with brine (3 mL), dried over anh. Na2SO4, filtered,
and evaporated under vacuum to obtain 1–2 mL of a solution
of isocyanate in DCM. To this solution were added DMF (4 mL), 4-[((1r,4r)-4-aminocyclohexyl)oxy]benzoic acid
hydrochloride (182 mg, 0.67 mmol), and Et3N (136 mg, 1.34
mmol). The mixture was stirred overnight at room temperature and the
solvent was then evaporated. The residue was dissolved in DCM (5 mL)
and washed with 2N HCl (3 mL). The organic phase was dried over anh.
Na2SO4, filtered, and evaporated under vacuum
to obtain benzoic acid 23 (240 mg, 72% yield) as a yellow
residue. The analytical sample was obtained by crystallization from
hot ethyl acetate/pentane mixtures, mp 253–254 °C. IR
(ATR): 3325, 2929, 2859, 1682, 1629, 1606, 1558, 1511, 1424, 1359,
1317, 1282, 1251, 1221, 1165, 1104, 1090, 1003, 938, 851, 772, 697,
642 cm–1. 1H NMR (400 MHz, MeOD): δ
1.31 [dq, J = 3.2 Hz, J′
= 13.2 Hz, 2 H, 3′(5′)-Hax], 1.54 [dq, J = 3.2 Hz, J′ = 12.8 Hz, 2 H, 2′(6′)-Hax], 1.81 [d, J = 12 Hz, 2 H, 10″(13″)-Hax)], 1.93–2.02 [complex signal, 4 H, 3′(5′)-Heq, 6″(12″)-Hax], 2.07–2.19
[complex signal, 8 H, 2′(6′)-Heq, 4-H, 6″(12″)-Heq, 10″(13″)-Heq], 3.23 [t, 2 H, J = 7.2 Hz, 5″(11″)-H], 3.49 (m, 1 H, 4′-H),
4.37 (m, 1 H, 1′-H), 6.95 [d, J = 8.8 Hz,
2 H, 3(5)-H], 7.09 [broad s, 4 H, 1″(4″)-H, 2″(3″)-H],
7.94 [d, J = 8.8 Hz, 2 H, 2(6)-H]. 13C
NMR (100.5 MHz, MeOD): δ 31.1 [CH2, C2′(6′)],
31.6 [CH2, C3′(5′)], 40.4 [CH2, d, 4JC-F = 1.9 Hz,
C6″(12″)], 41.1 [CH, d, 3JC-F = 13.3 Hz, C5″(11″)], 41.4 [CH2, d, 2JC-F =
20 Hz, C10″(13″)], 47.9 (CH2, d, 2JC-F = 17.9, C8″), 48.7
(CH, C4′), 57.6 (C, d, 3JC-F = 11.2 Hz, C7″), 76.0 (CH, C1′), 95.4 (C, d, 1JC-F = 177 Hz, C9″),
116.1 [CH, C3(5)], 123.9 (C, C1), 127.9 [CH, C2″(3″)],
129.1 [CH, C1″(C4″)], 132.8 [CH, C2(6)], 146.4 [C, C4a″(11a″)],
159.2 (C, NHCONH), 163.2 (C, C4), 169.8 (C, CO2H). HRMS:
calcd for [C29H33FN2O4–H]−; 491.2352; found, 491.2334.
Determination of IC50 sEHIs in
Human, Murine, and Rat Purified sEH
IC50 is the
concentration of a compound that reduces the sEH activity by 50%.
The IC50 values reported herein were determined using a
fluorescent-based assay (CMNPC as substrate).[16] The fluorescent assay was used with purified recombinant human,
mouse, or rat sEH proteins. The enzymes were incubated at 30 °C
with the inhibitors ([I]final = 0.4–100,000 nM)
for 5 min in 100 mM sodium phosphate buffer (200 μL, pH 7.4)
containing 0.1 mg/mL of BSA and 1% of DMSO. The substrate (CMNPC)
was then added ([S]final = 5 μM). Activity was assessed
by measuring the appearance of the fluorescent 6-methoxynaphthaldehyde
product (λex = 330 nm, λem = 465
nm) every 30 s for 10 min at 30 °C on a SpectraMax M2 (Molecular
Devices). Results were obtained by regression analysis from a linear
region of the curve. All measurements were performed in triplicate
and the mean is reported. t-TUCB, a classic sEHI,
was run in parallel and the obtained IC50s were corroborated
with reported literature values[37] to validate
the experimental results.
In silico
Study
MD Simulation Details
The parameters
for t-AUCB, 20, 22, and 23 for the MD simulations were generated within the ANTECHAMBER
module of AMBER 18[38] using the general
AMBER force field (GAFF),[39] with partial
charges set to fit the electrostatic potential generated at the HF/6-31G(d)
level by the RESP model.[40] The charges
were calculated according to the Merz–Singh–Kollman
scheme[41,42] using Gaussian 09.[43]MD simulations of sEH were carried out using PDB 5AM3 (crystallized with t-AUCB)[27] as a starting point.
For the MD simulations in the apo state, the t-AUCB was removed from the active site. The benzohomoadamanatane
derivatives corresponding to 20, 22, and 23 were manually prepared using the t-AUCB
structure as a starting point. Molecular docking calculations using
the standard parameters of the SwissDock web server were carried out
to assess the preferred orientation of 20, 22, and 23.[44,45] The coordinates of t-AUCB in PDB 5AM3 were a reference for placing compounds 20, 22, and 23 in molecular docking calculations.
From these coordinates, conventional MD simulations were used to explore
the conformational plasticity of sEH in the apo state
and in the presence of t-AUCB, 20, 22, and 23 bound in the active site. All simulations
were performed using the AMBER ff14SB force field.[46] Amino acid protonation states were predicted using the
H++ server (http://biophysics.cs.vt.edu/H++). The MD simulations have been carried with the following protonation
of histidine residues: HIE146, HIE239, HIP251, HID265, HIP334, HIE420,
HIE506, HIE513, HIE518, and HIP524.Each system was immersed
in a pre-equilibrated truncated octahedral
box of water molecules with an internal offset distance of 10 Å.
All systems were neutralized with explicit counterions (Na+ or Cl–). A two-stage geometry optimization approach
was performed. First, a short minimization of the positions of water
molecules with positional restraints on the solute by a harmonic potential
with a force constant of 500 kcal mol–1 Å–2 was done. The second stage was an unrestrained minimization
of all the atoms in the simulation cell. Then, the systems were gently
heated in six 50 ps steps, increasing the temperature by 50 K each
step (0–300 K) under constant-volume, periodic-boundary conditions,
and the particle-mesh Ewald approach[47] to
introduce long-range electrostatic effects. For these steps, a 10
Å cutoff was applied to Lennard-Jones and electrostatic interactions.
Bonds involving hydrogen were constrained with the SHAKE algorithm.[48] Harmonic restraints of 10 kcal mol–1 were applied to the solute, and the Langevin equilibration scheme
was used to control and equalize the temperature.[49] The time step was kept at 2 fs during the heating stages,
allowing potential inhomogeneities to self-adjust. Each system was
then equilibrated for 2 ns with a 2 fs timestep at a constant pressure
of 1 atm (NPT ensemble). Finally, conventional MD trajectories at
a constant volume and temperature (300 K) were collected. In total,
there were three replicas of 250 ns MD simulations for sEH in the
apo state and in the presence of t-AUCB, 20, 22, and 23, gathering a total of 3.75 μs of MD simulation
time. Each MD simulation was clusterized based on active site residues,
and the structures corresponding to the most populated clusters were
used in the non-covalent interactions analysis. We monitored the presence
of water molecules using the watershell function
of the cpptraj MD analysis program.[50]
Microsomal Stability
The human,
rat, and mice recombinant microsomes employed were purchased from
Tebu–Xenotech. The compound was incubated at 37 °C with
the microsomes in a 50 mM phosphate buffer (pH = 7.4) containing 3
mM MgCl2, 1 mM NADP, 10 mM glucose-6-phosphate, and 1 U/mL
glucose-6-phosphate-dehydrogenase. Samples (75 μL) were taken
from each well at 0, 10, 20, 40, and 60 min and transferred to a plate
containing 4 °C 75 μL of acetonitrile and 30 μL of
0.5% formic acid in water were added for improving the chromatographic
conditions. The plate was centrifuged (46,000g, 30
min) and supernatants were taken and analyzed by an ultraperformance
liquid chromatograph–tandem mass spectrometer (Xevo-TQD, Waters)
by employing a BEH C18 column and an isocratic gradient of 0.1% formic
acid in water: 0.1% formic acid acetonitrile (60:40). The metabolic
stability of the compounds was calculated from the logarithm of the
remaining compounds at each of the time points studied.
Solubility
A 10 mM stock solution
of the compound was serially diluted in 100% DMSO and 1 μL of
this solution was added to a 384-well UV-transparent plate (Greiner)
containing 99 μL of PBS. The plate was incubated at 37 °C
for 2 h and the light scattering was measured in a Nephelostar Plus
reader (BMG LABTECH). The data were fitted to a segmented linear regression
for measuring the compound solubility.
Permeability
The Caco-2 cells were
cultured to confluency, trypsinized, and seeded onto a 96-filter transwell
insert (Corning) at a density of ∼10,000 cells/well in DMEM
cell culture medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 1% penicillin/streptomycin. Confluent Caco-2
cells were subcultured at passages 58–62 and grown in a humidified
atmosphere of 5% CO2 at 37 °C. Following an overnight
attachment period (24 h after seeding), the cell medium was replaced
with fresh medium in both the apical and basolateral compartments
every other day. The cell monolayers were used for transport studies
21 days post seeding. The monolayer integrity was checked by measuring
the transepithelial electrical resistance (TEER), obtaining values
≥500 Ω/cm.[2] On the day of
the study, after the TEER measurement, the medium was removed and
the cells were washed twice with pre-warmed (37 °C) Hank’s
balanced salt solution (HBSS) buffer to remove traces of medium. Stock
solutions were made in dimethyl sulfoxide (DMSO) and further diluted
in HBSS (final DMSO concentration 1%). Each compound and reference
compounds (Colchicine, E3S) were all tested at a final concentration
of 10 μM. For A → B directional transport, the donor
working solution was added to the apical (A) compartment and the transport
media as receiver working solution was added to the basolateral (B)
compartment. For B → A directional transport, the donor working
was added to the basolateral (B) compartment and transport media as
receiver working solution was added to the apical (A) compartment.
The cells were incubated at 37 °C for 2 h with gentle stirring.At the end of the incubation, samples were taken from both donor
and receiver compartments and transferred into 384-well plates and
analyzed by ultraperformance liquid chromatography–tandem mass
spectrometry (UPLC–MS/MS). The detection was performed using
an ACQUITY UPLC/Xevo TQD System. After the assay, Lucifer yellow was
used to further validate the cell monolayer integrity, cells were
incubated with LY 10 μM in HBSS for 1 h at 37 °C, obtaining
permeability (Papp) values for LY of ≤10 nm/s, confirming the
well-established Caco-2 monolayer.
Cytotoxicity
in SH-SY5Y Cells
Cytotoxicity
was evaluated in the human neuroblastoma SH-SY5Y cell line (ATCC number:
CRL-2266). Cells were cultured in minimum essential medium/Ham’s-F12
(1:1, v/v) medium, supplemented with non-essential amino acids, 10%
fetal bovine serum, 1 mM glutamine, and 50 μg/mL gentamycin
(all reagents from Gibco, Invitrogen). For experiments, the cells
were seeded at 3 × 105 cells/mL (100 μL/well) in 96-well
plates (Nunc). After 24 h, the testing compounds were added concentrated
to triplicate wells to obtain the final different concentrations up
to 100 μM. Compounds were incubated for further 24 h. At termination,
cytotoxicity was analyzed by the propidium iodide (PI) fluorescence
stain assay. All compounds were tested in three independent experiments
using different cell passages. The PI assay measures cell death. PI
enters into the cells with damaged membranes and greatly increases
the fluorescence by binding to DNA. PI reagent (Molecular Probes)
at the final concentration of 7.5 μg/mL was added to the cells
and incubated for 1 h. The resulting fluorescence was measured by
a Gemini XPS Microplate reader (Millipore) at 530 nm excitation and
645 nm emission. The percentage of cell death induced by the treatments
was calculated from the fluorescence of treated cells (Ft) relative to that of control cells (Fmin) and cells incubated with Triton X100 (Fmax) as the 0 and 100% cell death, respectively [% = ((Ft – Fmin)/(Fmax – Fmin)) × 100].
Inhibition of hLOX-5
AA and 2′,7′-dichlorodihydrofluorescein
diacetate (H2DCFDA) were obtained from Sigma. Human recombinant
LOX-5 was purchased from Cayman Chemical. For the determination of hLOX-5 activity, the method described by Pufahl et al. was followed.[51] The assay
solution consisted of 50 mM Tris (pH 7.5), 2 mM EDTA, 2 mM CaCl2, 3 μM AA, 10 μM ATP, 10 μM H2DCFDA, and 100 mU/well hLOX-5. For the enzyme inhibition
studies, the compounds to be tested were added to the assay solution
prior to AA and ATP and were preincubated for a period of 10 min at
room temperature, after which AA and ATP were added. The enzymatic
reaction was carried out for 20 min and terminated by the addition
of 40 μL of acetonitrile. The fluorescence measurement, 485
nm excitation and 520 nm emission, was performed on a FLUOstar OPTIMA
(BMG LABTECH, Offenburg, Germany.). The IC50 is defined
as the concentration of compound that inhibits enzymatic activity
by 50% over the untreated enzyme control.
Cytochrome
P450 Inhibition Assay
The objective of this study was to
screen the inhibition potential
of the compounds using recombinant human cytochrome P450 enzymes CYP3A4
(BFC and DBF substrates) and probe substrates with fluorescent detection.
Incubations were conducted in a 200 μL volume in 96-well microtiter
plates (COSTAR 3915). The addition of the mixture buffer-cofactor
(KH2PO4 buffer, 1.3 mM NADP, 3.3 mM MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase), control supersomes,
the Standard inhibitor Ketoconazole (Sigma K1003), and previously
diluted compounds to plates was carried out by a liquid handling station
(Zephyr Caliper). The plate was then preincubated at 37 °C for
5 min in a 100 μL volume, and reaction was initiated by the
addition of a prewarmed enzyme/substrate (E/S) mix. The E/S mix contained
buffer (KH2PO4), enzyme (CYP), substrate 7-benzyloxytrifluoromethyl
coumarin (7-BFC), and dibenzylfluorescein (DBF) in a reaction volume
of 200 μL. Reactions were terminated after various times depending
on the substrate by addition of STOP solution (ACN/TrisHCl 0.5 M 80:20
(BFC) or 2 N NaOH for CYP3A4 (DBF). Fluorescence per well was measured
using a fluorescence plate reader (Tecan M1000 pro) and percentage
of inhibition was calculated.
hERG
Inhibition Assay
The assay
was carried out at a CHO cell line transfected with the hERG potassium
channel. 72 h before the assay, 2500 cells were seeded on a 384-well
black plate (Greiner 781091). The cell line was maintained at 37 °C
in a 5% CO2 atmosphere for 24 h and at 30 °C in a
5% CO2 atmosphere for 48 h plus. hERG activity was measured
by using the FluxorTM Potassium Ion Chanel Assay Kit (Thermo Fisher
F10016). Medium was replaced for 20 μL of loading buffer and
the cells were incubated for 60 min at RT, protected from direct light.
After incubation, the loading buffer was replaced for the assay buffer
and the compounds were incubated for 30 min at RT. 5 μL of a
stimulus buffer was added to each well and the fluorescence was read
(λex = 490 nm, λem = 525 nm) using
an imaging plate reader system (FDSS7000EX, Hamamatsu) every second
after the establishment of a baseline line.
Pharmacokinetic Study
24 male C57BL/6
mice (21 g approx.) were supplied by Envigo (Barcelona, Spain) (Ref
15131). During the experimental procedure, animals were identified
with a permanent marker (tail code numbers). Plasma samples were obtained
at 0, 0.25, 0.5, 1, 2, 4, 6, and 24 h post-dosing. Upon arrival, animals
were housed in groups of 3 animals/cage in polycarbonate maintenance
cages (type IIL; 365 × 207 × 140 mm, with a surface area
of 530 cm2) with absorbent bedding (Lignocel, JRS). Animals
were kept in an environmentally controlled room (ventilation of 10–15
air changes per hour, temperature of 22 ± 3 °C, and humidity
of 35–70%) on a 12 h light/dark cycle. They underwent a period
of at least 5 days of observation and acclimatization between the
date of arrival and the start of the procedure. During this period,
the animals were observed to check their general health state. A maintenance
diet was supplied by Harlan Interfauna Ibérica S.L. (2014 Harlan
Teklad Global Diets) and was provided to the animals ad libitum. Diet was analyzed by the manufacturer to detect possible contaminants.
Tap water was supplied by CASSA (Servei d’Aigües de
Sabadell) and was provided to the animals by bottles ad libitum. The animals were maintained in accordance with the European Directive
for the Protection of Vertebrate Animals Used for Experimental and
other Scientific Purposes (86/609/EU). Decree 214/1997 of 30th July.
Ministry of Agriculture, Livestock, and Fishing of the autonomous
government of Catalonia, Spain. Royal Decree 53/2013 of 1st February
(Spain) Animal care including environmental and housing conditions
conformed to the applicable standard operating procedures regarding
laboratory animals of Draconis Pharma S.L. All the experimental procedures
were approved by the Animal Experimentation Ethical Committee of Universitat
Autònoma de Barcelona (procedure number: 3718) and by the Animal
Experimentation Commission of the Generalitat de Catalunya (Catalan
Government) (DAAM:9590). Formulations were prepared on the day of
the study. Vehicle was 10% of 2-hydroxypropyl-β-cyclodextrin,
(CAS 128446-35-5) Sigma-Aldrich (Ref.332607-25G). 21 mice were intraperitoneally
administered with a single dose of 3 mg/kg of 22. The
volume of administration was 10 mL/kg. Animals were weighed before
each administration to adjust the required volume. Blood samples were
collected at different times post administration: 0, 0.25, 0.5, 1,
2, 4, 6, and 24 h. Three mice were not administered and referred as t = 0. Blood samples (0.5–0.8 mL) were collected
from anesthetized animals with isoflurane by cava vein puncture in
tubes containing K2-EDTA 5%. Blood samples were centrifuged at 10,000
rpm for 5 min to obtain plasma that was stored at −20 °C
until analysis. Analytical measurements were performed by LC–MS/MS.
Pharmacokinetics parameters were calculated with Phoenix 64 (WinNonlin).Forty-one male C57BL/6 mice (8-week-old; approximately 24 g) were
supplied by Envigo (Barcelona, Spain) (Ref 16512). During the experimental
procedure, the animals were identified with a permanent marker (tail
code numbers). Upon arrival, the animals were housed in groups of
8–9 animals/cage in polysulfone maintenance cages (480 ×
265 × 210 mm, with a surface area of 940 cm2) with
wire tops and wood chip bedding. The animals were kept in an environmentally
controlled room (ventilation, temperature 22 ± 2 °C, and
humidity 35–65%) on a 12 h light/dark cycle. They underwent
a period of 7 days of acclimatization between the date of arrival
and the start of the procedure. During this period, the animals were
observed to check their general health state. The maintenance diet
was supplied by Harlan Interfauna Ibérica S.L. (2018 Harlan
Teklad Global Diets). Diet was provided to the animals ad
libitum, but they were fasted overnight before the first
cerulein injection, and food was replaced after the last cerulein
injection. Tap water was supplied by CASSA (Servei d’Aigües
de Sabadell) ad libitum. The animals were maintained
in accordance with the European Directive for the Protection of Vertebrate
Animals Used for Experimental and other Scientific Purposes (86/609/EU).
Decree 214/1997 of 30th July. Ministry of agriculture, livestock and
fishing of the Autonomous Government of Catalonia, Spain. Royal Decree
53/2013 of 1st February (Spain). All the experimental procedures were
approved by the Ethical Committee on human and animal experimentation
(CEEAH) of Universitat Autònoma de Barcelona (UAB) (procedure
number: 4107) and by the Animal Experimentation Commission of the
Generalitat de Catalunya (Catalan Government) (DAAM: 10146). The test
item was dissolved in vehicle 10% 2-hydroxypropyl-β-cyclodextrin
(CAS 128446-35-5) Sigma-Aldrich (Ref 332607). The vehicle was prepared
the day before and kept at 4 °C. Pancreatitis induction: Mice
(n = 41) were weighed, identified by a distinct number
at the base of the tail, and fasted overnight. Cerulein (cerulein
and cerulein + 22 groups) (50 μg/kg, prepared in
0.9% NaCl) or vehicle (0.9% NaCl) (control group) were intraperitoneally
injected (V = 5 mL/kg) 12 consecutive times, at 1
h intervals (h = 0–11). Food was replaced
after the last injection. A satellite experiment was designed where
animals (n = 3) were distributed in control, cerulein,
and cerulein + 22-treated groups. Pancreatitis was induced
by seven injections of cerulein (or vehicle in the control group)
at 1 h intervals (h = 0–6). Treatments: the
test item was administered intraperitoneally in one injection to 22_03 (0.3 mg/kg) and 22_01 (0.1 mg/kg) groups
at 14 h after the first cerulein injection. Animals from the control
and cerulein groups received vehicle administration (10% 2-hydroxypropyl-β-cyclodextrin)
(V = 10 mL/kg). Extra groups were treated 2 h after
the first cerulein injection: 22 (0.3 mg/kg), control,
and cerulein groups (10% 2-hydroxypropyl-β-cyclodextrin). Study
end: 24 h after the first cerulein injection, animals were weighed
and anesthetized with isoflurane. Blood was collected from the vena
cava in an Eppendorf containing K2-EDTA and centrifuged at 10,000
rpm for 5 min for plasma collection. Plasma was stored at −80
°C until analysis. The mice were sacrificed by cervical dislocation
and the pancreases were dissected and weighed. Pancreases from three
animals were frozen in liquid N2 and stored at −80
°C until analysis. Pancreases from five mice were sectioned and
one part was placed in 10% formalin and sent to Anapath (Granada,
Spain) for histology analysis and the other was immediately placed
in RNAse-free eppendorfs, frozen in N2, and stored at −80
°C for gene expression assays.
Histologic
Analysis
Pancreatic
samples were treated with increasing grade alcohols and two xylol
baths and embedded in paraffin. They were subsequently cut using a
microtome and processed for staining. For the deparaffinization of
the samples, two xylene baths (10 min) and three alcohols were used
in decreasing solutions (100%, 90 and 70%) (5 min) and subsequently
stained with hematoxylin (5 min) and eosin (5 min). During the dehydration
process after staining with eosin, alcohols in increasing solution
(70, 96, and 100%) and xylene were used again. Finally, the samples
were mounted with DPX.Histologic scoring of pancreatic sections
was performed to grade the extent of pancreatic parenchymal atrophy,
vacuolar degeneration of cells, edema, hemorrhage, mononuclear inflammatory
cells, mononuclear inflammatory cells, polymorphonuclear inflammatory
cells, and necrosis. The assigned scores were the following: 0 (no
changes): when no lesions were observed or the observed changes were
within normality; 1 (minimal): when changes were few but exceeded
those considered normal; 2 (light): lesions were identifiable but
with moderate severity; 3 (moderate): important injuries but they
can still increase in severity; 4 (very serious): the lesions are
very serious and occupy most of the analyzed tissue. The lesions were
evaluated in the most affected lobes of all the pancreases. In the
case of assessment of atrophy, it was determined based on the percentage
of atrophied tissue as: 0 without atrophy; 1: 0–25% of atrophic
parenchyma; 2: between 25 and 50%; 3: between 50 and 75% and 4: between
75 and 100%.
Authors: C Morisseau; M H Goodrow; D Dowdy; J Zheng; J F Greene; J R Sanborn; B D Hammock Journal: Proc Natl Acad Sci U S A Date: 1999-08-03 Impact factor: 11.205
Authors: James A Maier; Carmenza Martinez; Koushik Kasavajhala; Lauren Wickstrom; Kevin E Hauser; Carlos Simmerling Journal: J Chem Theory Comput Date: 2015-07-23 Impact factor: 6.006
Authors: Juan Martín-López; Sandra Codony; Clara Bartra; Christophe Morisseau; María Isabel Loza; Coral Sanfeliu; Bruce D Hammock; José Brea; Santiago Vázquez Journal: Pharmaceuticals (Basel) Date: 2021-12-17
Figure 1
Figure 2
Figure 3
Scheme 1
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8