5-Functionalized indazoles as glucocorticoid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2010.04.012

An indazole based series of glucocorticoid receptor agonists is reported. The SAR exploration of this scaffold yielded compounds with nanomolar affinity for the glucocorticoid receptor with indications of selectivity for the preferred transrepression mech

Full text of DOI:10.1016/j.bmcl.2010.04.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3017–3020
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Functionalized indazoles as glucocorticoid receptor agonists
Mei Bai a, Grant Carr a, Russell J. DeOrazio a, Thomas D. Friedrich b, Svetlana Dobritsa a, Kevin Fitzpatrick a,
Peter R. Guzzo a, Douglas B. Kitchen a, Michael A. Lynch a, Denise Peace b, Mohammed Sajad a,
Alexander Usyatinsky a, Mark A. Wolf a,
*
a AMRI, 26 Corporate Circle, PO Box 15098, Albany, NY 12212-5098, USA
b Albany Medical College, 43 New Scotland Avenue, Albany, NY 12208, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2010
Revised 5 April 2010
Accepted 7 April 2010
Available online 11 April 2010
An indazole based series of glucocorticoid receptor agonists is reported. The SAR exploration of this scaf-
fold yielded compounds with nanomolar affinity for the glucocorticoid receptor with indications of selec-
tivity for the preferred transrepression mechanism; in vivo efficacy was observed in the mouse LPS
induced TNFa model for compound 28.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Glucocorticoid receptor agonists
Transrepression
Transactivation
Anti-inflammatory
Indazoles
Glucocorticoid receptor (GR) agonists are highly effective anti-
inflammatory drugs that have been used for more than 50 years.
All of the marketed GR agonists are modified steroids. Orally
administered GR agonists, such as dexamethasone (DEX, 1) and
prednisolone (PRED, 2) (Fig. 1), are highly efficacious in combating
inflammation; however, their use is tempered by the many side ef-
fects that all of these drugs induce.1 The risk of developing one or
more side effects such as osteoporosis, muscle wasting, skin thin-
ning and others, increases with the higher doses of GR agonists
and/or prolonged therapy.
Studies have revealed much about two GR mechanisms desig-
nated transactivation and transrepression.2 It is widely proposed
that the anti-inflammatory effects of glucocorticoid modulators
are largely mediated through transrepression. In the transrepres-
sion mechanism, ligand bound GR interacts with other transcrip-
with the expectation that they may provide a superior drug ther-
apy with reduced side effects for patients. There have been reports
by several drug discovery groups of selective glucocorticoid ago-
nists sometimes referred to as SEGRAs.3
A rational design approach was undertaken based upon known
interactions from crystal structures of GR with steroidal ligands
and the variety of non-steroidal ligands that had been reported.
This approach led to the discovery of hit 3 shown in Figure 1. While
only showing modest binding to the GR (IC50 = 2.1 lM), 3 provided
a good starting point for further exploration. Similar structures
were subsequently disclosed by another group4 working in this
area which has prompted us to disclose our results. To the best
of our knowledge this is the first presentation of SAR data and bio-
logical activity for this scaffold.
The crystal structures of both agonist (PDB: 1p93) and antago-
nist (PDB: 1nhz) forms of the glucocorticoid receptor5 allowed
integration of a docking model into the discovery effort. A picture
of 3 fitted to the modeled GR site is shown in Figure 2 below. A
tional factors, specifically AP-1 and NF
post-transcriptional activity. Many pro-inflammatory genes are
regulated through the AP-1 and NF B pathways, thus providing
jD, inhibiting their
j
the underlying rationale of how transrepression is a key to the
anti-inflammation properties of glucocorticoid modulators. Alter-
natively, it has been suggested that transactivation is responsible
for many unwanted side effects. Thus, we sought non-steroidal
glucocorticoid modulators which selectively act through the trans-
repression mechanism relative to the transactivation mechanism
OH
O
OH
R'
HO CH3
HO
H
N
N
R
H
F
O
1
3
dexamethasone , R = F, R' = Me
prednisolone 2, R = H, R' = H
F
* Corresponding author. Tel.: +1 518 512 2348.
E-mail address: mark.wolf@amriglobal.com (M.A. Wolf).
Figure 1. Known steroidal GR agonists and initial hit molecule.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.012

3018
M. Bai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3017–3020
Table 1
Structure–activity relationships at the 5-position
R
OH
N
N
F
F
Compound
R
GR binding assay IC50 (nM) mean SD
11
3
H
>10,000
2113
161 76
86 43
>3000
CH3
CF3
CF3
CF3
(+/À)-12ab
(À)-12a
(+)-12b
binding affinity using a fluorescence polarization (FP) competitive
binding assay.11
Figure 2. Model of initial hit 3 binding to GR.
Varying substituents on the quaternary carbon at the 5-position
led to profound changes in GR binding affinity. As can be seen in
Table 1 below the introduction of the trifluoromethyl group
(12ab) was extremely favorable for increasing binding affinity.
The trifluoromethyl moiety has also proven beneficial for other
GR ligands.12 The separate enantiomers of 12ab were isolated
using chiral HPLC. Testing showed that there was a preference
for one enantiomer, with the (À)-enantiomer (IC50 = 86 nM) being
most active.
Variation in the 5-arylmethyl side-chain focused on the phenyl
ring substituents. A subset of compounds illustrating the exem-
plary SAR at this position is found in Table 2 below. For ease of
comparison, all compounds listed in Table 2 are racemic mixtures.
Following an optimization strategy using the Topliss opera-
tional scheme13 (data not shown) it was discovered that 3-substi-
tuted phenyl rings generally demonstrated better GR binding
affinity as illustrated by comparison of compounds 13, 15, and
20. However, there were exceptions such as the chlorophenyl ana-
logues (16 vs 22) where substitution at the 2-position was favored.
3,4-Disubstitution with halogens in the 4-position were tolerated
(14, 18, and 19). Addition of a 4-fluoro substituent with 3-substitu-
tions at least maintained affinity seen in the corresponding 3-mon-
osubstituted analogues (16 and 18, 17 and 19).
preference for the fluorophenyl moiety to be in close proximity
with the Arg-611 and Gln-570 residues was observed. Close exam-
ination of the binding pocket shows a number of hydrophobic res-
idues (Met-646, Phe-623, Met-601, Leu-732, etc.) closely
encapsulating 3. Figure 2 shows 3 with a van der Waals surface
added to highlight its proximity to the surrounding amino acid res-
idues. Using our docking model, the tertiary alcohol of 3 forms a
hydrogen bond with Glu-642 (suggesting that this hydroxyl does
not correspond to the secondary hydroxyl at C-11 of DEX, which
has been shown to interact with Asn-564).6 The docking model
indicated that larger substituents off of the chiral carbon could take
advantage of increased hydrophobic interactions.
The synthetic routes illustrated in Scheme 1 were used to pre-
pare the indazole analogues.7 Benzaldehyde 4 was reacted with
4-fluorophenylhydrazine to give hydrazone 5. Hydrazone 5 was
then cyclized to provide indazole 6.8 The versatility of 5-bromo-
1-(4-fluorophenyl)-1H-indazole (6) allowed for the synthesis of
numerous analogues in as few as 5–6 synthetic steps. Key interme-
diate (7) was prepared by palladium catalyzed carbonylation.9 The
addition of organometallic reagents (e.g., Grignard or organolith-
ium) introduced the variable R group to provide secondary alcohols
8. Following oxidation of 8 the trifluoromethyl moiety was intro-
duced10 to prepare the targeted tertiary substituted alcohols 10.
These analogues were first tested to determine their human GR
Docking experiments of a virtual library exploring this class of
analogues indicated that compounds with 2-substituted aryl rings
H
F
H
F
Table 2
H
N
Br
Br
Br
a
b
Structure–activity relationships at 5-arylmethyl position
O
N
F
HO CF3
4
5
N
N
OHC
R
c
d
N
N
N
N
6
7
9
F
F
F
Compound
R
GR binding assay IC50 (nM) mean SD
12
13
14
15
16
17
18
19
20
21
22
23
4-F
161 76
2089
178
52
72 57
99
117
30
282
54
OH
O
R
OH
F3C
R
4-Me
3,4-Cl2
3-Me
3-Cl
3-CN
3-Cl-4-F
3-CN-4-F
2-Me
2-F
f
R
e
N
N
N
N
N
N
8
10
F
F
F
Scheme 1. Reagents and conditions: (a) 4-Fluorophenylhydrazine, diisopropyleth-
ylamine, CH2Cl2, rt, 16 h, 99%; (b) K2CO3, N-methylpyrrolidinone, 185 °C, 67%; (c)
Pd(Ph3P)4, NaCO2H, CO (250 psi), DMF, 110 °C, 37%; (d) RM THF, 0 °C, 1 h, 55–85%;
(e) PCC, CH2Cl2, rt, 3 h, 55–90%; (f) (i) (trifluoromethyl)trimethylsilane, THF, rt, 3 h;
(ii) TBAF, rt, 1 h, 35–77%.
2-Cl
2,3-Cl2
35
87 11

M. Bai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3017–3020
3019
Table 3
In vitro activity profile of leading compounds
HO CF3
HO CF3
HO CF3
N
N
N
N
N
N
N
F
F
Cl
Cl
CN
24
25
26
(-)-Enantiomer
(-)-Enantiomer
(-)-Enantiomer
F
F
F
HO CF3
HO CF3
N
N
N
N
F
Cl
CH3
Cl
27
(-)-Enantiomer
28
(+)-Enantiomer
F
F
Compound
GR affinity
Transrepression IL-6 assay
EC50 (nM) mean SD
Transactivation (MMTV-Luc)
50% Point (nM) % DEX (conc)
IC50 (nM) mean SD
% DEX
24
25
26
27
28
2
14 16
49 21
54 32
59 23
627 256
93 21
112 37
60
85
90
87
87
94
NA
ca. 1100
NA
NA
ca. 350
55
42% (3.3
57% (3.3
47% (3.3
47% (3.3
70% (3.3
99% (3.3
lM)
lM)
lM)
lM)
lM)
lM)
93
19
3
4
34
7
8
4
3.0
fit the receptor well. As a result both 2- and 2,3-disubstituted ana-
logues were prepared and screened. The 2,3-dichlorophenyl ana-
logue 23 demonstrated good binding affinity with an IC50 of 87 nM.
The most active racemates were separated to provide the indi-
vidual enantiomers. The active enantiomers, as determined by the
binding assay, were screened to determine their functional activ-
ity. Selectivity for transrepression over transactivation was exam-
ined using in vitro IL-614 and MMTV-luciferase (LUC)15 assays.
The agonist activity is reported as a percentage of the response in-
duced by DEX. Prednisolone is included for comparison and the re-
sults are found in Table 3.
ity, solubility, CYP inhibition, etc.) data not shown. The stability of
compound 28 in acid was examined and the enantiomeric purity of
compound 28 did not change upon exposure to 1 N HCl at 37 °C for
1 h; consequently it was selected for study in the inflammation LPS
challenge model. Figure 3A shows that 28 demonstrated significant
TNFa
reduction relative to control at 3 mg/kg (25% reduction). The
2500
2000
Initial results from the lead series showed that binding affinity
for GR did not always translate into the desired functional activity
(e.g., compound 24, Table 3). An increased understanding of the
SAR for the series drove improvements in activity seen in the trans-
repression (IL-6) assay (compounds 25–28) and a better translation
of binding affinity to functional IL-6 repression was observed.
These compounds all achieve P85% of the functional activity of
DEX in the IL-6 assay and are less active in the MMTV-LUC assay,
never reaching the same levels of activation seen with DEX or
PRED, indicating enhanced selectivity for transrepression over
transactivation versus current medications.
1500
*
1000
**
**
**
**
**
500
0
0.0 0.1 0.3 1.0 3.0 10.0 30.0 60.0 100.0
dose (mg/kg)
* p< 0.05 relative to control
** p< 0.01 relative to control
The five lead compounds (24–28) were profiled against a panel
of nuclear hormone receptors including estrogen (ER
a and ERb,
progesterone (PR-B), and androgen (testosterone) receptors.16 All
compounds showed >1000-fold selectivity for GR over these recep-
tors. Additionally, these compounds were tested for off-target
activity against 29 various receptors and ion channels.17 No signif-
150
125
100
75
icant off-target activities (% inhibition <50% at 1
l
M), including
M),18 were noted
*
interaction with the hERG channel (IC50 >10
for these compounds.
l
In order to demonstrate proof of concept for this series of non-
steroidal GR agonists, a mouse lipopolysaccharide (LPS) challenge
model of inflammation was employed. It is known that LPS chal-
lenge induces an upregulation of pro-inflammatory cytokines such
50
**
**
**
**
PRED
**
25
0
as TNF
a
in mice.19 Known GR agonists, such as PRED, have been
0.1
1
10
100
dose (mg/kg)
P < 0.05 relative to control
** P < 0.01 relative to control
shown to down regulate this expression resulting in decreased
inflammation.
Compound 28 possessed the most favorable in vitro profile of
the lead compounds, including ADME assays (ex. metabolic stabil-
*
Figure 3. Compound 28 in LPS challenge model.

3020
M. Bai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3017–3020
cells per well in 50 lL of the growth medium and incubated at 37 °C with 5%
maximum reduction observed was 68% at 60 mg/kg. At 30 mg/kg it
achieved similar results to that of PRED at 3 mg/kg. The dose re-
sponse curve fitting of this data (Fig. 3B) shows that 50% inhibition
of TNFa expression relative to control is obtained around 6 mg/kg.
This experiment demonstrates that 28 is effective at suppressing
CO2 for 24 h. Test compounds (10 mM in 100% DMSO) were serially diluted at
100Â assay concentrations in 100% DMSO and further diluted to 1.34Â assay
concentrations with Dulbecco’s Modified Eagle’s Medium (Fisher Scientific,
Cat. No. SH3002202) supplemented with 1.75% bovine serum albumin (Sigma,
Cat. No. A9647) and 1% penicillin-streptomycin solution (Fisher Scientific, Cat.
No. SV30010). The growth medium in the cell plates was replaced with 30 lL
of the assay medium containing test compounds, with samples done in
inflammatory mediators in vivo.
quadruplicates. After cells were pre-treated with compounds for 1 h at 37 °C,
10 lL of 4 ng/mL IL-1b (Cytoshop, Cat. No. CYT-208) in assay medium was
added to give a final IL-1b concentration of 1 ng/mL. Positive control wells
received IL-1b but no test compounds and represented maximum (or 100%)
production of IL-6. Background control wells did not receive IL-1bAdditional
In summary, (i) a rationally designed series has yielded com-
pounds with nanomolar affinity for the glucocorticoid receptor
with indications of selectivity for the preferred transrepression
mechanism; and, (ii) in vivo efficacy was observed in the mouse
control wells received either 100 or 2 nM DEX as
a standard treatment
LPS induced TNFa model for compound 28. In addition to the posi-
resulting in 100% or 50% inhibition of IL-6 production, respectively. The final
DMSO concentration in all wells was 1%. Plates were incubated for an
additional 24 h, and supernatants were harvested at the end of incubation and
diluted eightfold with Dulbecco’s Phosphate Buffered Saline (Fisher Scientific,
Cat. No. SH3025602). IL-6 levels in the supernatants were determined with a
Human Interleukin-6 Assay kit (Cisbio International, Cat. No. 62IL6PEB),
according to manufacturer’s instructions. IC50 values were determined using
XLfit4.1 curve fitting software. A cell viability assay using Alamar Blue™ was
duplexed with the IL-6 repression assay to eliminate cytotoxic compounds.
15. MDA-kb2 cells (ATCC, Cat. No. CRL-2713) were cultured in Leibovitz’s L-15
medium with phenol red (Cellgro, Cat. No. 10-045-CV), supplemented with
tive in vitro and in vivo results, the profiling of these compounds
against other nuclear hormone receptors, off-target receptors and
ion channels shows excellent selectivity. The data indicates that
these compounds represent an exciting new lead series for prepar-
ing selective glucocorticoid receptor agonists.
References and notes
10% Fetal Bovine Serum (FBS, Cellgro, Cat. No. 35-010-CV) and 2 mM
glutamine (Cellgro, Cat. No. 25-005-C1), at 37 °C, without CO2. Cells were
plated on 384-well plates at 10,000 cells per well in 40 L of L-15 medium
without phenol red (GIBCO, Cat. No. 21083-027), supplemented with 10% FBS
and 2 mM -glutamine, and incubated at 37 °C without CO2 for 5 h. Test
L-
1. (a) Schäcke, H.; Döcke, W. D.; Asadullah, K. Pharmacol. Ther. 2002, 96, 23; (b)
Schulz, M.; Eggert, M. Curr. Pharm. Des. 2004, 10, 2817.
2. (a) Schäcke, H.; Schottelius, A.; Döcke, W. F.; Strehlke, P.; Jaroch, S.; Shmees, N.;
Rehwinkel, H.; Hennekes, H.; Asadullah, K. Proc. Natl. Acad. Sci. 2004, 101, 227;
(b) Rhen, T.; Cidlowski, J. A. N. Eng. J. Med. 2005, 335, 1711.
l
L
compounds (10 mM in 100% DMSO) were serially diluted at 100Â assay
concentrations in 100% DMSO and further diluted to 5Â assay concentrations
with the above plating medium, supplemented with hydroxyflutamide (OHF,
Toronto Research Chemicals, Cat. No. H942475), an antagonist of the androgen
3. Representative references include: (a) Elmore, S. W.; Pratt, J. K.; Coghlan, M. J.;
Mao, Y.; Green, B. E.; Anderson, D. D.; Stashko, M. A.; Lin, C. W.; Falls, D.;
Nakane, M.; Miller, L.; Tyree, C. M.; Miner, J. N.; Lane, B. Bioorg. Med. Chem. Lett.
2004, 14, 1721; (b) Kuzmich, D.; Kirrane, T.; Proudfoot, J.; Bekkali, Y.; Zindell,
R.; Beck, L.; Nelson, R.; Shih, C.-K.; Kukulka, A. J.; Paw, Z.; Reilly, P.; Deleon, R.;
Cardozo, M.; Nabozny, G.; Thomson, D. Bioorg. Med. Chem. Lett. 2007, 17, 5025;
(c) Smith, C. J.; Ali, A.; Balkovec, J. M.; Graham, D. W.; Hammond, M. L.; Patel, G.
F.; Rouen, G. P.; Smith, S. K.; Tata, J. R.; Einstein, M.; Ge, L.; Harris, G. S.; Kelly, T.
M.; Mazur, P.; Thompson, C. M.; Wang, C. F.; Williamson, J. M.; Miller, D. K.;
Pandit, S.; Santoro, J. C.; Sitlani, A.; Yamin, T.-T. D.; O’Neill, E. A.; Zaller, D. M.;
Carballo-Jane, E.; Forrest, M. J.; Luell, S. Bioorg. Med. Chem. Lett. 2005, 15, 2926;
(d) Barker, M.; Clackers, M.; Copley, R.; Demaine, D. A.; Humphreys, D.; Inglis,
G. G. A.; Johnston, M. J.; Jones, H. T.; Haase, M. V.; House, D.; Loiseau, R.; Nisbet,
L.; Pacquet, F.; Skone, P. A.; Shanahan, S. E.; Tape, D.; Vinader, V. M.;
Washington, M.; Uings, I.; Upton, R.; McLay, I. M.; Macdonald, S. J. F. J. Med.
Chem. 2006, 49, 4216.
receptor also present in MDA-kb2 cells. 10
mixtures were added to the cells. Negative control wells received no test
compounds; positive control wells received DEX. The final DMSO
concentration in all wells was 1%; the final OHF concentration was 1 M.
Plates were incubated for an additional 20 h and then lysed with 30 L of
Luciferin lysis buffer containing 0.1 M Tris–HCl, pH 7.8, 2 mM MgCl2, 0.6%
Triton X-100 (Sigma, Cat. No. T9284), 10 mM DTT, 1 mM coenzyme
(Calbiochem, Cat. No. 234101), 0.3 mM ATP (Amersham, Cat. No. 27-1006-
01) and 280 g/mL luciferin (Sigma, Cat. No. L6882). Luminescence was
lL of 5Â test compound/5 lM OHF
2 lM
l
l
A
l
measured in a CLIPR plate reader (Molecular Devices).
16. (a) MDS Pharma provided the off target nuclear hormone receptor testing for
these compounds (cat. Nos. 226010, 226050, 268020, 285010).; ERa/ERb: (b)
Obourn, J. D.; Koszewski, N. J.; Notides, A. C. Biochemistry 1993, 32, 6229; PR-B:
(c) Carbajo, P.; Christensen, K.; Edwards, D. P.; Skafar, D. F. Endocrinology 1996,
137, 2339; Testosterone: (d) Chang, C.; Liao, S. J. Steroid Biochem. 1987, 27, 123;
(e) Traish, A. M.; Muller, R. E.; Wotiz, H. H. Endocrinology 1986, 118, 1327.
17. MDS Pharma provided broad screening in their ‘Hit Profiling Screen’. This panel
consists of the following 29 receptors and ion channels: Adenosine A1,
4. Mugge, Ingo A.; Burke, Michael J.; Ralph, Mark S.; Thomson, David S.;
Hammach, Abdelhakim.; Kowalski, Jennifer A.; Bentzien, Joerg M. WO2006/
135826.
5. Kauppi, B.; Jakob, C.; Farnegardh, M.; Yang, J.; Ahola, H.; Alarcon, M.; Calles, K.;
Engstrom, O.; Harlan, J.; Muchmore, S.; Ramqvist, A. K.; Thorell, S.; Ohman, L.;
Greer, J.; Gustafsson, J. A.; Carlstedt-Duke, J.; Carlquist, M. J. Biol. Chem. 2003,
278, 22748.
6. Bledsoe, R. K.; Montana, V. G.; Stanley, T. B.; Delves, C. J.; Apolito, C. J.; McKee,
D. D.; Consler, T. G.; Parks, D. J.; Stewart, E. L.; Wilson, T. M.; Lambert, M. H.;
Moore, J. T.; Pearce, K. H.; Xu, H. E. Cell 2002, 110, 93.
7. These routes provided racemic products that were separated by chiral HPLC to
provide single enantiomers. The typical purity standards met by these
compounds were >95% purity by HPLC analysis with diagnostic 1H NMR,
mass spectral and elemental analysis.
Adenosine A2A, Adrenergic
b1, Adrenergic b2, Calcium Channel L-Type (Dihydropyridine), Dopamine D1,
Dopamine D2S GABAA (Agonist Site), GABAA (Benzodiazepine, Central),
a1A, Adrenergic a1B, Adrenergic a2A, Adrenergic
,
Glucocorticoid, Glutamate (NMDA, Phencyclidine), Histamine H1, Imidazoline
I2 (Central), Muscarinic M2, Muscarinic M3, Nicotinic Acetylcholine, Nicotinic
Acetylcholine (Bungarotoxin-Sensitive, Neuromuscular), Opiate
l (OP3, MOP),
Phorbol Ester, Potassium Channel ([KATP]), Potassium Channel HERG,
Prostanoid EP4, Rolipram, Sigma r1, Sigma r2, Sodium Channel (Site 2),
Norepinephrine transporter.
8. Halley, F.; Sava, X. Synth. Commun. 1997, 27, 1199.
18. (a) Functional hERG activity was assessed using the patch clamp methodology
in HEK cells with testing by MDS Pharma Services. The test compounds were
tested in triplicate in a 5-point dose response curve.; (b) Zhou, Z.; Gong, Q.; Ye,
B.; Fan, Z.; Makielski, J. C.; Robertson, G. A. Biophys. J. 1998, 74, 230.
19. (a) Gonzales, J. C.; Johnson, D. C.; Morrison, D. C.; Freudenberg, M. A.; Galanos,
C.; Silverstein, R. Infect. Immun. 1993, 613, 970; (b) Amjad, A.; Thompson, C. F.;
Balkovec, J. M.; Graham, D. W.; Hammond, M. L.; Quraishi, N.; Tata, J. R.;
Einstein, M.; Ge, L.; Harris, G.; Kelly, T. M.; Mazur, P.; Pandit, S.; Santoro, J.;
Sitlani, A.; Wang, C.; Williamson, J.; Miller, D. K.; Thompson, C. M.; Zaller, D. M.;
Forrest, M. J.; Carballo-Jane, E.; Luell, S. J. Med. Chem. 2004, 47, 2441.
9. Schoenberg, A.; Heck, R. F. J. Am. Chem. Soc. 1974, 96, 7761.
10. Prakash, G. K. S.; Krishnamurti, R.; Olah, G. A. J. Am. Chem. Soc. 1989, 111, 393.
11. PolarScreen™ Glucocorticoid Receptor Competitor Assay Kit, Green
(Invitrogen, Cat. No. P2816). Activity curves were fitted and IC50 values
calculated using XLfit4.1 curve fitting software from IDBS Ltd.
12. Lee, T. W.; Proudfoot, J. R.; Tomson, D. S. Bioorg. Med. Chem. Lett. 2006, 16, 654.
13. Topliss, J. G. J. Med. Chem. 1977, 20, 463.
14. Normal human dermal fibroblast cells (Cambrex, Cat. No. CC-2511) were
cultured in Fibroblast Growth Medium
(Cambrex, Cat. No. CC-3132). Cells were plated on 384-well plates at 2500
2
with BulletKitÒ supplements