Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration

Article abstract of DOI:10.1016/j.steroids.2014.12.016

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC₅₀ = 0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC₅₀ = 6 nM) > 10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 μmol/kg significantly inhibited and at 1 μmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t_1/2 = 6.5 h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

Full text of DOI:10.1016/j.steroids.2014.12.016

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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Discovery of a novel isoxazoline derivative of prednisolone endowed
with a robust anti-inflammatory profile and suitable for topical
pulmonary administration
Q1 E. Ghidini a, , A.M. Capelli , C. Carnini , V. Cenacchi , G. Marchini , A. Virdis , A. Italia , F. Facchinetti
⇑
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Q2 a Chemistry Research and Drug Design Department, Chiesi Farmaceutici S.p.A., Parma, Italy
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Pharmacokinetic Department, Chiesi Farmaceutici S.p.A., Parma, Italy
Pharmacology and Toxicology Department, Chiesi Farmaceutici S.p.A., Parma, Italy
Nikem Research Srl, Baranzate di Bollate (Mi), Italy
Chiman Srl, via Reggio Calabria, 12 Rottofreno (PC), Italy
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a r t i c l e i n f o
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A novel glucocorticoids series of (GCs), 6
a
,9
a
-di-Fluoro 3-substituted C-16,17-isoxazolines was designed,
1
2
2
2
Received 4 June 2014
synthesised and their structure–activity relationship was evaluated with glucocorticoid receptor (GR)
binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with
in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nano-
molar inhibitory potency (IC50 = 0.84 nM) against TNFa-evoked IL-8 release in primary human airways
smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with
a potency (IC50 = 6 nM) > 10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) admin-
istration, Cpd #15, at 0.1 lmol/kg significantly inhibited and at 1 lmol/kg fully counteracted eosinophilic
infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to
be suitable for pulmonary topical administration given its sustained lung retention (t1/2 = 6.5 h) and high
pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In
summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treat-
ment of conditions associated with pulmonary inflammation such as asthma and COPD.
Ó 2015 Published by Elsevier Inc.
Received in revised form 27 November 2014
Accepted 15 December 2014
Available online xxxx
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Keywords:
Corticosteroid
Isoxazoline
Ovalbumin
Nuclear translocation
PK profile
2
3
9
0
GILZ
4
6
4
4
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1. Introduction
A considerable amount of research is aimed at discovering novel
steroidal GR agonists with high anti-inflammatory potency upon
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An important limitation of GCs therapy is that the desired anti-
inflammatory effects are accompanied by side effects such as loss
of muscle mass, redistribution of body fat, osteoporosis, diabetes,
glaucoma and depression [1]. In patients with asthma and chronic
obstructive pulmonary disease (COPD), the adverse effects of GCs
chronic use can be limited by topical pulmonary delivery via inha-
lation [2]. Nevertheless, a degree of systemic exposure inevitably
occurs which may raise safety concerns in elderly patients as well
as in patients requiring high dose regimen [3]. Hence, there is the
need to enhance local anti-inflammatory potency of topical GCs
while limiting their systemic exposure in order to minimize
unwanted side effects.
topical application and limited systemic exposure. Despite these
efforts, only few novel steroidal molecules showing significant
structural changes with respect to existing drugs have been devel-
oped [4,5]. The present study attempts to fill this gap by describing
the design, synthesis and pharmacological profile of a novel series
of 6a,9a-di-Fluoro 3-substituted isoxazolines. Cpd #15, in particu-
lar, proved to be a suitable compound for pulmonary topical
administration given its robust anti-inflammatory potency, pro-
longed lung retention and low systemic exposure upon intratra-
cheal administration.
2
. Experimental section
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2.1. Chemicals and reagents
Abbreviations: GCs, glucocorticoids; NO, nitric oxide; GILZ, glucocorticoid-
induced leucine zipper; ASMCs, airway smooth muscle cells.
⇑
Corresponding author. Tel.: +39 0521 279913; fax: +39 05212762 545567.
E-mail address: e.ghidini@chiesi.com (E. Ghidini).
All commercially available chemicals and solvents were pur-
chased from Aldrich-Sigma (St. Louis, MO). Steroidal derivatives
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http://dx.doi.org/10.1016/j.steroids.2014.12.016
039-128X/Ó 2015 Published by Elsevier Inc.
0
Please cite this article in press as: Ghidini E et al. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory
profile and suitable for topical pulmonary administration. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2014.12.016

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9
(compounds #1–18) were synthesized in our laboratory following
the route described in Scheme 1. Starting from commercially avail-
able derivative #21 and for #22, the reaction proceeded in ethyl
dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxazole-3,20-dione,
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0
0
Cpd #14; (16S,17R)-3 -bromo-6,9-difluoro-11b,21-dihydroxy-4 H-
pregna-1,4-dieno[16,17-d]isoxazole-3,20-dione, Cpd #15; (16S,
17R)-3 -methyl-6,9-difluoro-11b,21-dihydroxy-4 H-pregna-1,4-
dieno[16,17-d]isoxazole-3,20-dione, Cpd #16; (16S,17R)-3 -(4-
methoxyphenyl)-6,9-difluoro-11b,21-dihydroxy-4 H-pregna-1,
4-dieno[16,17-d]isoxazole-3,20-dione, Cpd #17; (16S,17R)-3 -phe-
nyl-6,9-difluoro-11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]
isoxazole-3,20-dione, Cpd #18.
Preparation of Cpd #2, Cpd #3 and Cpd #9 was already
described in literature. [7]
The purity of tested compounds determined by analytical UPLC
was >98%. The standards Dexamethasone (Chart 1) is commercially
available and was purchased from Acros Organics while Deflaza-
cort active metabolite (Chart 1) was synthesized following a liter-
ature method [8].
0
0
acetate and NaHCO , together with a few drops of water, by stirring
3
0
at room temperature for six days (Scheme 1; A). When derivatives
0
Cpd #21 were prepared by in situ chlorination of the corresponding
aldoximes with BTMAICl4 (benzyltrimethylammonium tetrachlo-
roiodate) [6] or bleach, the reaction proceeded in dry dichloro-
methane (DCM) and triethylamine (TEA) at room temperature for
3 h (Scheme 1; B). All reactions details are reported in the Support-
ing Information. The structures of these compounds are shown in
Table 1 and the steroidal drugs are:
0
0
0
0
(16S,17R)-3 -(4-chlorophenyl)-11b,21-dihydroxy-4 H-pregna-
0
1,4-dieno[16,17-d]isoxazole-3,20-dione, Cpd #1; (16S,17R)-3 -(4-
methoxyphenyl)-11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,
17-d]isoxazole-3,20-dione, Cpd #4; (16S,17R)-3 -methylacetate-
11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxazole-3,20-
dione, Cpd #5; (16S,17R)-3 -propyl-11b,21-dihydroxy-4 H-pregna-
1,4-dieno[16,17-d]isoxazole-3,20-dione, Cpd #6; (16S,17R)-3 -
methyl-11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxaz-
ole-3,20-dione, Cpd #7; (16S,17R)-3 -(hydroxymethyl)-11b,
21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxazole-3,20-
dione, Cpd #8; (16S,17R)-3 -hydroxy-11b,21-dihydroxy-4 H-
pregna-1,4-dieno[16,17-d]isoxazole-3,20-dione, Cpd #10; (16S,
17R)-3 -(thiophen-3-yl)-11b,21-dihydroxy-4 H-pregna-1,4-dieno
[16,17-d]isoxazole-3,20-dione, Cpd #11; (16S,17R)-3 -(furan-3-
yl)-11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxazole-3,
20-dione, Cpd #12; (16S,17R)-3 -(thiophen-3-yl)-6,9-difluoro-
11b,21-dihydroxy-4 H-pregna-1,4-dieno[16,17-d]isoxazole-3,
20-dione, Cpd #13; (16S,17R)-3 -(furan-3-yl)-6,9-difluoro-11b,21-
0
0
0
0
0
2
2
.2. Biological assay
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.2.1. Cell culture
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Murine macrophagic cell line (RAW264.7) was purchased from
ATTC (Manassas, USA) and cultured in RPMI 1640 medium (w/o
Phenol Red) supplemented with 10% FBS, 2 mM glutamine, 100 U
penicillin and 100
sphere of 5% CO at 37 °C.
PathHunter™ CHO-K1 GR and MR Cell Line stably expressing
EA-NLS-NRS and the ProLabel-tagged glucocorticoid and mineral-
corticoid receptor respectively were purchased from DiscoverX
CA, United States). Cells were cultured in F-12 Nutrient Mixture
HAM) supplemented with 10% Fetal Bovine Serum (Invitrogen)
plus 2 mM
100 g/ml Streptomycin, 300 g/ml Hygromycin B, and 500 g/ml
G418/Geneticin) in an atmosphere of 5% CO at 37 °C.
Primary human airway smooth muscle cells (ASMCs) were pur-
chased from LONZA (Basel, CH) and cultured in DMEM medium
supplemented with 10% Fetal Bovine Serum, 2 mM glutamine,
0
1
00
0
0
101
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105
106
lg/ml streptomycin (Invitrogen), in an atmo-
0
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2
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1
07
08
(
(
0
1
L-glutamine and antibiotics (100 U/ml Penicillin,
Table 1
Compounds series.
2
Compound
R
X, Y
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
1
2
3
4
5
6
7
8
p-Cl-Phenyl,
COOEt
COOH
p-OMe-Phenyl
2 3
CH OCOCH
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
F, F
100 U penicillin and 100
atmosphere of 5% CO at 37 °C.
lg/ml streptomycin (Invitrogen), in an
2
Propyl
Methyl
2
.2.2. Nitric measurement assay protocol
RAW264.7 cells were seeded in 0.3 ml RPMI (w/o Phenol Red)
containing 10% FBS in 48-well tissue culture plates at the density
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CH OH
2
9
Br
OH
10
11
12
13
14
15
16
17
18
4
of 7.5 ꢀ 10 cells/well and grown for 24 h at 37 °C with 5% CO
2
.
3-Thienyl
3-Furyl
3-Thienyl
3-Furyl
Br
Methyl
p-OMe-Phenyl
Phenyl
Then cells were treated with different concentration of corticoste-
1
1
6
roids (10- M-10- M, final DMSO concentration 0.1%) for 15 min.
before stimulation with lipopolysaccharide from Escherichia coli
100 ng/ml as final concentration) and incubated for 18 h in RPMI
w/o Phenol Red) supplemented with 10% FBS.
Accumulation of nitrite in the medium was measured by a col-
F, F
F, F
F, F
F, F
(
(
F, F
orimetric assay method based on the Griess reaction. Briefly,
Scheme 1. Compounds 1–18 were synthesized starting from enone, #19 or #20 and hydroximoyl chlorides derivatives #21 or hydroxycarbonimidic dibromide #22 via
,3-dipolar cycloaddition of nitrile oxides.
1
Please cite this article in press as: Ghidini E et al. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory
profile and suitable for topical pulmonary administration. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2014.12.016

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Chart 1.
1
1
1
1
1
1
1
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samples were reacted with 1% sulfanilamide, 0.1% naphthyl
ethylenediamine dihydrochloride, and 2.5% phosphoric acid at
room temperature for 10 min, and nitrite concentration was deter-
mined by absorbance at 540 nm in comparison with sodium nitrite
as a standard. Compound potencies were expresses as concentra-
tion able to inhibit the half maximal (50%) NO release [IC50] in
the dose–response curve obtained after stimulation with LPS.
VIC for the endogenous reference b-actin gene (ACTB) and 6-car-
boxyfluorescein (FAM) for GILZ or TAT genes.
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The two sets of primers-probes were the follows: set 1, ACTB-
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FW (forward) 5 -GGCGGCACCACCATGTAC-3 , ACTB-RE (reverse)
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5 -CAGGGCAGTGATCTCCTTCTG-3 ACTB probe5 -VIC-TGGCA TTG
CCGACAGG-3 ; set 2 GILZ-FW (forward) 5 -TGGCCATAGACAACAAG
ATCGA-3 , GILZ-RE (reverse) 5 -TCACAGCATACATCAGATGATTC
TTC-3 , GILZ probe 5 -FAM-AGGCCATGGATCTGG -3 .
0
0
0
0
0
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2.2.3. Glucocorticoid receptor (GR) and mineralcorticoid receptor (MR)
translocation assay protocol
The cell-based GR-translocation assay in Enzyme Fragment
Complementation format developed by DiscoveRx (Fremont, CA)
was employed to quantitatively measure GR nuclear translocation
[9]. PathHunter CHO-K1 GR and PathHunter CHO-K1 MR cells were
The selected primers and probes were subjected to Basic Local
Alignment Search Tool (BLAST) database searches to exclude any
sequence similarities. Real-time quantitative PCR was performed
using StepOnePlus™ Real-Time PCR System (Applied Biosystems).
All samples were run in triplicate in a final volume of 25
taining 12.5
L of 2ꢀ TaqMan Gene Expression PCR Master Mix,
300 nM of each primer, 250 nM of each probe and 4 L of RT reac-
lL con-
l
seeded in a 96-well plate at 15,000 cells/well in 100
lL medium
l
without antibiotics and 24hours later the compounds were added
tion, according to the manufacturer’s instructions (Applied Biosys-
tems). Amplification conditions for GILZ/b-actin were: 50 °C for
2 min and 95 °C for 10 min, followed by 50 cycles of 95 °C for
30 s and 60 °C for 1 min.
Relative expression of GILZ mRNA was calculated using the
2-(CT) comparative method, with normalization against the inter-
nal endogenous reference b-actin gene.
ꢁ
12
ꢁ6
(concentration ranging from 10 M to 10 M) for 3 h at 37 °C.
Luminescence, estimated as relative light units (RLU), was detected
by using a CENTRO LB 960 microplate reader (Berthold Technolo-
gies). Statistical analysis and determinations of EC50s were per-
formed by using Prism-version 3.0 Graphpad Software (San
Diego, CA).
1
1
1
1
1
1
1
1
1
1
1
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2.2.4. IL-8 release assay protocol
ASMCs were seeded in 0.5 ml DMEM containing 10% FBS in 48-
well tissue culture plates at the density of 10 cells/well and grown
2.2.6. Competitive binding assay
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Competitive binding assays to evaluate the affinity of the
compounds to human glucocorticoid receptor and human mineral-
corticoid receptor were performed in triplicate in a total volume of
4
for 24 h at 37 °C with 5% CO . Subsequently, cells were serum
2
3
3
starved for 18 h and treated with different concentration of corti-
160
lL, containing 5nM [ H]Dexamethasone or 4.5 nM [ H]
costeroids (final DMSO concentration 0.1%) for 60 min before stim-
ulation with TNF (0.1 ng/ml). After 18 h incubation in DMEM
serum free, the IL8 release in the supernatant was assayed using
ELISA kit (Invitrogen). Compound potencies were expressed as
concentration able to elicit half maximal inhibition of IL8 release
[IC50].
D-Aldosterone (Amersharm Pharmacia Biotech) and various concen-
trations (0–1000 nM) of test compounds as previously described
[10]. After 24 h incubation at 4 °C, unbound [ H]Dexamethasone
was removed by the treatment with DCC in TEDM buffer on ice. A
3
150 lL sample was pipetted into scintillation vial and 5 ml scintilla-
tion cocktail were added. By using, similar assay conditions were
adopted for determining aldosterone binding. Non-specific binding
was determined in the presence of 1000-fold excess of unlabelled
Dexamethasone or Aldosterone respectively. The radioactivity was
measured with scintillation counter (Beckman Instruments, Fuller-
ton, CA). IC50 values (concentrations at which 50% of specific binding
is displaced by the compounds) were determined from the best fit
lines derived by least square regression lines of competitive dis-
placement graph. The Ki values were calculated using the equation
of Cheng and Prusoff.
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2.2.5. Real time PCR analysis of GILZ gene expression
ASMCs were seeded in 0.1 ml DMEM containing 10% FBS in
96-well tissue culture plates at the density of 4x10 cells/well
3
and grown for 24 h at 37 °C with 5% CO . Then cells were starved
2
over-night in DMEM without FBS and then treated with different
concentrations of compounds (10-12M-10- M, final DMSO concen-
7
tration 0.1%) for 4 h before mRNA extraction.
Total RNA was isolated using TaqMan Gene expression Cells-to-
Ct kit (Applied Biosystems, Foster City, CA). Briefly, cells were lysed
in Cell Lysis solution containing DNAse I for 5 min, followed by
two-minute incubation with the stop solution. Reverse transcrip-
2.2.7. Animals
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Male Brown Norway rats (150–200 g) and male Sprague Dawley
rats (250 g) were purchased from Charles River Laboratories Italy
(Calco, Lecco). Prior to use animals were acclimated for at least
5 days to the local vivarium conditions (room temperature:
20–24 °C; relative humidity: 40–70%), having free access to stan-
dard rat chow and tap water. All the procedures were performed
tion reactions were performed using 10 lL of each cell lysate,
according to the manufacturer’s instructions. Two sets of prim-
ers-probes were designed for the human and rat cell lines using
the Primer Express Software version 3.0 (Applied Biosystems).
The chosen reporter fluorophores for TaqMan MGB probes were
Please cite this article in press as: Ghidini E et al. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory
profile and suitable for topical pulmonary administration. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2014.12.016

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in animal operating rooms in conformity to the Italian legislation
(D.L. vo 116/92) and the UE directive 2010/63/UE.
2.3. Modelling studies
316
The crystal structure of Dexamethasone in complex with
human GR was retrieved from Brookhaven Protein Databank
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2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
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2
2
2
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2.2.8. Intratracheal administration and pharmacokinetic
Compound #15 was intratracheally administered to male Spra-
gue Dawley rats.
(
PDB code: 1M2Z) [11] and utilized for binding site analysis using
SiteMap (Schrodinger9.1) and for docking studies using GlideSP
(
Schrodinger9.1) [12].
Before intratracheal administration of 1 lmol/kg of #15 as sus-
Finally, logP and pKa values were calculated with the use of
ACD/Labs (v12) [13].
pension at volume of 0.5 ml/kg (0.2% Tween 80 in 0.9% sodium
chloride solution as vehicle) animals were anesthetized with iso-
flurane or sodium thiopental, in case of terminal sampling. Rats
fasted from 1 h before the treatment. After i.t. treatment blood
and lung were collected as follows: 0.083, 0.5, 1, 2, 4, 6, 24 and
48 h. Blood were put in heparinised plastic tubes, kept in an ice-
water bath and then centrifuged within 0.5 h, for 3 min at
10,000ꢀg at 4 °C for plasma separation. Lungs were collected,
washed with 10 ml of 0.9% sodium chloride solution, then frozen
over liquid nitrogen. Plasma and lungs were stored at ꢁ80 °C until
3. Results
324
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3.1. Chemistry
A series of isoxazolines, Cpds #1–18 described in Table 1, was
prepared following the synthetic pathway summarized in
Scheme 1.
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331
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333
334
335
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337
338
339
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341
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343
the bioanalysis. 120
lL of plasma samples were purified adding
Intermediates
11b,21-dihydroxy-pregna-1,4,16-triene-3,20-
480 L of acetonitrile and vortexed for 30 s., then the obtained
l
dione (intermediate #19) and 6,9-difluoro-11b,21-dihydroxy-
pregna-1,4,16-triene-3,20-dione (intermediate #20) were pre-
pared respectively in four and two steps starting from commer-
cially available Prednisone 21-O-acetate and Difluprednate by a
method established in this laboratory [14]. For example, 1,3-dipo-
lar cycloaddition of 4-chlorophenylformonitrile oxide (generated
in situ by the treatment of 4-chloro-N-hydroxybenzimidoyl chlo-
samples were stored at ꢁ20 °C for 0.5 h and centrifuged at
3600ꢀg for 0.25 h. The supernatant was later evaporated under
N stream. The samples were reconstituted with 200 lL of 1% ace-
2
tic acid in acetonitrile and 1% acetic acid in water mixture (50:50,
v:v), vortexed for 30 s and injected into the LC/MS-MS system.
70
ine solution and acetonitrile mixture (50:50, v:v) to 1 g of rat lung,
were purified with 280 L of formic acid solution (0.2% in acetoni-
trile) and vortexed for 30 s. then the obtained samples were stored
at -20 °C for 0.5 h and centrifuged at 3600ꢀg for 0.25 h. 50 L of an
aqueous solution of formic acid solution (0.3%) were added to
150 L of the supernatant and then vortexed for 30 s. The obtained
samples (5 L for lung and 20 L for plasma) were injected into the
lL of homogenated lung samples, obtained adding 3 ml of sal-
ride with aqueous NaHCO
enone #19 gave a single adduct #1 in a good yield. Only the
16,17-double bond reacted with the various nitrile oxides and 1
NMR spectra are consistent with the proposed structures. The reg-
iospecificity of 1,3-dipolar cycloaddition of nitrile oxides to an
3
solution) [15] to an a,b-unsaturated
l
H
l
a,
l
b-unsaturated enone and the stereospecificity of the cycloaddition
to 16-ene steroid system are known [16,17].
l
l
LC/MS-MS system, Thermo Electron TSQ Quantum Access, as spec-
trometer and Accela HPLC system. The chromatographic analysis
was performed in gradient mode with Formic acid in water
(0.2%) (Solvent A) and Formic acid in acetonitrile (0.2%) (Solvent
3.2. In vitro and in vivo evaluation of novel isoxazolines derivatives
344
3.2.1. GR binding affinity
345
346
347
348
349
350
351
B), using Accucore (Thermo Fisher) C18 (50 ꢀ 2.1 mM) 2.6
umn equipped with a Thermo Fisher in-line filter Javelin (88200)
m,
lm col-
The affinity for the human GR receptor of this novel series of
isoxazolines was measured in a binding assay and compared with
Dexamethasone and Deflazacort as reference ligands. As reported
in Table 2, the great majority of the compounds exhibited Ki values
between 0.5 and 250 nM. In particular, the bromine derivative Cpd
#15 showed the lowest Ki value (0.5 nM).
and Accucore C18 defender guard cartridge 10 ꢀ 2.1 mM, 2.6
l
operating at 40 °C. The spectrometer was used in ESI positive ion
mode, monitoring the following transitions: 502 ? 462 (#15)
and 501 ? 293 (Fluticasone propionate, as IS).
The measured plasma concentrations were in the linear range:
0.48–533 ng/ml.
The measure lung concentrations properly adjusted to the cor-
responding weighs were in the linear range: 12.4–169680 ng/g.
3.2.2. GR nuclear translocation
352
353
354
355
356
357
358
359
360
The potency and efficacy of the isoxazoline derivatives in induc-
ing nuclear translocation were evaluated in an enzyme fragment
complementation format by using CHO-K1 PathHunters™ cells
[
8
18]. As reported in Table 2, EC50s values lie between 9 and
84 nM. In particular, the bromine derivative Cpd #15 stands out
as the most potent derivative in this assay with an EC50 value of
nM, while Dexamethasone showed an EC50 value of 25 nM
Table 2 and Fig. 1).
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
2.2.9. Ovalbumin-induced pulmonary eosinophilia in sensitised Brown
Norway rats
Male Brown-Norway rats were sensitized by intraperitoneal
injection of 1 ml suspension containing ovalbumin (OVA, 1 mg/
ml, Sigma Aldrich) and Al(OH)3 (100 mg/ml, Sigma Aldrich) for 3
consecutive days. Two weeks later the animals were nose-only
exposed to an aerosol of OVA solution (1% in saline). The non chal-
lenged-vehicle-control treated animals were sensitised to OVA but
exposed to aerosolized saline (sham). At 24 h after exposure either
to OVA or saline aerosol, animals were anaesthetised with sevoflu-
rane (4% in oxygen, Sevoflo, Abbott) and bronchoalveolar lavage
fluid (BALF) was collected and subjected to total and differential
cell counts. Cpd #15 and Dexamethasone were administered as
suspension (vehicle: saline containing 0.2% Tween 80) by the intra-
tracheal route 2 h before and 4 h after OVA aerosol.
9
(
3.2.3. Effect of varying concentrations of isoxazolines compounds on
the LPS-evoked NO release in RAW 264.7
361
362
363
364
365
366
367
368
369
370
371
372
RAW 264.7 is a murine cell line of immortalized peritoneal
macrophages which has been widely used for studying inflamma-
tory responses in macrophages. In particular, LPS-evoked nitric
oxide (NO) release is an index of inflammatory activation in these
cells.
RAW264.7 were stimulated for 24 h with LPS (100 ng/ml) after
1 h pre-incubation with different concentrations of compounds
tested (0.01–1000 nM).
Data were analysed using Graph Pad Prism™.The parametric
tests performed to determine statistical significance were an anal-
ysis of variance (ANOVA) with Dunnett’s post-test.
Among the isoxazolines tested, Cpd #6, Cpd #9, Cpd #14
and Cpd #15 showed to be more potent or equipotent to
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Table 2
In vitro data.
Cpd#15 IC50=6nM (1.0-37.4 nM)
DEX IC 50=115nM (82.6-159.6 nM)
100
Ki [nM]a
RAW NOb
Compound
GR
% Efficacy vs.
traslocation
EC50 [nM]
dexamethasone release IC50
(nM)
c
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
1
2
3
4
5
6
7
8
107.3 161.7
16
32
21
–
50
c
84.2 884.8
1630.8 327
c
c
c
249.5
c
85.2 236.2
66.6 212.4
9.4 192.3
55.3 272
22
41
52
25
67
135
c
0
c
-11
-10
-9
-8
-7
-6
-5
-4
9
2.5
>10000
22.6
69.4
c
c
c
c
c
10
11
12
13
14
15
16
17
18
–
Log[M]
31.7 126.3
59.7 243.9
29
28
80
66
91
76
40
42
Fig. 2. Inhibitory effect of increasing concentrations of corticosteroids isoxazolines
on the accumulation of nitrite induced by LPS. Concentration-dependent inhibition
by Dexamethasone (DEX) and Cpd #15 of LPS-evoked NO release in Raw 264.7 cells.
Each data point is the mean ± SD of a representative experiment performed in
triplicate.
2.4
3.2
0.5
68.6
56.4
9.0
53.7
6.1
1.5 129.4
–
–
–
3.0
2.5
48.8
22.9
d
Deflazacort
Dexamethasone
10.8
2.3
25
100
115
Table 3
Mineralocorticoid receptor nuclear translocation.
a
3
Concentration displacing 50% of GR bound [ H]Dexamethasone values repre-
sent the mean of at least two experiments. The standard deviation of the mean of
Compound
EC50 [nM]
% Efficacy vs. aldosterone
GR Ki is 618%.
#
#
#
#
9
37.6
76.5
66.6
6.1
20
30
19
50
50
b
Order of magnitude: ꢁ9.
13
14
15
c
No inhibition up to 1 lM.
d
The des-acetyl-derivative.
Dexamethasone
9.2
Cpd#15 EC50=9nM (7.3-11.2 nM)
DEX EC50=25nM (9.8-62.8 nM)
2.0×
1.5×
1.0×
5.0×
10 5
10 5
10 5
10 4
0
Cpd #15 EC50=6.1nM (5.1-7.4 nM)
DEX EC50=9.2nM (6.3-13.4 nM)
1.5×
1.0×
5.0×
10 6
10 6
10 5
0
Aldosterone EC50=0.41nM (0.26-0.65 nM)
-13
-11
-9
-7
-5
-13
-11
-9
-7
-5
Log[M]
Log[M]
Fig. 3. Steroid-evoked stimulation of MR nuclear translocation. Concentration–
response curves in CHO-K1 PathHunter™ cells incubated with Dexamethasone
(DEX), Cpd #15 and Aldosterone for 3hrs. Data are mean ± SD of a representative
experiment performed in triplicate.
Fig. 1. Steroid-evoked stimulation of GR nuclear translocation. Concentration–
response curves in CHO-K1 PathHunter™ cells incubated with Dexamethasone
DEX) or Cpd #15 for 3 h. Data shown are mean ± SD of a representative experiment
(
performed in triplicate.
run with cell culture medium containing charcoal stripped fetal
bovine serum to delete cortisol, that could be present in cell cul-
ture medium. Indeed, results comparable to the non-stripped
serum condition were obtained: EC50 = 7.2 nM (c.i. 6.8–7.6) for
Cpd #15 and EC50 = 7.5 nM (c.i. 7.2–7.7) for dexamethasone. Cpd
388
389
390
391
392
393
394
3
3
3
73
74
75
dexamethasone in inhibiting LPS-induced NO release from
RAW264.7, whereas the other compounds tested exhibited poor
inhibitory activity against NO release (IC5 > 1
0
lM) (Table 2, Fig.2).
#9, #13, #14 tested in the MR translocation assay appeared to be
3
3
3
3
3
3
3
3
3
3
3
3
76
77
78
79
80
81
82
83
84
85
86
87
3.2.4. MR nuclear translocation
4
–7-fold less potent than Cpd #15.
The human mineralocorticoid receptor (MR) is highly homolo-
gous to GR [19,20]. In order to determine receptor selectivity,
Cpd #15 was tested in a competitive binding assay and in a MR
nuclear translocation functional assay head to head with aldoste-
rone and dexamethasone.
Cpd #15 and dexamethasone showed a Ki for the mineralcorti-
coid receptor of 45.0 nM (c.i. 28–74) and 12.5 nM (c.i. 5.3–30.6)
respectively.
3.2.5. Effect of varying concentrations of isoxazoline compound Cpd
#15 on the TNF -evoked IL-8 release in ASMCs
395
396
397
398
399
400
401
402
403
a
In the airways smooth muscle cells (ASMCs) the secretion of IL-
8 induced by inflammatory mediators is repressed by glucocorti-
coids [21] through a direct inhibitory interaction of the GR with
activated transcription factors, such as NF-kB and AP-1 [22].
The anti-inflammatory effects of novel isoxazoline Cpd #15 was
As reported in Table 3, Cpd #15 showed potency and efficacy in
eliciting MR translocation greatly inferior to aldosterone and sim-
ilar to Dexamethasone (Fig. 3). The same experimental set up was
assessed by measuring its potency in inhibiting TNFa-induced IL-8
release in ASMCs. As shown in Fig. 4, Cpd #15 inhibited IL-8 release
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1
00000
Cpd#15 IC50=0.84nM (0.22-3.2 nM)
DEX IC 50=1.7nM (0.03-87.3 nM)
1
0000
7
5
5
lung
1
000
2
1
00
10
1
-
12 -11 -10 -9
-8
-7
-6
-5
-
-
25
plasma
Log[M]
0
6
12
Time (h)
18
24
75
Fig. 4. Inhibitory effect of increasing concentrations of corticosteroids isoxazolines
on IL-8 release induced by TNF . Concentration-dependent inhibition by Dexa-
methasone (DEX) and Cpd #15 of TNF (0.1 ng/ml)–evoked IL-8 release in ASMCs.
Fig. 6. Plasma (squares) and lung (triangles) levels after i.t. administration of Cpd
a
#15 (1 lmol/kg) to rats.
a
Data are mean ± SD of a representative experiment performed in triplicate.
3
.2.7. In vitro ADME and PK profile
418
419
420
421
422
423
424
425
426
427
cpd#15 EC50=0.17nM (0.06-0.6)
Cpd #15 showed a medium rate of clearance in rat hepatocytes
6
(
25.8
l
L/min 10 cells) and was stable in rat plasma (up to 5 h) as
7.5
5.0
2.5
0.0
DEX EC50=0.26nM (0.15-0.46)
well as in S9 lung homogenates (45% after 1 h).
Following intra-tracheal (i.t.) administration in rat, Cpd #15
(
1
lmol/kg) showed a Cmax value of 0.448 nmol/ml in plasma and
⁄
the exposure achieved was 0.232 nmol/ml h. In the lung, Cpd
#
5
15 levels peaked at 0.083 h reaching
5.37 nmol/g and its exposure (AUClast) was 147.78 nmol/g h, with
a
concentration of
⁄
a MRTlast of 5.35 h (Table 4). (See Fig. 6)
3
.2.8. Effect of #15 on OVA-induced eosinophilia in sensitised Brown
Norway rats
The effects of Cpd #15 and the reference compound Dexameth-
428
429
430
431
432
433
434
435
436
437
438
439
440
-
13 -12 -11 -10
-9
-8
-7
-6
asone in a rat model of asthma (OVA-induced pulmonary eosino-
philia) were investigated. Animals were dosed i.t. with Cpd #15
log[M]
(
0.1 ꢁ 1
lmol/kg) or Dexamethasone (1 lmol/kg) 2 h prior to and
Fig. 5. Dose–response curves for reference compounds Dexamethasone (DEX) and
isoxazoline Cpd #15 on mRNA GILZ induction in airway smooth muscle cells.
Relative GILZ mRNA levels were measured by Taqman real-time PCR and normal-
ized to b actin. Data are mean ± SD of a representative experiment performed in
triplicate.
4 h post OVA challenge.
Cpd #15 inhibited the OVA-induced increase in eosinophil
number in the BAL in a dose-dependent fashion (Fig. 7). The inhib-
itory responses ranged from 43% (0.1
mol/kg) and were statistically significant (p < 0.05 ꢁ p < 0.01)
at all tested doses. The inhibitory effect of 1 mol/kg Dexametha-
sone was superimposable to that of 1 mol/kg of Cpd #15.
lmol/kg) to 95% (1 and
3
l
l
4
4
04
05
with a sub-nanomolar potency (EC50 = 0.84 nM) comparable to
that of Dexamethasone (EC50 = 1.7 nM).
l
3.3. Modelling studies
441
406
407
408
409
410
411
412
413
414
415
416
417
3.2.6. Cpd #15 induces transactivation of the anti-inflammatory gene
GILZ in human airway smooth muscle cells
Part of the anti-inflammatory properties of glucocorticoids
depend also on their ability to trans-activate genes able to counter-
act inflammatory processes such as GC-induced leucine zipper
(GILZ) [23].
Following incubation of ASMCs with increasing concentration of
Cpd #15 or Dexamethasone, relative GILZ mRNA amounts were
measured by TaqMan real-time PCR and normalized with respect
to b-actin. As shown in Fig. 5, Cpd #15 displayed a potency
(EC50 = 0.17 nM) and efficacy in inducing GILZ mRNA (about 6-fold
induction over basal) comparable to those of Dexamethasone.
Overlay of Cpd #15 (GlideSP docked conformation) on Dexa-
442
443
444
445
446
447
448
449
450
451
452
methasone bound to the ligand binding domain of human GR is
shown in Fig. 8. The contour map highlights that the steroid bind-
ing site is mainly hydrophobic (yellow grids). An extended polar
region is located near R611 and N570 both involved in hydrogen
bond interactions with the 3 ꢁ C = O of Dexamethasone. Besides,
a small polar area (red and blue grids) is close to N564 which is
involved in a hydrogen bond interaction with 11-OH of the ligand.
Finally, the hydroxyl moiety of Dexamethasone at C17 is nicely
accommodated in a polar pocket and makes a hydrogen bond
interaction with Q642.
Table 4
PK data of Cpd #15.
⁄
⁄
C
max (nmol/ml or nmol/g)
0.448
AUClast (nmol/ml h or nmol/g h)
T
1/2 (h)
T
last (h)
MRTlast (h)
Plasma (i.t.)
Lung (i.t.)
0.232
147.78
1.59
6.47
6
1.09
5.35
55.37
24
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7
ring with X, Y equal to hydrogen showed very weak potency and
efficacy in the Raw assay and was not further profiled (unpub-
lished results). Cpd #1, bearing a p-chlorophenyl substituent at
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
0
C3 on the isoxazoline ring at the C16–17 position, displays only
moderate binding affinity (Ki = 262.96 nM). Substitution of the p-
0
chlorine moiety with p-methoxy group on the phenyl ring at C3 ,
as in Cpd #4 (Ki = 249.54 nM) slightly improves the affinity in
the binding assay. Replacement of the phenyl ring with either a
furyl, Cpd #12, or a thienyl, Cpd #11, did not improve the affinity
at GR (Ki = 59.66 and 31.73 respectively).
The relatively weak GR binding affinity of the compounds with
X, Y = Hydrogen was improved with the incorporation of two fluo-
rine atoms at C6, C9 on the corticosteroid core. These isoxazoline
derivatives showed a 10-fold increase in their binding affinity
while their potency did not change significantly.
The increase of the size and bulk of the para-substituent of phe-
nyl derivative led to a reduction in both potency and efficacy in the
GR nuclear translocation, e.g., Cpd #18.
These results were rationalized in light of the binding modes of
this compound series by using docking methods. As shown in
Fig. 8, the isoxazolidine ring of Cpd #15 maps well the same polar
region filled by the hydroxyl moiety at C20 of Dexamethasone,
while the bromine substituent is nicely accommodated in a small
hydrophobic pocket. The limited size of this cavity highlighted by
the yellow grid map suggests that bulkier groups like phenyl
Fig. 7. Compound #15 dose–response curve in the rat model of OVA-induced
eosinophilia. Test compounds were administered i.t. to sensitized Brown Norway
rats 2 h before and 4 h after OVA challenge. Each column represents the mean of
total eosinophil number in BAL and each bar represents SEM. Changes were
compared to the OVA challenged group (*) or to the sham challenged group ($)
⁄
⁄⁄
$$
using ANOVA followed by Dunnett’s test. p < 0.05 and p < 0.01, p < 0.01, n = 6–7
animals/group.
(Cpd #18), thienyl (Cpd #11) and furyl (Cpd #14) are not tolerated
due to their large steric hindrance which can be described with the
use of calculated Molecular Refractivity (MR) parameter [24]. Map-
ping of this pocket appears to be suitable for small and hydropho-
bic substituents such as Br (Cpd #9 and Cpd #15) and Me (Cpd #7).
Both are characterized by low MR values (Br: MR = 8.88; Me:
MR = 5.65) and medium lipophilicity (Br: cLogP = 1.73; Me:
cLogP = 2.53) [14]. When either steric bulk or polarity increases,
binding affinity significantly drops as observed for Cpd #6 (R = pro-
2
pyl: MR = 14.96, cLogP = 3.55), Cpd #8 (R = CH OH: MR = 7.19,
cLogP = 1.38) and Cpd #2 (R = COOEt: MR = 17.47, cLogP = 1.45)
and is completely lost for Cpd #3 which is likely charged at phys-
iological pH (calculated pKa = 2.74).
4.2. Biological profile Cpd #15
510
Fig. 8. Overlay of
a docking solution of Cpd #15 on the X-ray structure of
Dexamethasone (orange) bound to the ligand binding domain of human GR (1M2Z
PDB code) [11] (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.).
The most interesting compound among the novel isoxazoline
derivatives, described in this study, is the bromine derivative Cpd
15, which proved to be the most potent compound in the GR
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
#
binding assay and in the cell-based GR nuclear translocation assay.
Cpd #15 showed Ki values pointing out a 50-fold higher affinity for
GR vs. MR in the binding assay. The Ki ratio between MR and GR for
dexamethasone is about 5, suggesting that Cpd #15 is relatively
more selective than dexamethasone. The observation that Cpd
#15 interaction with MR is also associated with nuclear transloca-
tion, suggests that such compound acts as an agonist at the MR. In
order to evaluate its anti-inflammatory potency, we examined the
effects on the release of relevant inflammatory mediators. Cpd #15
dose-dependently inhibited the production of nitric oxide in LPS-
stimulated Raw 264.7 macrophages, with a potency superior to
the reference compound Dexamethasone. Inhibition of nitric oxide
release in Raw 264.7 macrophages occur with relatively high EC50
with respect to Ki values due likely to the mechanism of suppres-
sion that requires transcriptional and post-transcriptional down
regulation of the enzyme nitric oxide synthase [25]. It should also
be kept in mind that Raw 264.7 are immortalized cell lines which
are known to express relatively high levels of P-glycoproteins [26].
Upregulation of P-glycoprotein may cause drug resistance as well
as reduced glucocorticosteroid responsiveness [27]. On the other
hand, the repression of inflammatory responses by glucocorticoids
also involves the up-regulation of anti-inflammatory genes,
4
4
53
54
4. Discussion
4.1. SAR interpretation
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
Cpd #7 (Ki = 22.33 nM)) shows a comparable binding affinity to
the active metabolite of Deflazacort (see Chart 1, Ki = 10.84 nM).
0
Small alkyl substituents are preferred at the C3 position for the
0
binding affinity. In particular, C3 -methyl-isoxazoline derivative
0
shows better binding affinity than C3 -n-propyl-isoxazoline deriv-
ative Cpd #6 (Ki = 66.59 nM). When the methyl group is replaced
with a hydroxyl-methyl moiety, as in Cpd #8, the affinity is
reduced (Ki = 55.29 nM), which suggests that polar groups are
not tolerated. This is further confirmed by the lack of activity of
Cpd #10 (Ki = > 10,000). Improved affinity is obtained if the isox-
0
azoline ring is decorated with a bromine at C3 as in Cpd #9
(Ki = 2.46 nM).
The inhibitory effect of compounds with R equal to hydrogen
(see Scheme 1) on NO production in RAW 264.7 macrophage cells,
described in Park et al. [5], is 10-fold lower than Cpd #15.
In order to expand the SAR of new chemical series, aromatic and
heterocyclic moieties were introduced. The unsubstituted phenyl
Please cite this article in press as: Ghidini E et al. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory
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[
[
8] Yuan L; Yanchang L; Jinlu L; Chaohui S CN 101177443 A 20080514.
9] Fung P, Peng K, Kobel P, Dotimas H, Kauffman L. A homogeneous cell-based
assay to measure nuclear translocation using beta-galactosidase enzyme
fragment complementation. Assay Drug Dev Technol 2006;4:263–72.
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including GILZ, whose expression is up-regulated by steroids in
several cellular type including smooth muscle cells [28]. In human
ASMCs, Cpd #15 evoked a significant concentration-dependent
induction of GILZ expression and suppression of TNF
a-stimulated
[10] Wambach G, Casals-Stenzel J. Structure-activity relationship of new steroidal
aldosterone antagonists. Comparison of the affinity for mineralocorticoid
receptors in vitro and the antialdosterone activity in vivo. Biochem Pharmacol
production of IL-8 with a potency and efficacy comparable to dexa-
methasone. In both assays, Cpd #15 and dexamethasone show sub/
low nanomolar EC50 values close to respective Ki values.
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Having demonstrated that Cpd #15 is a potent anti-inflamma-
tory agent in vitro, and that its pharmacokinetic profile is suitable
for topical administration (pulmonary levels > 100-fold higher
than plasma levels upon i.t. delivery), we tested it in an in vivo
model of allergen-induced pulmonary inflammation. Ovalbumin
(OVA) inhalation to sensitized rats is a well characterized and rec-
ognized model for asthma [29]. This model is commonly used to
investigate the anti-inflammatory activity of compounds devel-
oped for the treatment of allergic inflammatory airway diseases,
such as corticosteroids and phosphodiesterase 4 (PDE4) inhibitors
[30–32]. Cpd #15, when administered via the intratracheal route,
proved to be as efficacious as Dexamethasone in counteracting
eosinophilic infiltration in the broncho-alveolar lavage following
[
[
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OVA challenge (ED50 < 0.3 lmol/kg).
[
In conclusion, through a rational drug design approach we iden-
tified Cpd #15, a novel isoxazoline with a potent and robust anti-
inflammatory profile which exhibits a pharmacokinetic and phar-
macodynamic profile suitable for topical pulmonary delivery.
Therefore, Cpd #15 could potentially be developed as an inhala-
ble agent for the treating of pulmonary inflammation such as
asthma and COPD.
[
[
[
[
[
[
5
64
Acknowledgments
5
5
65
66
We kindly thank Mr. Pier Tonino Bolzoni (Chiesi Farmaceutici,
Parma, Italy) for skilful technical support.
[
[
25] Söderberg M, Raffalli-Mathieu F, Lang MA. Regulation of the murine inducible
5
67
Appendix A. Supplementary data
nitric oxide synthase gene by dexamethasone involves
nuclear ribonucleoprotein (hnRNPI) dependent pathway. Mol Immunol
007;44(12):3204–10.
a heterogeneous
I
2
568
569
570
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2014.12.
016.
26] Roy KR, Arunasree KM, Dhoot A, Aparna R, Reddy GV, Vali S. C-Phycocyanin
inhibits 2-acetylaminofluorene-induced expression of MDR1 in mouse
macrophage cells: ROS mediated pathway determined via combination of
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Please cite this article in press as: Ghidini E et al. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory
profile and suitable for topical pulmonary administration. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2014.12.016