Discovery of 7-azaindole derivatives as potent Orai inhibitors showing efficacy in a preclinical model of asthma

Article abstract of DOI:10.1016/j.bmcl.2015.01.063

Synthesis and SAR of a series of 7-azaindoles as Orai channel inhibitors showing good potency inhibiting IL-2 production in Jurkat cells is described. Compound 14d displaying best pharmacokinetic properties was further characterized in a model of allergen induced asthma showing inhibition in the number of eosinophils in BALF. High lipophilicity remains as one of the main challenges for this class of compounds.

Full text of DOI:10.1016/j.bmcl.2015.01.063

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1217–1222
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 7-azaindole derivatives as potent Orai inhibitors
showing efficacy in a preclinical model of asthma
Cristina Esteve, Jacob González, Sílvia Gual, Laura Vidal, Soledad Alzina, Sonia Sentellas, Irene Jover,
⇑
Raquel Horrillo, Jorge De Alba, Montserrat Miralpeix, Gema Tarrasón, Bernat Vidal
Almirall Research Center, Almirall, Ctra. Laureà Miró 408, E-08980 St. Feliu de Llobregat, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and SAR of a series of 7-azaindoles as Orai channel inhibitors showing good potency inhibiting
IL-2 production in Jurkat cells is described. Compound 14d displaying best pharmacokinetic properties
was further characterized in a model of allergen induced asthma showing inhibition in the number of
eosinophils in BALF. High lipophilicity remains as one of the main challenges for this class of compounds.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 10 November 2014
Revised 26 January 2015
Accepted 27 January 2015
Available online 4 February 2015
Keywords:
Orai inhibitors
IL-2 inhibitors
LAD2
CRAC channel
OVA model
The regulation of intracellular calcium is a key element in the
transduction of signals into and within cells. Cellular responses
to growth factors, neurotransmitters, hormones and other signal
molecules are initiated through calcium-dependent processes.
Virtually all cell types depend upon the generation of cytoplasmic
calcium signals to regulate cell function or to trigger specific
responses. Cytosolic calcium signals control a wide array of cellular
functions ranging from short-term responses such as contraction
and secretion to longer-term regulation of cell growth and prolifer-
ation. These signals involve combination of calcium release from
intracellular stores such as the endoplasmic reticulum (ER), and
influx of calcium across the plasma membrane.1
In immune cells the molecular mechanisms of calcium signaling
are especially well characterized. Cell activation through immuno-
receptor engagement induces a rise in cytosolic calcium levels
mainly through a selective channel-based process known as
Store-operated calcium entry (SOCE). Calcium release-activated
calcium (CRAC) channels are a type of SOC channels present in B
and T-lymphocytes, NK cells, macrophages, DC and mast cells
among the immune cells. CRAC channels are characterized by an
extremely high ion selectivity for Ca2+ and a low conductance,
and are activated through a Ca2+ sensor protein in the ER called
stromal interaction molecule (STIM) that bind to Orai, the pore
forming unit of the CRAC channel in the plasma membrane. Upon
antigen binding to the T-cell receptor, the activation of phospholi-
pase C (PLC ) generates the lipid metabolite InsP3, which pro-
c
motes the release of Ca2+ from the ER stores. STIM proteins sense
the calcium depletion in the ER, oligomerize and redistribute into
discrete puncta located in junctional ER sites in close proximity
to the plasma membrane. STIM proteins directly interact with
Orai1 in the plasma membrane opening the CRAC channel and
allowing a sustained calcium entry across the plasma mem-
brane.2–4 Such a prolonged intracellular calcium increase activates
short term processes such as degranulation, and long term pro-
cesses involving gene transcription through NFAT to produce lipid
mediators, several cytokines Th1, Th2, and Th17, matrix metallo-
proteinases, all of which participate in the pathogenesis of autoim-
mune and inflammation-based diseases.
Based on this, Orai1 inhibition is expected to exert broad anti-
inflammatory activity by suppressing T cell activation and mast
cell degranulation, and may have therapeutic potential in all dis-
ease stage of respiratory diseases such as asthma.5,6
We envisaged the exploration of 7-azaindoles as an area of fur-
ther optimization around Orai inhibitors as bicyclic isosteric
replacements of the arylcarboxamide motif. This could provide a
new entry point to explore SAR towards compounds with
increased polarity versus highly lipophilic reference compounds
1 and 2 (measured logD7.4: 4.4 and 3.5, respectively) (Fig. 1).
Our initial SAR was based on exploring substitutions in position
2 of the 7-azaindole core ring keeping the 1-methyl-3-(trifluoro-
methyl)-1H-pyrazol-5-yl group in position 5 as this is a motif
⇑
Corresponding author. Tel.: +34 93 291 3996; fax: +34 93 312 8615.
E-mail address: bernat.vidal@almirall.com (B. Vidal).
http://dx.doi.org/10.1016/j.bmcl.2015.01.063
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

1218
C. Esteve et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1217–1222
CF3
O
F3C
N
Ar'
O
N
O
F
O
Ar
N
H
N
H
N
N
H
N
S
N
N
1
2
7-Azaindoles
YM-58483
Synta compound 66
Figure 1. Orai channel inhibitors.
N
N
F3C
OTf
O
B
N
N
N
4
N
F3C
b
O
F3C
Br
a
N
NH2
N
NH2
N
NH2
R
3
6
5
c
H
7a-g
N
N
N
F3C
N
R
d
2
F3C
R
N
H
N
N
NH2
9a-g
8a-g
Scheme 1. Reagents and conditions: (a) PdCl2dppf.DCM, Cs2CO3, dioxane–water, 110 °C, 35%; (b) NBS, acetonitrile, 0 °C to rt, 2 h, 30%; (c) CuI, (Ph3P)2PdCl2, Et3N, THF, 90 °C,
4 h, 68–97%; (d) for 9a–c: t-BuOK, N-methylpyrrolidinone, rt, 20–67%; for 9d–g; (i) 2,2,2-trifluoroacetic anhydride, dioxane, rt, 1 h, (ii) Et3N, DMF, 120 °C, 20 h, 25–30% (two
steps).
reported to be well tolerated in similar series of Orai inhibitors.7
Thus, a synthesis was devised in order to access molecules contain-
ing different substituents in position 2 of the 7-azaindazole core
(Scheme 1). Suzuki-type coupling reaction of commercially avail-
able boronate 3 with the triflate of 1-methyl-3-(trifluoromethyl)-
1H-pyrazole 4 provided aminopyridine 5. Bromination followed
by Sonogashira type coupling with a diverse set of alkynes 7a–g
provided key intermediates 8a–g that were converted into com-
pounds 9a–g using a base-mediated indolization reaction in
t-BuOK at room temperature or using a sequential trifluoroacetyla-
tion- cyclization sequence.8,9
The synthesis of 7-azaindole derivatives 14a–n is described in
Scheme 2. Chemoselective Sonogashira type coupling of
5-bromo-3-iodopyridin-2-amine (10) with 1-chloro-2-ethynyl-3-
fluorobenzene (11) provided compound 12. Base mediated cycliza-
tion with t-BuOK/NMP afforded intermediate 13. Suzuki type
coupling of intermediate 13 with the corresponding boronic acids
or boronates of R1 provided compounds 14a–n.
Compounds 9a–g were tested in a fluorescence-based assay of
store operated calcium entry in Jurkat cells.10 Furthermore, their
ability to inhibit NFAT induced transcription was monitored
through the evaluation of IL-2 released from Jurkat T cells.11 A first
round of optimization with a limited set of substitutions in posi-
tion 2 of the azaindole core (Table 1) led us to the 2,6-disubstituted
phenyl group as providing best potency in both assays. 2-Chloro-6-
fluorophenyl substituted compound 9b was a good starting point
with an IC50 of 66 nM in the calcium assay and an IC50 of 23 nM
in the IL-2 assay. When slightly polar groups (9f) or a benzyl group
(9g) were introduced, the activity inhibiting calcium influx and
IL-2 dropped dramatically. For compounds 9a–e we observed a good
correlation between the activity in the calcium and the IL-2 assays.
We then explored replacement of the 3-(trifluoromethyl)-1H-
pyrazol-5-yl group in position 5 with disubstituted aryl and
heteroaryl groups as well as bicyclic derivatives, maintaining the
2-chloro-6-fluorophenyl substitution in position 2 (Table 2) as is
was the best substitution providing additivity based on internal
F
F
Cl
F
F
Br
I
R1
Br
11
Br
Cl
N
NH2
N
H
N
H
N
NH2
N
a
b
N
c
Cl
Cl
10
12
13
14a-n
Scheme 2. Reagents and conditions: (a) CuI, PdCl2(PPh3)2, Et3N, DMF, 110 °C, 82%; (b) t-BuOK, N-methylpyrrolidinone, rt, 34%. (c) R1-B(OH)2 or R1-pinacolboronate,
PdCl2dppf.DCM, Na2CO3, EtOH-toluene, 90 °C, 70–85%.

C. Esteve et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1217–1222
1219
Table 1
Jurkat Ca2+ and IL-2 secretion inhibition of selected 7-azaindole derivativesa
N
F
N
F
F
R
N
H
N
Compound
R
Jurkat calcium inhibition IC50 (nM)
Jurkat IL-2 secretion inhibition IC50 (nM)
cLogP
LipEb
1 YM
2 Synta
—
—
75 40
82 24
10
10
1
2
4.1
3.3
3.0
3.8
Cl
Cl
9a
9b
160 17
260
1
4.6
2.2
66 39
23
3
4.8
2.4
F
F
9c
140 58
73 10
4.3
2.6
F
9d
9e
9f
310 140
260 44
>10,000
183 82
270 62
ND
4.4
4.0
2.8
2.1
2.6
O
<2.2
9g
1400 210
3000 258
4.3
1.6
a
IC50 values reflect mean +/À standard deviation of two or more determinations.
Lipophilic efficiency (pIC50—clogP).
b
Table 2
Jurkat Ca2+ and IL-2 secretion inhibition of selected 7-azaindole derivativesa
F
R1
N
H
N
Cl
Compound
R1
Jurkat calcium inhibition IC50 (nM)
129 48
Jurkat IL-2 secretion inhibition IC50 (nM)
cLogP
LipEb
1.2
F
F
F
14a
120 23
5.7
N
O
14b
14c
14d
14e
71
64
7
1
40 37
150 29
68 24
ND
4.9
5.8
4.2
3.6
2.2
1.4
2.6
<1.4
N
F
O
F
N
O
O
150 22
>10,000
N
O
N
(continued on next page)

1220
C. Esteve et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1217–1222
Table 2 (continued)
Compound
R1
Jurkat calcium inhibition IC50 (nM)
>10,000
Jurkat IL-2 secretion inhibition IC50 (nM)
cLogP
LipEb
<1.7
O
14f
ND
ND
3.3
HN
O
N
O
14g
10,000
3.9
4.5
1.1
2.3
N
O
S
N
H
14h
14i
155 15
180 76
O
S
O
O
N
550 250
10,000
119
ND
2
4.7
4.5
4.5
1.6
0.5
2.1
O
S
N
14j
O
O
S
O
14k
280 141
290 79
ND
N
H
14l
10,000
88 24
5.2
5.3
0
Cl
Cl
O
14m
117
5
1.8
N
O
F
14n
120 82
56 10
6.8
0.1
F
O
a
IC50 values reflect mean +/À standard deviation of two or more determinations.
Lipophilic efficiency (pIC50—clogP).
b
Table 3
In vitro profile of selected derivatives
Compound
I CRAC in HEK STIM1
Orai1 overexpressing cells IC50 (nM)
LAD2 degranulation hexosaminidase
Rat whole blood IL-2 secretion
inhibition IC50 (nM)15
Cytotoxicity CHO cells
IC50 M)
12
*
release IC50 (nM)14
(l
9b
14b
14d
<100
104
440
372 73
443 33
630 34
500
1300
740
>100
>100
>100
*
IC50 values reflect mean +/À standard deviation of two or more determinations.
Table 4
In vitro ADME profile of selected derivatives
findings (data not shown). As can be seen in Table 2, disubstituted
3-pyridyl groups (14a–d) provided the highest potency inhibiting
calcium in Jurkat cells with less than 2 fold drop between IL-2
and calcium inhibitory activity, except for compound 14c. Com-
pound 14b is the only example providing IC50 <100 nM in the cal-
cium and in the IL-2 assays. In an attempt to introduce more
polarity, pyridones were introduced in R1 (14e–f) resulting in a
complete loss of potency and demonstrating that N-methylpyri-
done or hydroxy pyridine groups are not tolerated in this position
of the molecule. Replacement of the pyridine ring of 14b by a
pyrimidine (14g) also resulted in a drop of potency similarly as
when basic groups were introduced (14l). These results indicate
that in this series, the room for SAR expansion to introduce polar
solubilizing groups is very limited. Similarly, N-methyl (14h) and
N,N-dimethyl (14i) 3-methylbenzenesulfonamides and sulfone
14k provided acceptable potencies whereas the sulfonylmorpho-
line derivative (14j) was inactive. Substitution with bicyclic groups
Compound
Rat/human metabolism (%)a
PAMPA [⁄10À6 cm/s]b
9b
14b
14d
<5/<5
10/15
10/<5
<0.05c
0.1
0.66
a
Percentage of metabolism in hepatic microsomes after 30 min of incubation at
37 °C in the presence of NADPH. Compound and protein concentration were set to
5
l
M and 1 mg/mL, respectively.
Passive permeability through a PAMPA membrane. Compound concentration:
b
20 lM.
c
The compound was not detected in the acceptor chamber.
in R1 such as 6-chlorobenzoxazole and 6-chloro-2,2-difluorobenzo-
dioxolyl groups (14m–n) was also tolerated, although these mole-
cules suffered from very high lipophilicity (clogP >5) and low
solubility (<1 lg/mL).

C. Esteve et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1217–1222
1221
Table 5
Wistar rat pharmacokinetic parameters of selected derivativesa,b
Compound
iv (1 mg/kg)
p.o (10 mg/kg)
t1/2 (h)
AUC(0–24) (ng h/mL)
Cl (mL/min/kg)
Vss (L/kg)
Cmax (ng/mL)
AUC(0–24) (ng h/mL)
F (%)
14b
14d
2.1 0.2
8.0 0.7
596 136
1824 279
24
8
0
1
4.2 0.4
153 57
807 415
2179 985
11219 3100
37 17
62 17
5
1
a
Mean values SD (n = 2).
b
Formulation: iv: 50% PEG400 + 10% ethanol (compounds 4 and 11) and 40% PEG300 + 10% ethanol (compound 9); p.o.: 0.5% methylcellulose + 0.1% Tween 80.
Compounds 9b, 14b and 14d were chosen to for further profil-
9% 68% 75% 100%
(*)
300000
250000
200000
150000
100000
50000
0
15000
12500
10000
7500
5000
2500
0
ing since they exhibited the most balanced potencies in the cal-
cium flux and IL-2 cellular assays. In terms of physicochemical
properties, the lead compounds selected displayed very low solu-
bility in water (<5 lg/mL), clogP >4 and LipE in a range of 2.2–2.4.
These compounds were profiled in further in vitro cellular
assays to determine their mechanism of action through CRAC cur-
rent inhibition, using an electrophysiology assay in HEK cells over-
expressing STIM1/Orai1 proteins,12 and the inhibition of mast cell
degranulation through the inhibition of IgE-induced hexosamini-
dase release in human LAD2 cells (human mast cell line).
(Table 3)13,14 Moreover, before testing the compounds in an
in vivo rat model, the inhibitory effect of the compounds on IL-2
secretion in rat whole blood was evaluated.15 All compounds
showed decreased potency in rat cells compared to that observed
in human Jurkat cells in IL-2 secretion (Tables 2 and 3). Compounds
g
g
e
g
k
k
kg
D
k
cl
/
/
/
g
i
g
g
g/
h
m
m
0
3
m
1
3m
0
Ve
1
a
x
e
Figure 2. Dose response effect on BALF eosinophil count and total plasma levels of
Compound 14d in OVA rat model. Results are expressed as the number of
eosinophils/mL in BALF (black dots), and total plasma levels (nM) (grey squares).
Median values with interquartile range of the 8 animals used for each group are
were not cytotoxic up to concentrations of 100 lM, indicating that
inhibitory effects observed on cellular assays are related to the
represented. Percentages reflect
% inhibition of Eosinophils vs vehicle treated
mechanism of action of the compounds.
animals. Statistical analysis was done with a one way ANOVA followed by a
Dunnett’s test (⁄p 6 0.05). Dotted line indicates plasma levels required to achieve
IC50 of IL-2 secretion from rat whole blood cells in vitro.
Very low metabolism was observed in both rat and human
microsomes in the presence of NADPH (Table 4). PAMPA perme-
ability results indicated lack of passive permeability for compound
9b and limited permeability for compounds 14b and 14d (Table 4).
Taking into consideration these results, compound 9b was dis-
carded for further characterization. All three compounds showed
rat plasma protein binding >99.9%.
The pharmacokinetic profile of compounds 14b and 14d ana-
logues was determined in rat (Table 5). Moderate clearance and vol-
ume of distribution were observed for compound 14b. Compound
14d had a significantly lower clearance, similar volume of distribu-
tion and increased terminal half-life. Both compounds displayed
good oral exposure, with bioavailabilities higher than 35%.
To assess in vivo pharmacological activity, compound 14d was
evaluated in the ovalbumin (OVA) induced model of allergic inflam-
mation in Brown Norway rats (Fig. 2).16–19 Three different doses of
compound 14d (3, 10 and 30 mg/kg) were orally administered bid,
1 h before and 6.5 h after OVA challenge. Dexamethasone was
included in the experiment as positive control. The anti-inflamma-
tory potential of compound 14d was evaluated on its ability to inhi-
bit airway cell infiltration in bronchoalveolar lavage fluid (BALF) at
the end of the experiment (24 h post OVA-challenge). A dose depen-
dent inhibition of eosinophils in BALF of 9%, 68% and 75% respec-
tively versus vehicle treated animals was observed. Plasma levels
of compound 14 at the end of the experiment proportionally
increased with dose and correlated with the inhibitory effect
observed. Total plasma levels are reported since compound free lev-
els could not be calculated due to very high plasma protein binding.
Moreover, rat whole blood IL-2 inhibition was used as surrogate
pharmacodynamic marker to contextualize the efficacy observed
in the rat OVA-challenged animals. This rat assay allows us to com-
pare efficacy of the compound in target cells of the same species of
the efficacy model. Although IL-2 inhibition is not linked with
eosinophil infiltration inhibition, it has been reported as a practical
measure of efficacy for cyclosporine A that, similar to Orai inhibi-
tors, interferes with the NFAT pathway.20 At the end of the exper-
iment, plasma levels below those required for rat whole blood IL-2
IC50 were observed in animals showing no efficacy at inhibiting
eosinophil infiltration. Animals having plasma levels >3-fold above
IC50 for rat IL-2 inhibition showed almost 70% inhibition of eosin-
ophils infiltration in BALF (Fig. 2).
In summary, we have described a series of 7-azaindole based
Orai inhibitors. The initial SAR was explored, demonstrating that
the nature and substitution of groups on both 2- and 5-positions
of the 7-azaindole ring are essential to calcium and IL-2 inhibitory
activity. Best molecules tend to be highly lipophilic, with limited
room for introduction of polar groups. After further profiling of
the most potent derivatives, compound 14d was selected as a good
tool for in vivo studies, as it is potent and displays good oral expo-
sure in rat. The efficacy observed with 14d reinforces the potential
of Orai channel inhibitors for the treatment of respiratory diseases
such as asthma. Further optimization with novel core rings to
achieve both an improvement in Orai inhibitory activity and lower
lipophilicity is essential for high quality lead compounds suitable
for oral administration.
Acknowledgements
The authors acknowledge the contribution of Dr. A. Kirshen-
baum and Dr. Y. Wu (NIH, Bethesda) and the US Public Health Ser-
vices for providing the LAD2 cell line.
References and notes
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Kraft, S.; Turner, H.; Fleig, A.; Penner, R.; Kinet, J.-P. Science 2006, 312, 1220.
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Hutchings, A. B.; Jouvin, M.-H.; Putney, J. W.; Kinet, J.-P. Nat. Immunol. 2008,
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C. Esteve et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1217–1222
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5. Parekh, A. B. Nat. Rev. Drug Disc. 2010, 9, 399.
6. Feske, S. Ann. N. Y. Acad. Sci. 2011, 1238, 74.
before execution of the voltage protocol. The QPatch system uses a 48-channel
parallel amplifier for recordings of whole-cell currents. Data were filtered at
1 kHz and sampled at 5 kHz. Current amplitude was measured at À80 mV. The
IC50 values were determined by normalizing the inhibition values obtained
from independent cells (minimum n = 3 cells per concentration) using Synta
7. Sweeney, Z. K.; Minatti, A.; Button, D. C.; Patrick, S. ChemMedChem 2009, 4, 706.
8. Rodriguez, A. L.; Koradin, C.; Dohle, W.; Knochel, P. Angew. Chem., Int. Ed. 2000,
39(14), 2488.
9. Majumdar, K. C.; Mondal, S. Tetrahedron Lett. 2007, 48, 6951.
10. Fluorescent calcium flux assay: Jurkat E6-1 cells were obtained from ATCC
(number TIB-152TM) and maintained as ATCC recommend; in medium RPMI-
1640 with 10% FBS at 37 °C and 5% CO2. Before the assay, Jurkat cells were
grown at a density of 2 million cells/mL in cell culture media (RPMI with 10%
FBS) in a T-175 flask at 37 °C and 5% CO2. The day of the assay, cells were
harvested by centrifugation (5 minutes at 1500 rpm) and the pellet were
suspended in assay buffer (HBSS w/o Ca2+ and Mg2+, 20 mM Hepes and 1 mM
MgCl2, pH 7.4) at a density of 5 million cells/mL. Calcium depletion was
initiated by the addition to the cell suspension of Thapsigargin at a final assay
(compound 2) at 30 lM as positive control. All experiments were done at room
temperature.
13. Kirshenbaum, A. S.; Akin, C.; Wu, Y.; Rottem, M.; Goff, J. P.; Beaven, M. A.; Rao,
K.; Metcalfe, D. D. Leuk. Res. 2003, 27, 677.
14. LAD2 cells degranulation assay: the human mast cell line LAD2, established at
the National Institutes of Health (NIH) was provided by Dr. Arnold
Kirshenbaum through a biological materials license agreement. Cells were
sensitized in its normal growth media (Complete StemPro-34 SFM, containing
StemPro-34 nutrient supplement; 2 mM glutamine and 100 ng/mL SCF) by
adding 100 ng/mL of biotin-labeled IgE overnight (37°, 5% CO2) in a humidified
atmosphere (90%). Sensitized LAD2 cells were washed with release buffer
(137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, 1.8 mM CaCl2,
1.3 mM MgSO4, 0.025% BSA) and adjusted to a density of 0.2 Â 106/mL. Cells
were transferred to the assay plate (10.000 cells/well) and pre-incubated with
drugs for 30 min, and then activated with 125 ng/mL of streptavidin for
30 min. After the activation, supernatants were removed and transferred to
another well for b-hexosaminidase determination. Cells were lyzed in 2 cycles
of freeze-thaw and they were incubated for 90 min at 37 °C with 1 mM b-
concentration of 10
lM. 20
l
L
of the cellular suspension containing
Thapsigargin (100.000 cells/well) were plated in 30
lL
of HBSS Ca2+ free,
previously dispensed into 384 well plates (corning 3711), and incubated at RT
for 15 min. Test compounds were diluted in DMSO 100% to get a 10À3 M stock
solution and serial dilutions 1/3 were performed in the same solvent. Then,
0.5
lL of test compounds were added to the cells and incubated at rt for 1 h.
Once this incubation was completed, 5
lL of dye solution (FLIPR Calcium 5
Express Kit, Molecular Devices), prepared as Molecular Devices recommended,
were added into the assay plates followed by an extra incubation period of 1 h
at room temperature. After the second incubation period, assay plates were
placed into the FLIPR Tetra from Molecular Devices. Fluorescence was
measured at a wavelength of 525 nm after the dye excitation at 485 nm. The
hexosaminidase substrate (p-nitrophenyl-N-acetyl-D-glucosamide, in citric
buffer, pH 4.5). Reaction was stopped with 0.4 M glycine solution (pH 10.0)
and absorbance was read at 405 nm.
initial baseline was measured during 20 s, and then 5
lL of a 25 mM calcium
15. Rat whole blood cells were diluted 1:4 in RPMI cell culture medium and
solution in assay buffer were added (2 mM final calcium concentration) except
for basal wells, where assay buffer without calcium was added. At this time,
changes in the cellular fluorescent were measured for an extra 80 s. Peak and
base line were taken for the calculation of the ratio (Peak/Base Line). Total,
basal and compound well ratio values were used to calculate percentage of
inhibition.
stimulated by Concanavalin A (ConA) at 62.5 lg/mL in 96-well plates with
increasing concentrations of the test compounds for 24 h. After that period,
and upon centrifugation supernatants were transferred to a new plate and IL-2
detection was performed using Rat IL-2 DuoSet (DY502 from R&D) following
the manufacturer instructions. A standard rat IL-2 curve was included for
quantification purposes.
11. In vitro assay of cytokine release from T-cells: Jurkat E6-1 cells were obtained
from American Type Culture Collection (ATCC) and maintained in complete
medium with 10% fetal bovine serum (FBS) at 37 °C/5% CO2. Cells were plated
16. Brown Norway rats (200-250 g) were sensitized with Ovalbumin/Aluminium
(OVA/Alum) i.p. (days 0, 14 and 21) and challenged with OVA aerosol on day 28
(Devilbiss).Compound 14d was formulated in D-alpha-tocopheryl polyethylene
in 100
l
L of Dubelcco’s Modified Eagle Medium (DMEM) with 1% FBS in a 96
glycol 1000 succinate (TPGS) 11.7%, PEG-400 40%, propylen glycol 15%, water
33.3% and was dosed at 3, 10 and 30 mg/kg orally one hour prior to OVA
challenge and 6.5 h after challenge (to sustain compound levels). Eight animals
were included per treatment group. 24 h after OVA challenge, bronchoalveolar
lavage fluid (BALF) was collected to determine cell counts (Sysmex). Plasma
samples were analyzed by UPLC-MS after protein precipitation.
17. Garlisi, C. G.; Falcone, A.; Hey, J. A.; Paster, T. M.; Fernandez, X.; Rizzo, C. A.;
Minnicozzi, M.; Jones, M.; Billah, M. M.; Egan, R. W.; Umland, S. P. Am. J. Respir.
Cell Mol. Biol. 1997, 17, 642.
18. Underwood, S. L.; Raeburn, D.; Lawrence, C.; Foster, M.; Webber, S.; Karlsson, J.
A. Br. J. Pharmacol. 1997, 122(3), 439.
19. Underwood, S. L.; Haddad, E.-B.; Birrell, M. A.; McCluskie, K.; Pecoraro, M.;
Dabrowski, D.; Webber, S. E.; Foster, M. L.; Belvisi, M. G. Br. J. Pharmacol. 2002,
137, 263.
well plate at a density of 1 Â 106 cells/well and 50
lL of test compound was
added. Cells were then stimulated by the addition of 50 mL of
phytohemagglutinin (PHA) (final concentration 20 lg/mL) and incubated at
37 °C/5%CO2 for 20 h. On the following day, the supernatants were collected
and assayed for IL-2 levels by ELISA according to the manufacturer’s protocols.
The IC50 value was calculated as the concentration at which 50% of secreted IL-
2 in vehicle is inhibited.
12. A CRAC assay optimized for compound screening was set up based on the one
described by Fierro and Parekh in RBL-1 cells in (Fierro, L.; Parekh, A. B. J.
Membrane Biol. 1999, 168, 9) adapted to an automated QPatch HTX platform at
Evotec AG, using a BPS Bioscience generated cell line containing HuSTIM/
HuOrai in HEK293 cells. Activation of ICRAC currents was achieved by passive
depletion of Ca2+ stores by application of 10 mM EGTA in the internal buffer.
Cells were held at a holding potential of 0 mV. Every 3 s, a voltage ramp from
À100 to +100 mM (100 ms duration) was executed. Series resistance and
20. Stein, C. M.; Murray, J. J.; Wood, A. J. J. Clin. Chem. 1999, 45(9), 1477.