Aryl aminopyrazole benzamides as oral non-steroidal selective glucocorticoid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2008.10.128

Aryl aminopyrazole amides capped with N-alkylbenzamides 13-16 are selective glucocorticoid receptor agonists. 2,6-Disubstituted benzamides have prednisolone-like potency or better in vitro. Good oral exposure was demonstrated in the rat, with compounds wi

Full text of DOI:10.1016/j.bmcl.2008.10.128

Bioorganic & Medicinal Chemistry Letters 19 (2009) 158–162
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aryl aminopyrazole benzamides as oral non-steroidal selective
glucocorticoid receptor agonists
Heather A. Barnett a, Diane M. Coe a, Tony W. J. Cooper a, Torquil I. Jack b, Haydn T. Jones a,
Simon J. F. Macdonald a, Iain M. McLay c, Natalie Rayner a, Rosemary Z. Sasse d,
Tracy J. Shipley b, Phil A. Skone b, Graham I. Somers a, Simon Taylor b, Iain J. Uings b,
James M. Woolven c, Gordon G. Weingarten b,
*
a Respiratory CEDD, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
b Immuno-Inflammation CEDD, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
c Computational Analytical and Structural Sciences, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
d Screening & Compound Profiling, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Aryl aminopyrazole amides capped with N-alkylbenzamides 13–16 are selective glucocorticoid receptor
agonists. 2,6-Disubstituted benzamides have prednisolone-like potency or better in vitro. Good oral
exposure was demonstrated in the rat, with compounds with lower lipophilicity, for example N-hydroxy-
ethyl benzamides (e.g., 16e).
Received 9 September 2008
Revised 28 October 2008
Accepted 29 October 2008
Available online 5 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
GR
Glucocorticoid
Aminopyrazole
Glucocorticoid receptor (GR) agonists such as prednisolone 1
(Fig. 1) or fluticasone esters 2 have potent anti-inflammatory and
immunosuppressive properties. There has been considerable effort
in recent years to try to produce selective anti-inflammatory drugs
which act as GR ligands that suppress the expression of inflamma-
tory cytokines (transrepression activity) but do not affect genes
associated with typical steroid side effects (transactivation activity)
such as glucose intolerance, muscle wasting, skin thinning and oste-
oporosis.1–3 Boehringer–Ingelheim have demonstrated that a mole-
cule with such dissociated pharmacology BI-115 3, had an improved
side effect profile over 1 in a mouse model.3 Another cause of off-tar-
get pharmacology with some GR agonists is cross-reactivity with
other members of the nuclear receptor (NR) family, such as the
progesterone receptor (PR) androgen receptor (AR) and mineralo-
corticoid receptor (MR). Selectivity in this respect has also been
achieved with some non-steroidal compounds.4,5
assays, whereas 5b, in which R is cyclopentyl, is selective for TR.
In this series, we found that the aryl aminopyrazole group is a good
replacement for the steroidal cyclohexadienone A-ring. We5 and
other groups6–8 report the use of an aryl indazole, reminiscent of
that present in cortivazol 4a. The X-ray crystal structure of deac-
ylcortivazol 4b bound into the GR ligand binding domain (LBD)9
showed that the space occupied by the steroidal A-ring in the ac-
tive site, can open up to accommodate the arylpyrazole structure.
Although the THN series had attractive pharmacology, it was
structurally complex and more hydrophobic than typical oral
drugs. We thus envisaged replacing the THN portion by an aryl
group linked to the remainder of the molecule by a hydrophilic lin-
ker such as an amide, to give molecules 13–16 (Scheme 1) with
physicochemical properties more appropriate for oral drugs. The
aryl group was designed to occupy the hydrophobic pocket popu-
lated by the 17
a
ester group in FP and FF 2, and R1 the position
We have previously reported a series of GR agonists containing
a tetrahydronaphthalene (THN) group, for example, 5a,b, in which
both NR and transrepression (TR) selectivity can be achieved.4,5
The angular alkyl group R in 5a,b is crucial for agonist activity
and it was designated an agonist trigger. Compound 5a, in which
R is ethyl, is a full agonist in both TR and transactivation (TA)
occupied by the trigger group R in the THN series.
Molecular modelling of a typical example, the 2,6-dichloro-
benzamide 13h, supported this concept. The structural information
about the GR LBD from our recently published fluticasone furoate
structure10 was used to provide the starting point for the docking
experiments used to help guide this work. The docking models
were created using FLO+ which allows flexibility of side chains
during docking.5 The model created for this series is compared in
Figure 2a–c with the previous model developed for arylpyrazoles5
* Corresponding author.
E-mail address: ggw1517@yahoo.com (G.G. Weingarten).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.128

H. A. Barnett et al. / Bioorg. Med. Chem. Lett. 19 (2009) 158–162
159
to open the epoxide ring and then with hot aqueous sodium
hydroxide to give a benzylaminodiol from which the benzyl group
was removed by hydrogenolysis to furnish the aminodiol 7. The
aminopyrazole acid 8 was coupled to 7 using HATU to give 9.
The terminal hydroxyl was tosylated, following which, treatment
with polymer-bound carbonate in THF gave the epoxide 10. The
trigger group was installed by opening the epoxide 10 with an
alkylamine to give 11. In order to prepare compounds incorporat-
ing a hydroxyethyl trigger group, the hydroxyl group of ethanol-
amine was protected either as a TBDMS ether (by treatment with
TDMSCl and imidazole in dichloromethane) or as a tBu ether, and
the protecting group was removed by conventional means as a
final step. Amine 11 was capped with a benzoic or heteroaromatic
acid to give compounds 13–16. HATU was used as the coupling
reagent unless the benzoic acid was 2,6-disubstituted, in which
case it was usually necessary to use an acid chloride.12
F
O
S
H
O
O
HO
O
R
OH
HO
HO
N
CF3
OH
OH
F
H
H
N
H
H
H
F
F
O
O
2a R= Et (FP)
2b R= 2-furoyl (FF)
1
3
O
RO
N
CF3
N
OH
HO
N
H
R
H
OH
NH2
O
N
H
H
N
4a R=Ac
4b R=H
5a R= Et
5b R= cyclopentyl
Alternatively, a benzoyl chloride 17 could first be reacted with
the trigger amine to give an N-alkylbenzamide 18 which after
deprotonation with sodium hydride, added to the aminopyrazole
epoxide 10 to give compounds 13–16. The synthesis could be
shortened by treating the epoxytosylate 6 in dioxan with an
N-alkylbenzylamine followed by ammonia to give the diamine
12. This was then coupled to the aminopyrazole acid 8 and the
benzyl group was removed by hydrogenolysis to yield the last
stage intermediate 11.
The more active compounds were separated into their enantio-
mers by preparative chiral HPLC.13 The two enantiomers are desig-
nated E1 and E2, indicative of the order in which they eluted.
Circular dichroism showed that there was no consistency in the
order in which the two enantiomers eluted.
Figure 1. Examples of some steroidal and non-steroidal GR agonists.
and with the structure for fluticasone furoate 2b. In Figure 2a,
showing the docking model for 13h, N2 of the arylpyrazole moiety
shows H-bonding to Gln570, and the central hydroxyl group is
seen H-bonding to Asn564; S configuration is predicted at this cen-
tre. Figure 2b shows a superimposition of the model for 13h and
the FF crystal structure. The similar positioning of the furoate
group and the dichlorophenyl group can be seen. Both the model
and crystal structure have Gln642 and Tyr735 positioned similarly
allowing access to the steroid 17a pocket. In addition, the opening
of the receptor to accommodate the arylpyrazole moiety is appar-
ent, through shifts in Arg611 and Gln570 relative to their positions
in the steroidal structure in which Gln570 H-bonds to the 3-keto
group. In Figure 2c the models for 13h and the THN 5a are super-
imposed. The ethyl trigger groups are seen to be very similarly
placed. Gln642 and Tyr735 can be seen to adopt a different posi-
tion for 13h compared to 5a, in order to accommodate the dichlo-
rophenyl group. In contrast to FF 2b and the THN 5a, the model
shifts the Arg611 side chain out of H-bond contact with Gln570
for 13h, apparently caused by interaction with the para-fluoro
substituent.
The compounds were assayed for their ability to bind to GR using
competition experiments with fluorescent-labelled dexametha-
sone. A functional GR agonist assay was carried out using human
A549 lung epithelial cells engineered to contain a secreted placental
alkaline phosphatase gene under the control of the NFjB dependent
ELAM promoter. This assay allows determination of the ability of
compounds to repress transcription.14 Transactivation activity was
assayed using human A549 lung epithelial cells engineered to con-
tain a renialla luciferase gene under the control of the distal region
of the LTR from the mouse mammary tumor virus (MMTV).5 Com-
pounds were tested for nuclear receptor selectivity with respect to
PR and AR by similar binding assays and functional assays using an
MMTV-luciferase reporter in monkey kidney CV1 cells.14,15
The synthesis of compounds 13–16 is shown in Scheme 1. We
recently reported the synthesis of a versatile doubly electrophilic
a
-trifluoromethyl-a
-tosyloxymethyl epoxide synthon 6.11 Com-
pound 6 was treated with a slight excess of benzylamine in dioxan
N
N
F
HO
O
N
NH2
O
CF3
CF3
CF3
N
a,b,c
e,f
CF3
N
F
O
8
HO
N
HO
NH2
N
F
N
H
OH
OH
OTs
H
NH2
O
NH2
O
d
7
6
9
10
O
Cl
O
NHR'
N
N
R' CF3
N
CF3
g
k
h
F
N
F
g
N
Ar
N
R'NH
N
H
O
H
R
OH
R
OH
NH2
O
O
NH2
13 - 16
11
17
18
j,c
13 Ar = substituted Ph,
14 Ar = heteroaromatic,
15Ar = 2,6-dichlorophenyl, R' = CH2CH2OH
R'= Et
R'= Et
i
R' CF3
N
Ph
NH2
OH
16 Ar = 2,6-difluorophenyl
12
Scheme 1. Synthesis of 13–16. Reagents and conditions: (a) PhCH2NH2, dioxan, rt; (b) 2 M NaOH, dioxan, reflux; (c) H2/Pd(OH)2/C, ethanol; (d) HATU, DIPEA, DMF, rt; (e) TsCl,
pyridine; (f) MP-CO3 or PS-CO3, THF; (g) R1NH2, THF; (h) ArCO2H, HATU, DIPEA, DMF or ArCOCl (sometimes prepared in situ from ArCO2H, oxalyl chloride and cat DMF),
DIPEA, THF; (i) N-alkylbenzylamine, dioxan followed by ammonia in dioxan; (j) 8, HATU, DIPEA, DMF; (k) NaH, DMF then 10.

160
H. A. Barnett et al. / Bioorg. Med. Chem. Lett. 19 (2009) 158–162
tively low in order to avoid high plasma protein binding, CYP450
inhibition and poor aqueous solubility. The strategy used to
achieve this was to introduce heteroatoms into the phenyl substit-
uents, the phenyl core and the trigger group.
Potency was enhanced by ortho substituents, but at the cost of
increased lipophilicity. Analogues with polar substituents, for
example, SO2Me 13g were less potent. A second ortho substituent
enhanced potency further, so that 2,6-dichlorobenzamide 13h
had NFjB pIC50 9.3, and 2,6-difluorobenzamide 13i, with a less
severe increase in clogP, was 5 times as potent as prednisolone.
Where the second ortho substituent reduced clogP as in the
sulphone 13j, potency enhancement was compromised. In 13j,
containing two dissimilar ortho substituents, restricted rotation
in the tertiary amide group caused atropisomerism. Because of this
complication such molecules were not progressed further. In con-
trast to the unsubstituted benzamide, the less active enantiomers
of ortho-substituted benzamides had some measurable agonism,
albeit typically partial (compare 13d and 13e). The more active
2,6-dichlorobenzamide enantiomer 13h had the S configuration.
This was determined from our recently reported X-ray crystal
structure of 13h in the GR LBD.20 Comparison of CD spectra of
active enantiomers of other examples with that of 13h, indicated
that they have the same S stereochemistry. All compounds showed
excellent nuclear receptor selectivity with PR and AR binding
pIC50 < 5 and pEC50 < 6 in cellular agonism assays.
No potency enhancement was seen with meta substituents on
the phenyl ring. Para substitution as in 13m, significantly reduced
binding and potency, nullifying gains made by 2,6-disubstitution in
a 2,4,6-trisubstituted benzamide (compare 13o with 13h). Hetero-
aromatic amides were active either with an ortho substituent in
molecules such as 14a,b, or where in a bicyclic amide, for example,
indole 14c, the heterocycle forms the ortho substituent. Potency
was highly dependent on the position of the heteroatom as seen
in pyridines 14a,b. Two heteroatoms were generally not tolerated.
Different trigger groups were examined in an array of 2-chlo-
robenzamides, and the better ones as 2,6-difluorobenzamides
(Table 2). The agonist potency rank order was Et > 2-fluoroeth-
yl > n-Pr > 2-hydroxyethyl > Me ꢀ CH2CHMe2, cyclopropyl > nBu,
H. Compounds with larger and branched alkyl trigger groups were
less potent (and had higher clogP). In contrast to the THN series,
none of the trigger groups gave potent compounds with TR selec-
tivity. The power of the 2,6-disubstitution potency enhancement
was such that even molecules which lacked a trigger group, for
example, 16a were active, but these were partial agonists. The
hydroxyethyl group had a profound effect on lowering clogP but
this came at the cost of reduced potency. However the 2,6-difluo-
robenzamide 16e struck a good balance with clogP of 2.64 and
prednisolone-like potency.
Figure 2. (a) The receptor docking model for the benzamide 13h. (b) Superimpo-
sition of the model for 13h (gold) and the FF 2b (green) crystal structure. GR
residues in magenta for 13h and cyan for FF 2b. (c) Superimposition of the models
for 13h (gold) and 5a (green). GR residues in magenta for 13h and cyan for 5a.
DMPK profiles were also obtained routinely. CYP450 inhibition
potential was assayed in a fluorimetric assay with Cypex cDNA ex-
pressed isoforms.16 Human plasma protein binding was determined
by an ultrafiltration method17 and human serum albumin (HSA)
binding was determined by a fast gradient HPLC method on an
immobilised HSA HPLC column.18 PK studies were carried out on
selected compounds using adult male Sprague–Dawley rats.19
The prototype molecule, the N-ethylbenzamide 13a, was a full
agonist, about a quarter as potent as prednisolone 1. In the TR
Table 3 shows the P450 inhibition and plasma protein binding
profiles of selected examples. In general, it was found that mole-
cules with clogP < 3 were likely to have P450 inhibition IC50 > 3 lM
for all the five isoforms tested. The P450 inhibition profiles were
different for a pair of enantiomers, so that for example, 13e had
IC50 > 4 lM for all isoforms while its enantiomer 13d was 2–3
times more potent. The hydroxyethyl trigger was particularly
effective at lowering clogP, but even molecules with clogP > 3 with
this trigger group, for example, 15 had a satisfactory P450 inhibi-
tion profile. Plasma protein binding was also clogP dependent
and the hydroxyethyl trigger group was particularly effective in
reducing plasma protein binding. Thus 16e with both the advan-
tage of a low lipophilicity and a hydroxyethyl trigger was only
68% bound to human plasma protein.
Table 4 shows rat PK data for selected examples. This series typ-
ically had moderate clearance and volume of distribution and
moderate or good oral bioavailability in rats. The strategies em-
ployed for lowering lipophilicity by adding heteroatoms raised
NFjB assay it had pIC50 7.3 and in the TA MMTV assay it had
pEC50 7.3. On testing the individual enantiomers, we found that
one enantiomer bound more strongly than the other, consistent
with modelling predictions, and hence the potency resided largely
in this enantiomer. Thus the more slowly eluting enantiomer 13c
(E2) had GR binding pIC50 7.9 and NF
lent to prednisolone 1), while the faster eluting enantiomer 13b
(E1) had GR binding pIC50 6.9 and NF B pIC50 < 5 (Table 1).
jB pIC50 8 (which is equiva-
j
It became apparent from the first arrays of structural analogues
of the prototype molecule, that the lipophilicity needed to be rela-

H. A. Barnett et al. / Bioorg. Med. Chem. Lett. 19 (2009) 158–162
161
Table 1
Biological data for N-ethyl arylamides (13, 14).
c
d
f
Compound
(13) Ph substituents (14) Ar =
Enanta
clogPb
GR binding pIC50
NF
8
7.3
<5
8
j
B pIC50
NFj
B maxe (%)
MMTV pEC50
MMTV maxg (%)
1
Prednisolone
–
–
–
2-Cl
7.9
7.3
6.9
7.9
nd
100
88
–
94
95
75
98
99
101
104
103
96
92
–
76
100
75
99
100
7.3
7.3
<5.2
7.6
8.2
6.5
7.7
6.8
8.7
8.1
7.6
6.6
7.3
<5.2
6.5
7.3
6.1
7.4
8.5
81
104
–
108
113
70
130
120
120
122
128
125
106
–
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
14a
14b
14c
rac
E1
E2
E1
E2
E2
E1
E2
E2
rac
rac
rac
rac
rac
rac
rac
E1
3.08
3.08
3.08
3.87
3.87
3.3
1.86
4.61
3.46
2.62
3.87
4.04
3.87
4.04
5.33
2.73
2.72
3.37
8.5
7
2-Cl
2-F
nd
7.9
7.2
8.5
8.1
7.3
7.8
8.1
6.5
7.7
8.2
7.4
nd
7.9
7.7
9.3
8.6
8.0
7.5
7.7
<5
7
2-SO2Me
2,6-DiCl
2,6-DiF
2-Cl,6-SO2Me
3-Cl
2-Cl,3-F
4-Cl
2-Cl,4-F
2,4,6-TriCl
2-Cl-pyrid-3-yl
3-Cl-pyrid-2-yl
5-Indolyl
56
86
74
93
7.6
6.6
8
E1
8.2
9
112
a
Data shown for more active enantiomer where this was available or otherwise for racemate.
Calculated using daylight.
Generally n = 1.
Mean potency for n P 2 SD 0.34 using the pooled variance from the tested compounds.
Mean asymptotic maximum relative to dexamethasone SD 8.5% using the pooled variance from the tested compounds.
Mean potency for n P 2; SD 0.23 using the pooled variance from the tested compounds.
b
c
d
e
f
g
Mean asymptotic maximum relative to dexamethasone SD 13.6% using the pooled variance from the tested compounds.
Table 2
Biological data for N-alkyl 2,6-difluorobenzamides (16).
Trigger R1
Enanta
clogPb
GR binding pIC50
NF
jB pIC50
NF
80
95
104
96
j
B maxe (%)
MMTV pEC50
MMTV maxg (%)
c
d
f
Compound
16a
16b
13i
16c
16d
16e
H
Me
Et
nPr
CH2CH2F
CH2CH2OH
rac
rac
E2
rac
rac
E2
2.28
2.94
3.46
3.99
3.19
2.64
6.4
7.1
8.1
7.8
7.7
7.8
6.5
7.3
8.6
7.8
8.0
7.7
<5.2
6.6
8.1
7.6
7.3
7.3
112
122
128
105
119
94
94
a–g
Notes as for Table 1.
Table 3
P450 inhibitiona and binding to human plasma proteinb (PPB) or human serum albuminc (HSA) for selected compounds.
Compound
R1
Substituents
clogP
1A2 (l
M)
2C19 (
lM)
2C9 (
lM)
2D6 (lM)
3A4 (lM)
HSA (%)
PPB (%)
13g
16e
14b
16b
16d
13i
Et
2-SO2Me
2,6-DiF
3-Cl-pyrid-2-yl*
2,6-DiF
2,6-DiF
2,6-DiF
2,6-DiCl
2-Cl
1.86
2.64
2.73
2.94
3.19
3.46
3.78
3.87
3.87
3.99
4.61
P40
100
100
100
100
100
100
100
100
P40
100
87
40
100
49
8
18
20
8
23
13
14
10
4
3
3
4
10
3
96
100
100
36
20
74
54
27
34
44
3
76
4
100
0.5
2
100
2
81
81
83
nd
93
93
89
nd
nd
94
95
nd
68
85
nd
93
97
93
nd
nd
nd
99
CH2CH2OH
Et
Me
CH2CH2F
Et
15
CH2CH2OH
13d
13e
16c
13h
Et
Et
Pr
Et
2-Cl
2,6-DiF
2,6-DiCl
20
5
7
4
0.5
0.7
0.8
89
a
n P 1.
n P 2.
n = 1.
Ar=.
b
c
*
the polar surface area; nevertheless such molecules showed im-
proved oral exposure. Examples of this are molecules with a
hydroxyethyl trigger 16e or an ortho-sulphone substituent 13g,j,
or where phenyl was replaced by pyridine 14b. However, com-
pounds with higher clogP, for example, 16c had higher clearance
and volume of distribution resulting in lower oral exposure as well
as having a low free fraction.
In summary, we have shown that the aryl aminopyrazole phar-
macophore discovered in the context of our THNs can be linked to
a benzamide bearing a trigger group to give potent agonists, selec-
tive for GR over other NRs, which have oral-drug-like lipophilicity
and good oral exposure of free drug. These molecules are full
agonists and do not display dissociated TR/TA pharmacology. The
molecule that emerged with the best overall profile is 16e. A full

162
H. A. Barnett et al. / Bioorg. Med. Chem. Lett. 19 (2009) 158–162
6. Thompson, C. F.; Quraishi, A. A.; Ali, A. Bioorg. Med. Chem. Lett. 2005, 15, 2163.
Table 4
Pharmacokinetics of selected compounds in rat19
.
7. Mugge, I. A.; Burke, M. J.; Ralph, M. S.; Thomson, D. S.; Hammach, A.; Kowalski,
J. A.; Bentzien, J. M. WO 2006/135826.
8. Berger, M.; Rehwinkel, H.; Schaecke, H.; Lepistoe, M.; Edmann, K. WO 2008/
052808.
9. Suino-Powell, K.; Xu, Y.; Zhang, C.; Tao, Y.; Tolbert, W. D.; Simons, S. S., Jr.; Xu,
H. E. Mol. Cell. Biol. 2008, 28, 1915.
10. Biggadike, K.; Bledsoe, R. K.; Hassell, A. M.; Kirk, B. E.; McLay, I. M.; Shewchuk,
L. M.; Stewart, E. L. J. Med. Chem. 2008, 51, 3349.
11. Keeling, S. P.; Campbell, I. B.; Coe, D. M.; Cooper, T. W. J.; Hardy, G. W.; Jack, T.
I.; Jones, H. T.; Needham, D.; Shipley, T. J.; Skone, P. A.; Sutton, P. W.;
Weingarten, G. G.; Macdonald, S. J. F. Tetrahedron Lett. 2008, 49, 5101.
12. a Full preparative details and spectroscopic characterisation of compounds in
Scheme 1 described in: Barnett, H. A.; Campbell, I. B.; Coe, D. M.; Cooper, A. W.
J.; Inglis, G. G. A.; Jones, H. T.; Keeling, S. P.; Macdonald, S. J. F.; Mclay, I. M.;
Skone, P. A.; Weingarten, G. G.; Woolven, J. M. WO 2007/144327.; b Barnett, H.
A.; Campbell, I. B.; Coe, D. M.; Cooper, A. W. J.; Inglis, G. G. A.; Jones, H. T.;
Keeling, S. P.; Macdonald, S. J. F.; McLay, I. M.; Skone, P. A.; Weingarten, G. G.;
Woolven, J. M. WO 2008/000777.; c Campbell, I. B.; Coe, D. M.; Cooper, A. W. J.;
Inglis, G. G. I.; Jones, H. T.; Keeling, S. P.; Macdonald, S. J. F.; Skone, P. A.;
Weingarten, G. G. WO 2008/074814.
Compound
R1
Substituents
Cl (mL/min/kg)
Vss (L/kg)
%F
13a
13i
Et
Et
–
21
27
36
21
8
32
23
17
9
2.5
2.8
5.3
2.6
0.9
4
2.8
3.6
2.6
12
36
16
39
44
25
48
77
62
2,6-DiF
2,6-DiF
2,6-DiF
2,6-DiF
16c
16d
16e
13d*
14b*
13g*
13j
nPr
CH2CH2F
CH2CH2OH
Et
Et
Et
Et
2-Cl
3-Cl-pyrid-2-yl**
2-SO2Me
2-Cl,6-SO2Me
*
Data for racemate.
**
Ar=.
in vitro and in vivo study in which it is compared with a steroid
will be reported.
13. Chiral HPLC was carried out on Chiralcel OD columns eluting with heptane and
ethanol or isopropanol mixtures.
Acknowledgments
14. Salter, M.; Biggadike, K.; Matthews, J. L.; West, M. R.; Haase, M. V.; Farrow, S.
N.; Uings, I. J.; Gray, D. W. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007, 293, L660.
15. Trump, R. P.; Blanc, J. B.; Stewart, E. L.; Brown, P. J.; Caivano, M.; Gray, D. W.;
Hoekstra, W. J.; Willson, T. M.; Han, B.; Turnbull, P. J. Comb. Chem. 2007, 9, 107.
16. The cytochrome P450 inhibitory potential was determined against CYP1A2,
2C9, 2C19, 2D6, and 3A4 using bactosomes expressing the appropriate
cytochrome P450 (Cypex Ltd, Dundee, UK). The following probes were used:
(3-butyryl-7-methoxycoumarin 7-ethoxyresorufin (1A2), 7-methoxy-4-
trifluoromethylcoumarin-3-acetic acid (2C9), 3-butyryl-7-methoxycoumarin
(2C19), 4-methylaminomethyl-7-methoxycoumarin (2D6), and diethoxy-
fluorescein (3A4). The inhibitory activity was determined by a fluorescent
plate reader methodology using a seven-point IC50 curve. The variability of the
assay is within 2-fold of the reported IC50 values.
We thank David Brown and Margaret Clackers for the in vitro
pharmacology assays, David Lugo and James Gray for the in vivo
DMPK studies, Nicola Lindsay for the P450 inhibition assays, Klara
Valko and Shenaz Nunhuck for the HSA binding assay and Eric
Hortense, Graham Foster and Steve Jackson for preparative chiral
chromatography.
References and notes
17. Taylor, S.; Harker, A. J. Pharm. Biomed. Anal. 2006, 41, 299.
18. Valko, K.; Nunhuck, S.; Bevan, C.; Abraham, M. H.; Reynolds, D. P. J. Pharm. Sci.
2003, 92, 2236.
19. PK studies were carried out using adult male Sprague–Dawley rats, n P 2. The
dose formulation for both intravenous and oral administration was 10% DMSO/
45% polyethylene glycol 200/45% water (v/v) administered at 1 mg/kg and at a
concentration of 0.2 mg/mL for both routes. Blood samples were collected at
timepoints up to 12 h after administration and were analysed by LC–MS/MS.
The variability on the bioanalytical assay for the determination of plasma
concentrations is considered to be 20%.
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