Novel ketal ligands for the glucocorticoid receptor: In vitro and in vivo activity

Article abstract of DOI:10.1016/j.bmcl.2005.03.027

A novel series of selective ligands for the human glucocorticoid receptor is described. Structure-activity studies focused on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands fo

Full text of DOI:10.1016/j.bmcl.2005.03.027

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2926–2931
Novel ketal ligands for the glucocorticoid receptor: in vitro
and in vivo activity
Cameron J. Smith,a,* Amjad Ali,a James M. Balkovec,a Donald W. Graham,a
Milton L. Hammond,a Gool F. Patel,a Gregory P. Rouen,a Scott K. Smith,a
James R. Tata,a Monica Einstein,b Lan Ge,b Georgianna S. Harris,b Theresa M. Kelly,b
Paul Mazur,b Chris M. Thompson,b Chuanlin F. Wang,b Joanne M. Williamson,b
Douglas K. Miller,c Shilpa Pandit,c Joseph C. Santoro,c Ayesha Sitlani,c
Ting-ting D. Yamin,c Edward A. OÕNeill,d Dennis M. Zaller,d
Ester Carballo-Jane,e Michael J. Forreste and Silvi Luelle
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Immunology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
eDepartment of Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 5 January 2005; revised 4 March 2005; accepted 7 March 2005
Available online 29 April 2005
Abstract—A novel series of selective ligands for the human glucocorticoid receptor is described. Structure–activity studies focused
on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands for
GR and have partial agonist profiles in functional assays for transactivation (TAT, GS) and transrepression (IL-6). Of these com-
pounds, 27, 28, and 35 were evaluated further in a mouse LPS-induced TNF-a secretion model. Compound 28 had an ED50 of
14.1 mg/kg compared with 0.5 mg/kg for prednisolone in the same assay.
Ó 2005 Elsevier Ltd. All rights reserved.
A variety of inflammatory diseases such as asthma,
rheumatoid arthritis, CrohnÕs disease, and allergic rhini-
tis are treated by synthetic glucocorticoids such as
dexamethasone (1) and prednisolone (2) (Fig. 1).1,2
Associated with these diseases is an increased expression
of pro-inflammatory cytokines such as interleukin-6 (IL-
6), and tumor necrosis factor-a (TNF-a).2 The produc-
tion of the inflammatory cytokine IL-6 has been shown
to be mediated by the glucocorticoid receptor (GR) at
the transcriptional level.2 Glucocorticoid use inhibits
the production of inflammatory cytokines but unfortu-
nately, is also associated with many side effects including
osteoporosis, hyperglycemia, lipid redistribution, and
muscle atrophy.3 Some of these effects arise from tran-
Keywords: Glucocorticoid; Dissociation; Transrepression; Transacti-
vation; Inflammation.
*
Corresponding author. Tel.: +1 732 594 2529; fax: +1 732 594
9545; e-mail: cameron_smith@merck.com
Figure 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.027

C. J. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2926–2931
2927
scription of genes such as tyrosine amino transferase
(TAT), a metabolic gene involved in gluconeogenesis
primarily found in the liver,4 and glutamine synthetase
(GS), an enzyme involved in the regulation of skeletal
muscle that catalyzes the conversion of glutamate to glu-
tamine.3 Glucocorticoid-mediated induction of TAT
and GS causes the side effects hyperglycemia and muscle
wasting, respectively.3 Consequently, it is of therapeutic
interest to discover selective GR ligands that retain anti-
inflammatory properties but have diminished side effect
profiles, and a number of laboratories are active in this
field.5–14
The mechanism3,15,16 of glucocorticoid action is initiated
by binding of the ligand to GR. The resulting GR/ligand
complex can then dimerize and bind to DNA sequences
known as glucocorticoid response elements (GREs) thus
initiating the transcription of genes associated with the
side effects of glucocorticoid use. Alternatively, as a
monomer, the GR/ligand complex can bind to transcrip-
tion factors such as nuclear factor jb (NFjb) and acti-
vator protein-1 (AP-1), thereby repressing their effect on
transcription of pro-inflammatory cytokines like IL-6
and TNF-a. The development of anti-inflammatory
drugs with reduced side effect profiles may be facilitated
by the identification of compounds that repress tran-
scription of inflammatory cytokines without activating
GREs. Such compounds are often referred to as Ôdisso-
ciatedÕ compounds.16
˚
Scheme 1. Reagents and conditions: (a) HOCH2CH2OH, p-TsOH, 4 A
mol sieves, rt, 30 min, 93%; (b) HCO2Et, NaH, C6H6, rt, 3 h, 93%; (c)
4-fluorophenyl hydrazine, NaOAc, HOAc, rt, 24 h, 41%; (d) 6 N HCl,
THF, 65 °C, 5 h, 100%; (e) diol, p-TsOH, C6H6, reflux, 12–24 h, 50–
95%.
selectively protected as ketals 9 and 1025 then formylated
to give 11 and 12, respectively. Condensation with 4-flu-
orophenylhydrazine then afforded N-arylpyrazole inter-
mediates 13 and 5. Hydrolysis of ketals 13 and 5
required 6 N aq HCl in THF at 65 °C to afford ketones
14 and 15. Substituted ketals 16 and 17 were synthesized
from the appropriate ketone 14 or 15 and substituted
diol by heating at reflux with catalytic p-toluenesulfonic
acid in benzene under a Dean–Stark apparatus. Diols
were commercially available except where noted and
were either racemic or enantiopure. If diastereoisomers
were produced they were separated using flash chroma-
tography and/or chiral HPLC and characterized using
NMR spectroscopy (2D-NOESY studies).
The development of pyrazolosteroids such as cortivazol
(3) and fluoro-cortivazol (4) evolved from the clinically
successful steroids dexamethasone (1) and prednisolone
(2) (Fig. 1).17,18 These compounds have an N-aryl pyraz-
ole ring fused to the A-ring of the steroid backbone.
This structural moiety gave these compounds enhanced
GR binding potency and selectivity over other nuclear
hormone receptors and improved in vivo activity. Struc-
tural diversity between synthetic glucocorticoids occurs
mainly in the region of space occupied by the C- and
D-rings. In an effort to prepare compounds that bind
selectively to GR and show improved anti-inflammatory
properties in vivo, compounds were designed with an N-
aryl pyrazole moiety fused to the conventional A- and
B-rings of a steroid backbone, and structural diversity
in the region of space usually occupied by the C- and
D-rings.
Initially, compounds were assayed for levels of binding
potency in a competition binding assay with radio-
labeled dexamethasone for the a-isoform of the human
GR.19 Steroid receptor selectivity was determined by
counter-screening against other nuclear hormone recep-
tors including progesterone (PR), mineralocorticoid
(MR), and estrogen (ER-a and ER-b) receptors.19
The functional activity of compounds in either activa-
tion or repression of transcription was evaluated using
cell-based assays. Repression of transcription or Ôtrans-
repressionÕ was determined by measuring IL-6 inhibi-
tion in both human A549 lung epithelial cells and
We recently reported19 a ketal intermediate (5) (Fig. 1)
used in the synthesis of a series of GR ligands that
was found to bind to GR with a reasonable affinity. Be-
cause clinically relevant glucocorticoids that contain a
ketal functionality are known (e.g., triamicinolone ace-
tonide (6) (Fig. 1)20), we wished to investigate this obser-
vation further. We decided to synthesize a series of
ketals varying first the ketal substituents and later the
ketal and B-ring sizes.
mouse
thioglycolate-elicited
peritoneal
exudate
cells.26–28 Activation of transcription or Ôtransactiva-
tionÕ was determined by measuring induction of TAT
in human HepG2 cells,4,29 and GS in both human skel-
etal muscle cells and mouse C2C12 cells.30–32 The
above cell-based assays were used to guide SAR devel-
opment with the goal of minimizing transactivation
and maximizing transrepression effects. Both human
and mouse cell lines were used for in vitro screening
The key intermediate ketones required were synthesized
as shown in Scheme 1 starting with either enantiopure
Hajos–Parrish ketone (7, five-membered B-ring, n =
1)21,22 or Wieland–Miescher ketone (8, six-membered
B-ring, n = 2),23,24 respectively. These ketones were

2928
C. J. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2926–2931
because the in vivo LPS-induced TNF-a secretion
model33 used to further characterize compounds that
showed reasonable levels of potency and dissociation
between transactivation and transrepression assays,
was conducted in a mouse.
transrepression assays. Bis-propenyl ketal 23 was of
comparable potency to 22 in the transrepression assays
but increases in the levels of transactivation of TAT
and GS were observed. Substituents such as phenyl
(compound 24) and increasing the ketal ring size to 6
(25 vs 18) were surprisingly well tolerated.
The first ketals investigated were synthesized using the
6,6-A,B-ring system (n = 2). Initially, symmetrically
disubstituted 1,2- and 1,3-diols were chosen for ketal
synthesis to eliminate the potential for formation of
multiple ketal diastereoisomers, thus complicating puri-
fication and analysis. In general, GR was not tolerant of
bulky, polar substituents and smaller lipophillic groups
were preferred. A comparison of the GR binding po-
tency and IL-6 repression for compounds 18, 19, 20,
and 21 showed that the optimal chain length was ethyl
(Table 1). The addition of allylic double bonds (com-
pound 22) increased potency in both the binding and
A selection of these compounds were synthesized in the
6,5-A,B-ring system (n = 1, Table 1). In general these
compounds were found to have comparable GR binding
and transactivation potency relative to those in the 6,6-
A,B-ring system but were slightly more potent in the
transrepression assays. Compounds 27 and 28 showed
good levels of dissociation between the transactivation
and transrepression assays. The bis-propenyl ketal (29)
was about as potent as dexamethasone in the trans-
repression assays, but suffered from higher levels of
transactivation of GS and TAT than other ketals in this
Table 1. SAR of disubstituted ketals
Compd
R
m
n
hGRa
hTATb,c
hGSd,c
hIL-6e,c
mIL-6f,c
IC50 (nM) EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex
2
—
—
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
—
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
13.8
14
211
81
5
n.m.i
n.m.i
8
32
97
2
4.5
n.d.h
n.d.h
19
102
5.7
n.d.h
n.d.h
78
n.m.i
n.m.i
8.7
95
25h
47h
79
n.m.i
n.m.i
81
5
H
Me
n.d.g
n.m.i
n.m.i
n.d.g
n.d.g
n.d.g
n.d.g
n.m.i
n.m.i
n.d.g
n.d.g
n.d.g
n.d.g
164
n.d.g
n.m.i
n.d.g
29
7h
18
19j
20l
21l
22
23m
24
25
13
26
27k
28
29m
30
31
32
10
n.m.i
11
58
54h
14
25
6
21h
32
10h
Et
n-Pr
5.5
8.9
22
n.d.h
n-Bu
Vinyl
1-Propenyl
Ph
1
n.d.h
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
17
92
21
65
1.2
2.5
10
12
16
n.m.i
n.m.i
3
5.3
7.8
67
37
79
n.d.h
n.d.h
28
23h
27h
69
26
66
Me
8.3
73
0
n.d.h
n.d.h
n.d.h
36
40h
17h
40h
62
H
Me
0
24
6
3
n.d.g
24
30
75
Et
6.6
8.0
2.7
26
11
11
74
3
7
Vinyl
1-Propenyl
CH2OCH3
Ph
5
2.6
6.5
58
93
61
56
45
23
0
7.6
111
69
n.d.g
n.d.g
n.d.g
n.d.g
8.3
62
67
10
60
10
19
9
10
n.d.g
10
85
72
Me
8.4
0
7.5
58
45
a Data reported is derived from duplicate wells and three independent experiments. Mean IC50 values were determined from 10-point, one-third log
concentration response curves and standard errors were 620%.
b Human tyrosine amino transferase (TAT) assay in HepG2 cells.
c Experiments were run in duplicate. EC50 values were determined from eight-point, one-third log concentration response curves and standard errors
were 620%.
d Human glutamine synthetase (GS) assay in skeletal muscle cells.
e Human IL-6 assay in A549 lung carcinoma cell line.
f Mouse macrophage IL-6 assay in peritoneal exudate cells harvested by gavage from C57B1/6 mice.
g n.d.: not determined; EC50 values not reported in cases where % dex 6 35%.
h % single point inhibition at 3 lM, hence EC50 not determined.
i n.m.: not measured.
j Compound synthesized from 22 by catalytic hydrogenation over 10% Pd/C in EtOAc.
k Compound synthesized from 28 by catalytic hydrogenation over 10% Pd/C in EtOAc.
l Diol synthesized by Sharpless asymmetric dihydroxylation from the corresponding trans-olefin as in Ref. 34.
m Diol synthesized by method adapted from Refs. 35 and 36.

C. J. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2926–2931
2929
Table 2. SAR of six-membered ketals
Compd R1 R2 R3 R4 R5 hGRa
hTATb,c
hGSd,c
hIL-6e,c
mIL-6f,c
IC50 (nM) EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex
33
34
32
35
36
37
38
39
H
H
H
H
H
H
H
Me
H
H
Me
H
H
18
21
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
4
4
9
8
9
8
2
5
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
0
15
0
0.48
0.53
7.5
46
35
58
60
64
73
44
59
n.d.h
n.d.h
45
38h
20h
72
Me
H
H
H
Me
H
Me
H
H
8.4
Me
Me
H
Me
H
7.1
3.1
7
13
54
69
H
Me
H
4
26
12
12
25
19
64
H
Me
H
28
6
n.d.h
n.d.h
18
35h
39h
77
H
Me Me
Me
2.2
2.1
0
15
Me Me
H
Legend as for Table 1.
series. Groups such as methoxymethyl (30) and phenyl
(31) were also tolerated.
functional activity appeared to be optimal with com-
pounds containing two methyl groups (32, 35, 36, and
37). A comparison of the asymmetrically substituted ke-
tals 38 and 39 showed that although both compounds
have similar GR binding potencies, transrepression activ-
ity was higher for ketal 39, which has an extra methyl
group projecting behind the plane of the molecule as
drawn. This trend was not followed with a comparison
of ketals 33 and 34, as ketal 33, with its solitary ketal
methyl group projecting in front of the plane of the mol-
ecule was more potent in the transrepression assays.
Increasing the ketal ring size to 6 in the 6,5-A,B-ring sys-
tem was tolerated well (m = 1, n = 1, Table 1). Com-
pound 32 retained good levels of dissociation and thus
warranted the further investigation of similar six-mem-
bered ketals on the 6,5-A,B-ring system (Table 2). A ser-
ies of six-membered methyl substituted ketals were
synthesized both varying the number of methyl groups
and substitution pattern and stereochemistry. These
compounds were synthesized from either racemic or
enantiopure 1,3-diols and where necessary, any ketal
diastereoisomers produced were separated by chroma-
tography and characterized by NMR spectroscopy
(2D-NOESY studies). Levels of GR binding potency in-
creased with the number of ketal methyl groups but
The effect of number of substituents and stereochemistry
was also investigated in the case of five-membered ketals
and the 6,5-A,B-ring system (Table 3). These com-
pounds were synthesized from either racemic or enantio-
pure 1,2-diols and the ketal diastereoisomers were
Table 3. SAR of monosubstituted five-membered ketals
Compd R1
R2
R3
R4
hGRa
hTATb,c
hGSd,c
hIL-6e,c
mIL-6f,c
IC50 (nM) EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex EC50 (nM) % dex
40
41
42
43
44
45
46
47
H
Vinyl
Ph
H
H
H
H
H
H
H
H
5.8
8.5
5.6
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
7
13
7
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
n.d.g
0
5
0
7
0
0
0
0
9.0
36
168
91
52
70
H
H
n.d.h
25
21h
47
Vinyl
Ph
H
H
183
57
H
H
12
2
n.d.h
n.d.g
n.d.h
n.d.h
n.d.h
2h
n.d.h
n.d.h
n.d.h
n.d.h
n.d.h
46h
27h
42h
35h
49h
H
Vinyl
Ph
H
38
26
4
32
H
H
13
4
0h
H
H
Vinyl 49
Ph 65
18h
20h
H
H
H
8
Legend as for Table 1.

2930
C. J. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2926–2931
separated by chiral HPLC and characterized by NMR
spectroscopy (2D-NOESY studies). Although these
compounds had far less functional activity than their
disubstituted counterparts in Table 1, there was a defi-
nite trend in potency and functional activity where com-
pounds with the R-group projecting back behind the
plane of the compound as drawn (40–43), were more po-
tent than compounds where the R-group is projected in
front of the plane (44–47). It therefore appears that in
the case of five-membered ketals, substitution at the car-
bon on the rear of the molecule has a greater contribu-
tion to potency than ketal substitution on the carbon on
the front face of the molecule.
was indicative of anti-inflammatory activity in animal
systems (Table 5). The mouse in vitro data for IL-6
transrepression and GS transactivation is also included
illustrating that these compounds are indeed dissociated
in vitro in mouse cells. Also shown is the pharmacoki-
netic data for 28 and 35. Compound 28 had a clearance
of 114 mL/min/kg, a half life of 2.3 h and an oral bio-
availability of 14%. In the LPS induced TNF-a secretion
model, compounds 28, 35, and 27 at an oral dose of
30 mg/kg showed good in vivo inhibition of TNF-a
secretion relative to that of dexamethasone, albeit at
an 100-fold lower oral dose of 0.3 mg/kg. A dose-depen-
dent inhibition of TNF-a secretion after oral dosing of
compound 28 relative to that of prednisolone is shown
in Figure 2. Compound 28 had an ED50 of 14.1 mg/kg
compared with 0.5 mg/kg of prednisolone in the same
assay.
A subset of the ketals was counter-screened against
other nuclear hormone receptors (PR, MR, and ER-a
and ER-b) to determine cross-reactivity. As shown in
Table 4, all of the compounds examined had excellent
selectivity and affinity toward the glucocorticoid
receptor.
In conclusion, a novel series of potent and selective non-
steroidal ligands has been discovered. The conventional
C- and D-rings of steroidal glucocorticoids can be re-
placed with a variety of substituted ketals of differing
ring size and the B-ring can be reduced in size to a
A subset of the above compounds was evaluated in an
LPS induced TNF-a secretion model33 in mice to deter-
mine whether the functional activity observed in vitro
Table 4. Binding selectivity toward GR relative to other nuclear
hormone receptors
Compd
hGRa
(nM)
hPRa
rMRa
hERaa
(lM)
hERba
(lM)
(lM)
(lM)
13
5
73
14
>1
>1
>10
7.56
>10
>10
7.40
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
26
28
22
35
24
>1
>1
8.0
1.2
7.1
>1
n.m.b
a Data reported is derived from duplicate wells and three independent
experiments. Mean IC50 values were determined from ten-point, one-
third log concentration response curves and standard errors were
620%.
b n.m.: not measured.
Figure 2. LPS induced TNF-a secretion model data for compound 28.
Table 5. In vivo data
Compd
mGSa,b
mIL-6c,b
IV PKd
PO PKe
% Inhibition
of TNF-af
EC50
(nM)
% dex
EC50
(nM)
% dex
Clg (mL/
min/kg)
Vdss
T1/2
AUCNj
AUCNj
F%k
h
i
(L/kg)
(h)
(lM h)
(lM h)
28
35
27
n.d.l
n.d.l
n.d.l
5
25
17
61
54
36
56
69
62
114.0
118.3
n.m.m
15.3
27.2
n.m.m
2.3
3.0
n.m.m
0.4
0.4
n.m.m
0.1
0.1
n.m.m
14.3
23.0
n.m.m
96.8
80.8
97.8
a Mouse glutamine synthetase (GS) assay in C2C12 cells.
b Experiments were run in duplicate. EC50 values were determined from eight-point, one-third log concentration response curves and standard errors
were 620%.
c Mouse macrophage IL-6 assay in peritoneal exudate cells harvested by gavage from C57B1/6 mice.
d IV pharmacokinetic study at a dose of 2 mg/kg.
e PO pharmacokinetic study at a dose of 10 mg/kg.
f Inhibition of TNF-a secretion reported for an oral dose of 30 mg/kg relative to that produced by dexamethasone at a dose of 0.3 mg/kg.
g Clearance.
h Volume of distribution.
i Half life.
j Normalized area under curve.
k Oral bioavailability.
l n.d.: not determined; EC50 values not reported in cases where %dex 6 35%.
m n.m.: not measured.

C. J. Smith et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2926–2931
2931
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five-membered ring. Symmetrically disubstituted ketals
have better profiles than their corresponding monosub-
stituted analogs. These compounds retain comparable
GR binding potency and steroid receptor selectivity in
vitro to that of marketed glucocorticosteroids. The few
compounds that showed elevated levels of transactiva-
tion also showed accompanying increases in levels of
transrepression. Generally however, most of the com-
pounds tested showed partial agonism in the transre-
pression assays and little to no agonism in the
transactivation assays. Although anti-inflammatory po-
tency in vivo is much reduced relative to marketed glu-
cocorticoids, these compounds show improved degrees
of dissociation between functional transactivation and
transrepression activities in vitro. Currently, mouse
models for characterizing the side effects of glucocorti-
coid use and consequently levels of dissociation in vivo
are not well established.6 Therefore, it remains to inves-
tigate whether these compounds will show a decrease in
the usual side effects of glucocorticoids when compared
with conventional steroids.
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