2-(1H-Pyrazol-4-yl)acetic acids as CRTh2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.03.093

High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antagonists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.03.093

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3349–3353
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-(1H-Pyrazol-4-yl)acetic acids as CRTh2 antagonists
Miriam Andrés, Mónica Bravo, Maria Antonia Buil, Marta Calbet, Jordi Castro, Teresa Domènech,
Peter Eichhorn, Manel Ferrer, Elena Gómez, Martin D. Lehner, Imma Moreno, Richard S. Roberts , Sara
Sevilla
⇑
Almirall R&D Centre, Laureano Miró, 408-410, 08980 Sant 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:
High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antago-
nists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar
inhibitors.
Received 14 February 2013
Revised 19 March 2013
Accepted 22 March 2013
Available online 2 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
CRTh2
Prostaglandin D2
Antagonist
Pyrazole
Plasma protein binding
Prostaglandin D2 (PGD2) is derived from the metabolism of ara-
chidonic acid by cyclooxygenases and downstream PGD2 syn-
thases. In the immune system, PGD2 is mainly produced by mast
cells1 but also although at lower levels, by macrophages and Th2
lymphocytes.2 PGD2 binds to three different receptors, the throm-
boxane type prostanoid receptor (TP),3 the PGD2 receptor (DP, also
known as DP1)4 and the chemoattractant receptor homologous
molecule expressed on Th2 lymphocytes (CRTh2, also known as
DP2).5 CRTh2 is a Gi coupling GPCR, signaling through reduction
of intracellular cyclic adenosine monophosphate (cAMP) and cal-
cium mobilization and it is involved in the chemotaxis of Th2 lym-
phocytes, eosinophils and basophils.6 CRTh2 also inhibits the
apoptosis of Th2 lymphocytes7 and stimulates the production of
IL4, IL5, and IL13,8 cytokines involved in important biological re-
sponses such as eosinophil recruitment and survival, mucus secre-
tion, airway hyper-responsiveness and immunoglobulin E (IgE)
production among others. Therefore, molecules that antagonize
the pro-inflammatory PGD2 effects mediated by CRTh2 on key cell
types associated with allergic inflammation (basophils, eosinophils
and Th2 lymphocytes) should have a potential benefit in related
pathologies.
acid indomethacin 1 inhibited the binding of PGD2 to CRTh2,11
and that the thromboxane inhibitor, the indolepropionic acid Ram-
atroban (BaynasÒ) 2 was also a fairly potent antagonist of CRTh2
(Fig. 1).12
While robust structural information on the receptor remains
elusive, a general preference for an arylacetic acid pharmacophore
has emerged. Furthermore, these structures may be conceptually
broken into three areas (Fig. 2):9b
(1) The acetic acid ‘head’ group. The chain length affects binding
affinity, with acetic acid chains generally best. Alpha-substi-
tution tends to lose potency and common bioisosteres of
carboxylic acids (for example tetrazoles or acyl sulfona-
mides) are not well tolerated.
(2) The ‘core’ ring. This may be monocyclic or bicyclic, and with
the exception of a few cases where activity is lost, no clear
preference has emerged in this position.
(3) The ‘tail’ group. This position seems to be the most open in
terms of structure–activity relationship (SAR) and a great
number of variations in chain length, directionality and
polarity have been published.
Several recent reviews have highlighted the progress and most
advanced series of CRTh2 antagonists.9,10 Many of the chemical
series owe their origins to the observations that the indoleacetic
We sought to take advantage of known SAR around the indole-
acetic acid pharmacophore but applying a conceptual ring expan-
sion of the indole core, revealing a substituted pyrazole (Fig. 2).
Two possible ring-openings were considered: this letter addresses
our work on pyrazole-4-acetic acids 3, while the work on pyrazole-
1-acetic acids 4 is described elsewhere.13
⇑
Corresponding author. Tel.: +34 93 291 7540; fax: +34 93 291 3420.
E-mail address: rick.roberts@almirall.com (R.S. Roberts).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.093

3350
M. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3349–3353
R5
O
tBuO2C
R5
O
O
R5
NH
tBuO2C
HO
O
i
ii
H
N
HO
O
O
O
O
S
R3
R3
N
N
N
R3
O
7
8
9
F
v
Cl
MeO
iii
R5
Indomethacin 1
Ramatroban 2
R5
tBuO2C
tBuO2C
+
Figure 1. Chemical structures of Indomethacin 1 and Ramatroban 2.
R1
N
N
N
N
R3
R3
R1
10b
10a
iv
HO
O
HO
O
X
HO
O
R5
R5
iv
"tail"
"head"
Y
R1
R
R4
HO2C
HO2C
N
N
N
Z
R5
R5
N
R1
N
N
N
N
R3
R3
R1
"core"
11b
11a
3
4
This work
Described elsewhere
Scheme 1. Representative syntheses of final compounds. Reagents and conditions:
(i) NaH, THF, 0 °C; tert-butyl bromoacetate, rt; (ii) NH2NH2ÁH2O, AcOH, rt; (iii) alkyl
halide, K2CO3, DMF, rt or NaH, THF, 0 °C; sulfonyl chloride, rt or aryl boronic acid,
Cu(OAc)2, O2, pyridine, CH2Cl2, rt; (iv) TFA, CHCl3, rt; (v) R1NHNH2, AcOH, rt.
Figure 2. Conceptual opening of the indole core to reveal the pyrazoleacetic acids.
A high throughput screening (HTS) campaign of our compound
collection14 revealed compound 5 as a micromolar hit. The meth-
oxypyrazole motif appeared a potential metabolic weak-spot of
the molecule so we directly synthesized and characterized di-
methyl analogue 6. Encouragingly, this compound was more active
followed by acidic deprotection led to the desired pyrazoles 11.
Alternatively, the diketones 8 could be cyclised directly with the
appropriate hydrazine to give 10, followed by acid deprotection.
For asymmetrical pyrazoles (R3 – R5), the step to incorporate
the R1 group inevitably led to the formation of both regioisomers
10a and 10b.17 According to the conceptual ring opening of
Fig. 2, the desired isomer generally required R3 = aryl and
R5 = methyl.18 In this case, functionalisation of pyrazoles 9 gave
the desired regioisomer 10a as the major product, typically in a ra-
tio of ꢀ90:10. Direct cyclisation of diketones 8 with aryl hydra-
zines (R1 = aryl) also gave regioisomer 10a as the major products.
Only in the case of direct cyclisation with alkyl hydrazines (R1 = al-
kyl) did the selectivity change, giving the undesired compounds
10b as the major isomer. The separation of the regioisomers 10a
and 10b required careful chromatography in each case and the ste-
reochemistry of the products was confirmed by nuclear Overhaus-
er effect (NOE) studies in many cases.
than the original hit 5 in the blockade of radio-labelled [35S]-GTP
cS
binding (Table 1).15 Functional activity was confirmed through the
eosinophil shape change assay (ESC) in human whole blood
(hWB),16 showing a modest 10-fold drop in activity, a shift
undoubtedly due to binding to plasma proteins. The physicochem-
ical properties were acceptable at this stage for an oral compound
so we proceeded to profile the pharmacokinetics in rat. Compound
6 showed low clearance, and good exposure after intravenous dos-
ing. The oral bioavailability was low, but as this was only a hit
compound, we were encouraged to continue.
The general synthetic route is outlined in Scheme 1. The appro-
priate diketones 7 were alkylated with tert-butyl bromoacetate
and cyclised with hydrazine to give the pyrazoles 9. Functionalisa-
tion of the pyrazole NH (alkylation, arylation or sulfonylation)
From the general synthetic route, we first synthesized a direc-
ted set of analogues of general formula 12, keeping the synthesis
simple by using a symmetrical pyrazole (R3 = R5 = Me). The SAR
Table 1
of a set of 30 compounds in the GTP
to be quite tight: only 4 (13%) compounds showed moderate activ-
ity of <1 M, with a further 6 (20%) compounds between 1 and
M.
A recent letter by Pfizer of a directed library of pyrazole-4-acetic
cS binding assay turned out
Pyrazole HTS hit 5 and dialkyl analogue 6
Ph
l
HO
O
5
l
HN
R5
O
5 R5 = OMe
6 R5 = Me
N
N
acids also highlighted a general absence of activity for this sub-
structure.19 However, this was in contrast to some very potent pyr-
azoles of similar general structure exemplified by Boehringer
Ingelheim.20
Property
GTP S binding IC50 (nM)
5
6
c
1600
230
When we investigated further, we saw that there was a clear
separation of the tail SAR between the pyrazole series and reported
indole-based compounds (Fig. 4). However, in the case of the
ortho-sulphonyl benzyl tails (23–25) the activity was maintained
in the pyrazole derivatives and we observed very good potencies.
Compound 25 was used as the starting point for further SAR
investigations around the tail, and we found a clear preference
for the sulfone linker (Table 2). The next most active linker after
the SO2 group was the methylene 27. It is interesting to consider
that in these linked bisaryl systems, the conformational preference
for both sulfone and methylene is similar, with the two aromatic
rings positioned orthogonal to each other.22 Carbonyl and oxygen
linkers are markedly different, favouring conformations in which
ESC hWB IC50 (nM)
2100
3/6
0.5/0.8
4.2/0.9
Caco-2 AB/BA (Papp  10À6 cm/s)
Solubility (mg/ml) pH 1/7.4
pKaa/logD7.4
b
iv Pharmacokineticsc
AUC0–24 h (ng⁄h/ml)
Clearance (ml/min/kg)
3364
2.5
1.3
0.14
12%
t
(h)
½
Vss (l/kg)
d
Foral
a
b
c
Potentiometric.
Shake-flask.
Rat iv dose 0.5 mg/kg.
Rat oral dose 1 mg/kg.
d

M. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3349–3353
3351
Table 2
Table 4
SAR of the sulfone variations
Profile of pyrazole 25
HO
O
HO
O
S
O
Q
N
N
O
N
N
25
25-30
Property
GTP S binding IC50 (nM)
25
Compd
Q
GTPcS IC50 (nM)
c
7
2400
98.8/>99.9
13/17
0.003/0.95
4.2/0.7
25
26
27
28
29
30
SO2
CH2SO2
CH2
C@O
bond
O
7
490
100
1400
5000
2000
ESC hWB IC50 (nM)
PPB%a (rat/human)
Caco-2 AB/BA (Papp  10À6 cm/s)
Solubility (mg/ml) pH 1, 7.4
pKab/logD7.4
c
iv Pharmacokineticsd
AUC0–24 h (ng⁄h/ml)
Clearance (ml/min/kg)
1955
8.5
4.8
1.6
73
t
(h)
½
both aryl rings try to achieve the maximum planarity with respect
to the linker group. In accordance, these linkers 28 and 30 respec-
tively lost orders of magnitude of potency with respect to 25.
Hence we reasoned that the position of the terminal aromatic
group was important for binding and was generally maintained
in subsequent molecules.
Vss (l/kg)
e
Foral
%
a
b
c
Plasma protein binding, rapid equilibrium dialysis.
Potentiometric.
Shake-flask.
Rat iv dose 1 mg/kg.
Rat oral dose 10 mg/kg.
d
e
With the tail linker group fixed as sulfone, we carried out fur-
ther SAR around the other positions of the tail (Table 3). Replace-
ment of the terminal phenyl group with a cycloalkyl group
(sulphonamides 32 and 33) lost an order of magnitude of potency.
More radical changes such as a strongly basic group at the termi-
nus (34) or a small terminal group (35) lost even more potency.
We also saw that alpha-substitution adjacent to the pyrazole core
(36) showed a dramatic drop in potency. Here the loss was attrib-
uted to one (or both) of the following reasons: steric clash of the
alpha-methyl group with the pyrazole-5-methyl disrupted the rel-
ative conformation between the pyrazole and ring X. Alternatively,
the proximity of the alpha-methyl to the sulfone linker was suffi-
cient to disturb the relative conformation between the X and Y
rings, a sensitive part of the molecule as we had already observed.
Finally replacement of the proximal phenyl ring with a pyrrolidine
(37) lost all activity. Clearly in this series, the SAR was very sensi-
tive to conformational changes of the tail group, with both the
rings X and Y preferably aromatic and requiring the correct relative
orientation between both themselves and the pyrazole core.
With a tight SAR emerging, we profiled compound 25 to evalu-
ate the series (Table 4).23 The first detail that emerged was the al-
most complete loss of potency in the human whole blood ESC
assay. This was followed up with plasma protein binding (PPB)
measurements: in human plasma, the true extent of binding was
hard to measure and even diluting the plasma to 10% concentra-
tion, the extent of binding as measured by equilibrium dialysis
was around 99.9%. Protein binding of this order was seen as an is-
sue. While the compound is bound to plasma proteins, it is essen-
tially shielded from the various clearance mechanisms of the body,
and an attractive overall PK profile may result. However, the bound
compound is also impeded from interacting with the target recep-
tor: large circulating levels of an inactive compound (or very long-
lived but very very low free concentrations) lead to little.24 In
terms of physical chemical properties, 25 showed properties ex-
pected for a lipophilic carboxylic acid. Permeability and, due to
the presence of the carboxylate at physiological pH, solubility were
good. At low pH, re-protonation to the carboxylic acid gave a lipo-
philic, neutral molecule and so solubility dropped away. Both the
PPB and PK profile in rat were acceptable and revealed a well ab-
sorbed, low clearance compound with a long terminal half-life.
With the narrow SAR of the tail favouring the ortho sulfone
group, we examined the remaining position open to substitution
at the pyrazole 3-position, in an attempt to at least moderate the
excessive human PPB (Table 5). Lipophilic groups in this position
maintained huge shifts in potency in the absence or presence of
plasma proteins. Only the polar pyridyl substitution (39) was capa-
ble of maintaining high potency in the presence of albumin. In this
Table 3
SAR of ortho-sulphone tails
case we used a substitute assay of running the GTPcS binding in
HO
O
X
the presence of 1% human serum albumin (HSA). This assay al-
lowed a much higher throughput than the ESC assay and we al-
ways observed a good correlation between the two (data not
shown).
2
S
O
1
Y
N
N
O
Rα
31-37
At this point, through the tail SAR we had found an optimum
substitution pattern in the ortho-sulfonylbenzyl tail. This in turn
had led to an optimisation of the substituents of the pyrazole core,
resulting in potent inhibitor 39. However, it was evident that these
substitution patterns of both tail and core were quite specific and
that we were left with little further room for manoeuvre. We could
however see parallels with a series of thiazole-4-acetic acid com-
pounds reported by 7TM,25 in which the equivalent R3 group of
the core was also 4-pyridyl. The tail groups of this series were
dibenzyl derivatives, so we decided to pursue this line of
investigation.
a
Compd
R
X
Y
GTPcS IC50 (nM)
25
31
32
33
34
35
36
H
H
H
H
H
H
Me
Ph
Ph
4-F-Ph
4-Morpholinyl
1-Piperidinyl
1-Piperazinlyl
NMe2
7
36
89
5-OMe-Ph
Ph
Ph
4-F-Ph
Ph
66
5000
850
2900
Ph
Ph
2
37
H
Ph
Inactive
N
1

3352
M. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3349–3353
Table 5
Table 6
SAR around the pyrazole R3 group
SAR around branched tail groups
R
HO
O
HO
O
R5
S
O
N
O
N
N
Rα
R3
N
R3
38-40
47-53
Compd
R3
GTP
c
S IC50 (nM)
ESC hWB IC50 (nM)
a
Compd
R3
R5
R
R
GTPcS IC50 (nM)
24
25
38
39
40
Me
Ph
4-Cl–Ph
4-Pyr
OH
81
7
24
4
1700
2400
8100
66a
47
48
49
50
51
52
53
4-pyr
4-pyr
4-pyr
2-F-4-pyr
4-OH-3-pyr
Ph
Me
Me
H
Me
Me
Me
Me
4-F–Ph
H
4-F
4-F
4-F
46
1900
290
4-F–Ph
4-F–Ph
4-F–Ph
4-F–Ph
4-F–Ph
160
1000
4-F
310
4-F
4-F
Inactive
1100
a
Measured by surrogate assay: GTPcS binding in presence of 1% human serum
albumin (HSA).
Ph
4-SO2Me
5000
R5
NH
R5
NH
R5
NH
R5
O
Br
R3
Br
Br
ii
iv
i
para
R
meta
R =
N
N
N
R
3 = H
HO
O
R3
O
R3
I
I
-hal
-alkyl, alkoxy
-CONR2
-NHCONHR
-NHSO2R
-Ar
7
41
43
42
44
45
iii
N
N
0,1
iii
iii
12
R5
R5
R5
v
ii
Br
R1
R1
R1
N
N
Figure 3. Directed set of phenyl and benzyl-substituted pyrazole analogues.
N
N
N
N
R3
R3
46
Cl
Cl
vi
HO
HO
O
HO
HO
O
R5
R5
Cl
Cl
tBuO2C
HO2C
vii
R1
R1
N
N
N
N
S
O
S
O
N
N
N
O
R3
R3
O
N
10a
11a
13
14
1600 nMb
52 nMa
Scheme 2. Representative syntheses of final compounds. Reagents and conditions
(i) NH2NH2ÁH2O, EtOH, rt; (ii) NBS, DMF, 55 °C; (iii) NaH, DMF, rt; alkyl halide; (iv)
NIS, DMF, 115 °C; (v) aryl boronic acid, PdCl2(dppf), dioxane, 80 °C; (vi) Pd(dba)2, Q-
Phos, tBuO2CCH2ZnBr, THF, microwave irradiation, 100 °C; (vii) HCl, dioxane, rt.
O
N
O
N
N
N
N
15
16
21 nMc
5000 nMb
For some of these compounds, alternative synthetic routes were
useful (Scheme 2). If the correct diketone 7 was available, this was
cyclised with hydrazine (41) and then alkylated and brominated
(in either order) to give the bromopyrazole 44. Alternatively, to al-
low introduction of the R3 group at a later stage of the synthesis
the iodobromopyrazoles 45 were synthesised. Alkylation
(45?46) proceeded with very high regioselectivity in this case.
The R3 group was then introduced via a Suzuki reaction. With
the purified regioisomer of the bromopyrazole 44 in hand, the ace-
tic acid head group was introduced via a palladium-catalysed Negi-
shi reaction under microwave conditions. Finally, acid
deprotection was carried out with HCl in dioxane, as when the
R1 tail group was benzhydryl, standard TFA treatments also
cleaved the tails.
In terms of this new SAR, the 4-pyridyl group at the pyrazole 3-
position was preferable (Table 6). Again, we found that small
changes to the first compound of the series lost activity. For exam-
ple, extending the hydrogen-bond acceptor capacity of the pyridyl
group to a pyridone (51) lost all potency. In the end, the benzhy-
dryl tail motif was unable to reach the same levels of potency as
seen with the sulfone tails.
HO
O
N
HO
O
HO
O
N
N
N
N
N
17
N
N
13 nMa
(130 nM)b
18
19
>5000 nMb
870 nMb
F
SO2Me
SO2Me
SO2Me
HO
HO
O
N
HO
HO
O
HO
HO
O
O
N
N
N
N
20
21
22
7 nMa
5000 nMb
inactiveb
F
F
O
N
O
SO2Ph
SO2Ph
SO2Ph
N
N
N
N
23
5 nMd
24
25
81 nMb
7 nMb
(16 nM)b
Indole-based core
Pyrazole cores
A selection of the compounds throughout the series, namely 24,
25, 27, 30, 32, 40 and 47 were also tested for their selectivity
against both the DP1 receptor and the thromboxane A2 receptor.
Figure 4. Comparative potency data (nM) between pyrazole and indole-based
cores. aKi data taken from Ref. 9b and references therein. bInternally measured
c
S IC50 data. cIC50 data taken from Ref. 21. dESC IC50 data taken from Ref. 9a and
All compounds were selective, showing <40% inhibition at 10
in all cases.
lM
GTP
references therein.

M. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3349–3353
3353
Invest. Drugs 2010, 19, 947; (d) Burgess, L. Annu. Rep. Med. Chem. 2011, 46,
119.
11. Hirai, H.; Tanaka, K.; Takano, S.; Ichimasa, M.; Nakamura, M.; Nagata, K. J.
Immunol. 2002, 168, 981.
12. Sugimoto, H.; Shichijo, M.; Iino, T.; Manabe, Y.; Watanabe, A.; Shimazaki, M.;
Gantner, F.; Bacon, K. B. J. Pharmacol. Exp. Ther. 2003, 305, 347.
13. Andrés, M.; Bravo, M.; Buil, M. A.; Calbet, M.; Castillo, M.; Castro, M.; Eichhorn,
P.; Ferrer, M.; Lehner, M. D.; Moreno, I.; Roberts, R. S.; Sevilla, S. Eur. J. Med.
Chem. 2013, manuscript in preparation.
We also characterised the dissociation kinetics of some of these
compounds, following some reports of the potential for slow off-
rate compounds from the CRTh2 receptor.26 Briefly, membranes
were incubated for 1 h with the test compound at 10 Â IC50. We
then initiated compound dissociation by adding a huge excess of
PGD2 (100
pound dissociated, re-binding was prevented by mass-action law.
We could then perform the standard GTP S assay at intervals event
l cS) so that once the test com-
M and 0.1 nM 35S-GTP
14. Approximately 100 K compounds were screened by homogeneous cAMP assay
in 384-well format using the cAMP dynamic 2 jumbo kit (Ref. 62AM4PEJ,
c
to follow the loss of binding activity over time. Using this assay,
benzyl-substituted compounds 24 and 25 showed rapid dissocia-
tion half-lives of 2 and 12 min respectively. Benzhydryl analogue
47 was somewhat slower with a half-life of around 30 min.
In conclusion, HTS identified the pyrazole-4-acetic acid sub-
structure as a micromolar hit. The SAR of the scaffold was investi-
gated, showing significant differences in some case to the SAR of
reported indole acetic acid CRTh2 antagonists. SAR was able to
identify 25 as a very potent inhibitor in vitro. The extremely high
PPB of this series was a mitigating factor for the further develop-
ment of these compounds. Pyridyl analogue 39 was seen as an
attractive alternative. The SAR uncovered around the pyrazoles
was of interest for other CRTh2 antagonist series which will be re-
ported in due course.
CisBio) using HTRF energy transfer technology.
observed.
15. 4-8 lg of membranes obtained from CHO.K1 cells stably over-expressing
A hit rate of 0.45% was
CRTh2 were pre-incubated with the compound to be tested for 1 h, followed
by incubation with 50 nM PGD2 and 0.1 nM guanosine [gamma-
thio]triphosphate ([35S]-GTP
c
S) in incubation buffer (20 mM HEPES, 10 mM
MgCl2, 100 mM NaCl, 10
l
M GDP, 10 g/ml saponine and either 0.2% BSA or
l
1% HSA as appropriate) for 2 h at rt. The reaction was terminated by filtration
using GF/C plates pre-treated with 20 mM HEPES, 10 mM MgCl2, 100 mM
NaCl and 0.1% BSA, washing 6 times with buffer (20 mM NaH2PO4, 20 mM
Na2HPO4). The plates were dried and scintillation buffer Optiphase was added.
The radioactivity retained in the filter was counted using a Microbeta liquid
scintillation counter. Compound IC50s were determined using Excel XL-fit for
calculations. Antagonism was observed by the reduction of retained
radioactivity from control. Agonism could be measured using
assay set-up but without the 1 h pre-incubation and without the addition of
PGD2.
a similar
16. Whole blood obtained from consenting donors was mixed with heparin and
kept in rotation until use. For compound treatment, 90
with 10 l of test compound solution and incubated for 10 min at room
temperature followed by addition of 10 l 500 nM PGD2 for exactly 4 min at
ll of blood was mixed
The structures of the set of pyrazoles 12 of Figure 3 are con-
tained in the Supplementary data supplied with this manuscript.
l
l
37 °C. The reaction was stopped by placing the tubes on ice and adding of
0.25 ml of ice cold 1:5 diluted CellFix (BD Biosciences). Red blood cells were
lysed in two steps by adding 2 ml of lysis solution (150 mM ammonium
chloride, 10 mM potassium hydrogencarbonate), incubating for 20 and 10 min
respectively and centrifuging at 300g for 5 min at 6 °C. The pellet was re-
Acknowledgments
The authors thank the following for their valuable assistance in
the experimental procedures: Laura Estrella, Manel de Luca, José
Luís Gómez, Encarna Jiménez, Francisco Jiménez, Juan Navarro, Isa-
bel Pagan and Sílvia Petit.
suspended in 0.2 ml fixative solution and immediately analysed on
a
FACSCalibur (BD Biosciences). Eosinophils were gated on the basis of
autofluorescence in the FL2 channels and shape change of 600 cells was
assayed by forward scatter and side scatter analysis. Compound IC50s were
determined using Excel XL fit for calculations.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
093.
References and notes
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3.47 (s, 2H), 5.53 (s, 2H), 6.54 (d, J = 7.03 Hz, 1H), 7.29–7.36 (m, 1H), 7.40 (t,
J = 7.42 Hz, 2H), 7.53 (d, J = 7.42 Hz, 2H), 7.60–7.71 (m, 4H), 7.72–7.80 (m, 1H),
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(15 min) retention time 8.18 min. LRMS: m/z 447 (M+1).
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