Novel glucocorticoids containing a 6,5-bicyclic core fused to a pyrazole ring: Synthesis, in vitro profile, molecular modeling studies, and in vivo experiments

Article abstract of DOI:10.1016/j.bmcl.2007.03.103

Chemistry was developed to synthesize the title series of compounds. The ability of these novel ligands to bind to the glucocorticoid receptor was investigated. These compounds were also tested in a series of functional assays and some were found to displ

Full text of DOI:10.1016/j.bmcl.2007.03.103

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3354–3361
Novel glucocorticoids containing a 6,5-bicyclic core fused
to a pyrazole ring: Synthesis, in vitro profile, molecular
modeling studies, and in vivo experiments
Christopher F. Thompson,a,* Nazia Quraishi,a Amjad Ali,a Ralph T. Mosley,a
James R. Tata,a Milton L. Hammond,a James M. Balkovec,a Monica Einstein,b Lan Ge,b
Georgianna Harris,b Terri M. Kelly,b Paul Mazur,b Shilpa Pandit,b Joseph Santoro,b
Ayesha Sitlani,b Chuanlin Wang,b Joanne Williamson,b Douglas K. Miller,c
Ting-ting D. Yamin,c Chris M. Thompson,c Edward A. O’Neill,c Dennis Zaller,c
Michael J. Forrest,d Ester Carballo-Janed and Silvi Luelld
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 Immunology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 15 December 2006; revised 28 March 2007; accepted 30 March 2007
Available online 5 April 2007
Abstract—Chemistry was developed to synthesize the title series of compounds. The ability of these novel ligands to bind to the
glucocorticoid receptor was investigated. These compounds were also tested in a series of functional assays and some were found
to display the profile of a dissociated glucocorticoid. The SAR of the 6,5-bicyclic series differed markedly from the previously
reported 6,6-series. Molecular modeling studies were employed to understand the conformational differences between the two series
of compounds, which may explain their divergent activity. Two compounds were profiled in vivo and shown to reduce inflammation
in a mouse model. An active metabolite is suspected in one case.
Ó 2007 Elsevier Ltd. All rights reserved.
Treatment of inflammatory disease remains a serious
medical challenge. Although a variety of pharmaceutical
agents are available to treat inflammation, none are
ideal and the search continues for new methods to com-
bat this affliction. Glucocorticoids (e.g., dexamethasone
and prednisolone) constitute one of the most powerful
and effective classes of agents for treating inflammation.
However, these drugs suffer from serious side effects.1–4
In this active state, GR can suppress pro-inflammatory
cytokines, such as IL-6 and TNF-a, leading to relief
from inflammation.1–4 However, the activated GR also
upregulates a number of cellular processes that produce
undesired side effects. This is particularly true when GCs
are administered systemically over prolonged periods of
time. Such extended treatment with artificially high lev-
els of GCs leads to a situation mimicking Cushing’s syn-
drome (endogenous corticoid excess) with complications
including muscle wasting, osteoporosis, and hyperglyce-
mia due to increased gluconeogenesis.5
Glucocorticoids (GCs) target the glucocorticoid recep-
tor (GR), which is a nuclear hormone receptor. Upon
binding to a GC ligand, GR undergoes a conforma-
tional change and translocates to the nucleus of the cell.
Both the desired anti-inflammatory effects and the unde-
sired side effects of GCs are mediated via GR. However,
recent scientific evidence indicates that anti-inflamma-
tory processes are mediated through a GR–GC monomer
interacting directly with proinflammatory transcription
factors including AP-1 and NF-jB. This process
is termed transrepression. In contrast, side-effects
Keywords: Glucocorticoid; Dissociation; Transrepression; Transacti-
vation; Inflammation.
*
Corresponding author. Tel.: +1 732 594 9537; fax: +1 732 594
9490; e-mail: christopher_thompson@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.103

C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
3355
apparently result from a GR–GC dimer binding to DNA
and upregulating certain metabolic genes, which is
known as transactivation.6–15 This hypothesis regarding
the dual nature of GR has allowed scientists to entertain
the possibility that a ‘dissociated’ GC could be
developed.16 A GC that favors the monomeric GR–GC
complex over the dimeric state could retain the desired
anti-inflammatory effects of GCs while eliminating side
effects. The discovery of such a dissociated GC has been
the goal of our research program.
and, more importantly, functional activity in the trans-
activation assay were similar. Based on this general
trend of improved transrepression activity for com-
pounds containing a 5-membered B-ring, an explora-
tion of this core structure was undertaken with
hydroxy-aromatic and heteroaromatic substituents on
the B-ring. It was also believed that compounds with
a 5-membered B-ring should have better pharmacoki-
netic properties as there are fewer sites available for
metabolism in the smaller ring. Hydroxy-aromatic
and heteroaromatic substituents have now been exam-
ined in the 6,5-system and the findings are reported in
this Letter.
Several recent papers from these laboratories have de-
scribed a novel GC platform consisting of a truncated
steroid (A and B rings) with an appended p-fluorophe-
nyl pyrazole.17–19 The p-fluorophenyl pyrazole provides
specificity for GR over other steroid receptors. Substit-
uents on the B-ring were tuned to modulate transre-
pression and transactivation activities and attempt to
dissociate the two parameters. Examples have been re-
ported in which the B-ring was substituted with an aryl
or heteroaryl group and a hydroxyl moiety.17a In these
cases, the A,B-ring system was a classical steroid 6,6-
ring system (6,6-system). More recently, ketal substitu-
ents were examined on the B-ring.17c In addition to the
traditional 6-membered B-ring, a 5-membered B-ring
was also explored. In this work, a general trend ap-
peared in which compounds with a 5-membered B-ring
had better activity in the transrepression assays than
those with a 6-membered B-ring.17c However, binding
The synthesis of these novel GCs is outlined in Scheme
1. An initial attempt was made to adopt the route used
for synthesis of the 6,6-system to our 6,5-platform. This
strategy worked well for conversion of the known Ha-
jos-Parrish ketone (1) to intermediate 2, which contains
the requisite p-fluorophenyl pyrazole. At this point in
the synthesis, an attempt was made to convert ketone
2 to aldehyde 3 as had been done previously for the
6,6-template.17a However, the transformation was prob-
lematic. Conversion to an enol-ether intermediate via a
Wittig reaction and hydrolysis to the aldehyde pro-
ceeded in poor yield. Furthermore, equilibration of the
diastereomeric mixture of aldehyde products to the de-
sired S-configuration at the newly formed stereocenter
was unsatisfactory. Therefore, an alternative protocol
Scheme 1. Synthesis of glucocorticoids with 6,5-ring system.

3356
C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
was employed.20 Conversion of 2 to alkene 4 using a
Wittig reaction was quantitative. Hydroboration pro-
ceeded cleanly with a selectivity of ꢀ25:1 to give 5,
which was oxidized to 3 in good yield via a Swern
reaction.
little TA. Unfortunately, the thiophene substituents in
the 6,5-series did not display the balanced mouse/human
profile seen in the 6,6-series (i.e., compound 10). Com-
pounds 11 and 12 had almost no activity in the mouse
cell lines and therefore could not be evaluated in an
in vivo mouse model of inflammation.
As seen previously in the 6,6-series, aryl and heteroaryl
lithium reagents added smoothly to aldehyde 3 provid-
ing secondary alcohol products. However, the stereo-
chemical outcome of the addition reaction differed
greatly between the 6,5- and 6,6-series. Whereas addi-
tions to the 6,6-aldehyde had provided the R-diastereo-
mer (b-hydroxyl) almost exclusively, additions to the
6,5-aldehyde gave nearly equal amounts of the R and
S isomers at the newly formed stereocenter. The selectiv-
ity in the addition was never better than 2:1 and often
closer to 1:1. The diastereomers were separated by either
silica gel chromatography or chiral HPLC in more
challenging cases.21
Having obtained disappointing results with monocyclic
substituents in the 6,5-series, attention was turned to
bicyclic substituents at C1, which had displayed favor-
able profiles in the 6,6-series (Table 2). The benzothi-
ophene ring system had been one of the best
substituents in the 6,6-series, and investigation of the
6,5-series was begun with this moiety. Whereas a
connection to either the 2- or the 3-position of the ben-
zothiophene ring had yielded active GR ligands in the
6,6-series (compounds 13, 16, and 17), a striking differ-
ence between the two points of attachment was observed
in the 6,5-series. Forming a linkage between the B-ring
and the 2-position of the benzothiophene ring led to
compounds that were functionally inactive (compounds
14 and 15). However, attachment of the benzothiophene
at the 3-position led to a large improvement in potency
and functional activity; these were the first GCs ob-
served in the 6,5-series with >50% of dexamethasone’s
activity in both the mouse and human TR assays. Com-
pound 18, bearing an R configuration at the hydroxyl
center, had similar potency to compound 19 (S hydro-
xyl) in the GR binding assay and the two compounds
had comparable EC50’s in the mouse and human IL-6
TR assays. However, when their efficacy was measured
relative to dexamethasone, compound 18 was found to
have a larger window of dissociation. Compound 18
achieved >70% of dexamethasone’s activity in both
TR assays, and <35% of dexamethasone’s activity in
the two TA assays. Compound 19 had >90% TR relative
to dexamethasone, but also nearly 60% TA in both
mouse and human species. Thus, the trend previously
observed for GCs, in which compounds with the stron-
gest functional activity showed little dissociation, was
continued.17 Having obtained promising results for the
benzothiophene diastereomers, exploration continued
of bicyclic ring systems. A naphthyl substituent yielded
similar results: attachment at the 2-position was not tol-
erated, leading to complete loss of functional activity
(compounds 20 and 21), while linkage to C1 of the
naphthyl ring led to a pair of potent diastereomers
(compounds 22 and 23). As in the benzothiophene ser-
ies, although both 22 and 23 bound to GR with near
equal affinity, the diastereomer with an S configuration
at the hydroxyl center (23) showed greater functional
activity, but a smaller window of dissociation. Finally,
a benzofuran moiety was investigated, although in this
case only attachment at the 3-position was undertaken.
Again, both diastereomers showed strong binding affin-
ity for GR and good functional activity. Interestingly, in
this case, the diastereomer with an S configuration at the
hydroxyl center (25) had a larger window of dissocia-
tion, particularly in the assays using human cell lines.
Following synthesis, the capacity of these novel GC
ligands to bind GR was tested by measuring their ability
to displace radiolabeled dexamethasone. The GCs were
then further evaluated in a series of in vitro assays.22 In
order to determine the extent to which the GCs were dis-
sociated, markers for transrepression (TR) and transac-
tivation (TA) were measured in both mouse and human
cell lines.23 In the human A549 lung carcinoma cell line,
repression of the inflammatory cytokine interleukin-6
(IL-6) relative to a dexamethasone control was mea-
sured.24 IL-6 was also used to evaluate TR in a mouse
cell line. To discern the TA activity of these compounds,
tyrosine amino transferase (TAT) activity was measured
in human cells. TAT is a hepatic gene known to be
upregulated by GCs.25 In mouse cell lines, TA was ana-
lyzed by investigating the levels of glutamine synthetase,
an enzyme associated with protein metabolism in
muscle.26
The investigation of GC ligands containing the 6,5-A,B-
ring system began with the synthesis of compounds con-
taining monocyclic substituents at the C1 position that
had shown potency in the 6,6-system; p-fluorophenyl
and thiophene rings were evaluated. The in vitro data
for these compounds are shown in Table 1, with results
from previously reported compounds in the 6,6-series in-
cluded for comparison.17 Striking differences in the
activity of the two series were apparent. The 6,5-p-fluor-
ophenyl analogs (8 and 9) bound much more weakly to
GR than their 6,6-system counterparts (6 and 7), dis-
placing less than 65% of [3H]dexamethasone in our bind-
ing assay. While compound 6 was a moderately potent,
well-dissociated, benchmark compound for our pro-
gram, its counterpart, compound 8, displayed almost
no functional activity in both mouse and human cell
lines. Compound 9 was more active but still fairly weak,
particularly in the mouse assays, and was not deemed to
be of interest, especially in light of the weak GR binding
that was observed. The results obtained for the thio-
phene substituent were also unsatisfying. Diastereomers
11 and 12 bound to GR with reasonable affinity and had
promising, dissociated profiles in the human cell lines
with >75% of dexamethasone’s activity in TR and very
Having synthesized compounds with a range of mono-
cyclic and bicyclic hydroxy-aromatic and heteroaromat-
ic substituents in the 6,5-series, it was evident that the

C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
3357
Table 1. Binding of GCs with monocyclic aromatic substiuents at C1 to GR; transrepression and transactivation in mouse and human cell lines
R
H
O
O
Me
Me
HO
Me
HO
Me
OH
OH
N
OH
OH
N
F
n
H
H
n=1; 6,5-series
n=2; 6,6-series
H
H
F
H
O
O
compounds 6-25
dexamethasone
prednisolone
Compound
n
R
GRa
hIL-6b
hTATc
mIL-6d
mGSe
IC50 (nM) EC50 (nM) %dex EC50 (nM) %dex EC50 (nM) %dex EC50 (nM) %dex
Prednisolone
—
—
13.8
5.3
4.5
9.7
16.5
nd
102
67
48
26
58
81
77
80
24
nd
nd
nd
nd
nd
nd
nd
82
25
15
3
5.7
95
74
70
19
62
74
44
ia
3.4
nd
nd
nt
89
31
23
nt
21
21
19
nt
F
F
F
F
6
2
2
1
1
2
1
1
22
HO
HO
HO
7
10.4
17.4*
12.0*
5.1
6.5
nd
392
23
8
9
6
7
nd
nd
nd
nt
HO
HO
S
S
S
10
11
12
1.0
21
28
17
7
HO
HO
8.0
145
ia
28
2.1
nd, not determined; EC50 values were not determined in cases where %dex 6 35%. ia, inactive; %dex <10%. nt, not tested. , Inflection point rather
*
than IC50. At the maximum dose tested (10 lM) for these two compounds only 55–65% of [3H]dexamethasone was displaced from GR; >90% of
[3H]dexamethasone was displaced in the binding assay for all other compounds reported here.
a Binding to hGRa receptor determined by displacement of [3H]dexamethasone.
b Human IL-6 assay in A549 lung carcinoma cell line.
c Activity in human tyrosine amino transferase (TAT) assay in HepG2 cells.
d Mouse IL-6 assay in peritoneal exudate cells harvested from C57BI/6 mice.
e Mouse glutamine synthetase (GS) assay in C2C12 cells.
SAR was much tighter than in the 6,6-series. The trend
observed in the ketal work did not follow in the hydro-
xyl series. Rather than seeing improved transrepression
upon moving from the 6,6- to the 6,5-core, many com-
pounds bearing the same substituents had very little
activity, although a select few had the desired profile.
In an attempt to better understand these results, molec-
ular modeling was employed. To explore the structural
impact of B ring size on activity, we chose to study the
conformational preferences for a subset of compounds
that exhibited disparate activity profiles, the fluorophe-
nyls and the 2- or 3-benzothiophenes. The compounds
with an R configuration at the hydroxyl group, 6,
8, 13, 14, 16, and 18, were studied first. The two
benzothiophenes, 16 and 18, were then compared to
their analogs, 17 and 19, which have an S configuration
at the hydroxyl group. Two hundred conformations
were generated for each of the compounds using an
implementation of distance geometry, which incorpo-
rates the theory and algorithm as previously described.27
Each set of conformers was then energy minimized using
MMFFs with a distance-dependent dielectric of 2r with-
out explicit solvent.28 Alignment of all the compounds
onto the pyrazole moiety common to each provides
the basis for our discussion.
The presence of the axial methyl at what would corre-
spond to the C10 position in the steroid core forces
the pendant aromatics to position the lowest energy con-
formers on the opposite face of the tricyclic system; the
a face using the steroid frame of reference. In all of the
6,6-systems, the most stable orientation of the pendant
aromatic is one in which the aromatic is most removed
from the axial methyl; ꢀ160° angle on the dihedral de-
fined by the four atoms starting at the C10 position
and ending with the aromatic. Conversely, in the

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C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
Table 2. Binding of GCs with bicyclic aromatic substiuents at C1 to GR; transrepression and transactivation in mouse and human cell lines
Compound
n
R
GRa
hIL-6b
hTATc
mIL-6d
mGSe
IC50 (nM) EC50 (nM) %dex EC50 (nM) %dex EC50 (nM) %dex EC50 (nM) %dex
Prednisolone
—
2
—
13.8
29.1
4.5
19
102
66
24
nd
82
22
5.7
42
95
66
3.4
89
41
S
S
S
13
HO
1209
14
15
16
1
1
2
HO
HO
HO
69.1
nd
ia
34
ia
ia
ia
ia
21
nd
ia
22
ia
ia
ia
689
ia
nd
nd
nd
33
S
S
S
S
5.1
1.5
2.1
3.1
18
86
nd
54
78
HO
HO
HO
17
18
19
2
1
1
6
5
94
71
92
356
43
29
62
11
16
14
99
73
92
3
100
33
nd
nd
36
10
675
58
20
21
22
1
1
1
86
nd
nd
48
14
13
80
ia
ia
ia
28
ia
ia
46
ia
ia
78
ia
ia
68
ia
ia
36
HO
198
5
ia
HO
HO
nd
HO
23
1
6.4
33
87
703
65
32
88
94
71
O
O
HO
HO
24
25
1
1
2.3
3.9
3
5
83
78
1624
1315
57
21
13
33
83
81
39
92
88
56
nd, not determined; EC50 values were not determined in cases where %dex 6 35%. ia, inactive; %dex <10%. nt, not tested. At the maximum dose
tested (10 lM) for these two compounds only 55–65% of [3H]dexamethasone was displaced from GR; >90% of [3H]dexamethasone was displaced in
the binding assay for all other compounds reported here.
a Binding to hGRa receptor determined by displacement of [3H]dexamethasone.
b Human IL-6 assay in A549 lung carcinoma cell line.
c Activity in human tyrosine amino transferase (TAT) assay in HepG2 cells.
d Mouse IL-6 assay in peritoneal exudate cells harvested from C57BI/6 mice.
e Mouse glutamine synthetase (GS) assay in C2C12 cells.
6,5-systems (except for compound 19 which maps to the
6,6-system in this instance), the energetically favored
position of the aromatic is proximate to the axial methyl
at ꢀ65° angle along the same dihedral as above. In the
case of the p-fluorophenyl compounds 6 and 8 and the
2-benzothiophenes 13 and 14, the difference in energy
between the lowest energy conformer and the next
higher group is ꢀ1.5 kcal/mol with a barrier to rotation
between the two of the order of 2–4 kcal/mol.29 For the
3-benzothiophenes 16 and 18, the difference between the

C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
3359
Figure 1. Superposition of the lowest energy conformers representing
compounds 6 (light green), 8 (pink), 13 (cyan), 14 (orange), 16 (white),
and 18 (purple) aligned to the pyrazole common to each. The
compounds with the least activity (8 and 14) are depicted in wire.
Figure 2. Superposition of the lowest energy conformers for the 6,6-
and 6,5-benzothiophene diastereomers. These are the R hydroxyl
group compounds 16 (white) and 18 (purple) and the S hydroxyl group
compounds 17 (yellow) and 19 (green). The benzothiophene ring of 19
has two isoenergetic conformers, one of which is shown as wire.
lowest energy conformer group and the next higher is
ꢀ2.5 kcal/mol with a similar rotational barrier to that
noted above.
Table 3. In vivo efficacy
Compound
(dose)
Absolute inhibition Inhibition of TNFa
of TNFa (%) relative to prednisolone (%)
In examining the superposition of the lowest energy con-
former for the six compounds with an R configuration at
the hydroxyl group (depicted in Fig. 1), it becomes clear
that one might first correlate activity with the rotation
angle about the bond connecting the aromatic to the
fused ring system. The compounds that favor placement
of the pendant aromatic near the axial methyl group at
C10, the 6,5-ring systems, suffer the largest drops in
binding and activity. The exception to this is when the
compound can extend its aromatic group into the space
occupied by the pendant aromatics in the 6,6-system. As
shown in Figure 1, compound 18 is capable of this feat
and, although not modeled but by extension, com-
pounds 22 and 24 could potentially achieve this as well.
The other ‘rescue’ of potency for the 6,5-system is by
inverting chirality of the hydroxyl group from R to S,
which, as noted above, causes the benzothiophene of
the 6,5-compound 19 to occupy the region of space
coincident with activity as depicted in Figure 2. The
inversion of chirality and concomitant potency enhance-
ment in the 6,5-ring system might also apply to
compounds 23 and 25.
Prednisolone 71
(3 mpk)
100
18 (30 mpk) 80
19 (30 mpk) 73
113
103
ingly, the two diastereomers differed markedly in their
PK. Dosed intravenously in a mouse, compound 19
had a normalized AUC of 1.7 lM h kg/mg, a clearance
(Cl) of 22.3 ml/min/kg, a half-life of 7.8 h, and an oral
bioavailability (F) of 34%; the type of strong PK param-
eters envisioned for this 6,5-series. In contrast, com-
pound 18 had a normalized AUC of only 0.4 lM h kg/
mg, an extremely high Cl of 106.8 mL/min/kg, a half-life
of 3.1 h, and an F of 19%. The relatively poor PK of 18
was quite disappointing as this was a fairly well-dissoci-
ated compound in vitro, of great interest to the pro-
gram. Furthermore, the result of the PK experiment
Although, as rationalized by molecular modeling, a
number of analogs lost activity in moving from the
6,6- to the 6,5-series, some were still quite potent. We
decided to profile two of these compounds, 18 and 19,
in vivo. Initial results in a mouse anti-inflammatory
model that measures inhibition of LPS-induced TNFa
were promising (Table 3).30 Both compounds were sim-
ilar to prednisolone in their ability to reduce inflamma-
tion, albeit at a higher dose (30 mpk vs 3 mpk).
Encouraged by this result, we determined the pharmaco-
kinetic (PK) profile of these two compounds. Surpris-
Figure 3. Compound 26.

3360
C. F. Thompson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3354–3361
G., Limbird, L. E., Gilman, A. G., Eds.; McGraw-Hill:
New York, 2001; pp 1649–1677.
2. Charmandri, E.; Kino, T.; Chrousos, G. P. Ann. N. Y.
Acad. Sci. 2004, 1024, 1.
3. Coghlan, M. J.; Elmore, S. W.; Kym, P. R.; Kort, M. E.
Curr. Top. Med. Chem. 2003, 3, 1617.
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with 18 called into question the impressive PD result and
raised the suspicion that there might be an active metab-
olite. Exposure of compound 18 to mouse liver micro-
somes resulted in rapid formation of
a major
metabolite with a molecular weight greater than the
parent by 32 mass units.31 Although the identity of this
species was not proven, compound 26 (Fig. 3) was
hypothesized to be the metabolite. Compound 26 could
be prepared in low yield (ꢀ15%) by treatment of com-
pound 18 with m-CPBA. Compound 26 was profiled
in vitro and shown to be very potent and not dissoci-
ated.32 This finding highlights yet another difficulty of
working in the dissociated glucocorticoid field: due to
subtle structural differences between compounds that
have a significant window of dissociation and those that
do not, one must ensure that not only is a parent com-
pound well dissociated, but that any active metabolites
are as well.
6. Schulz, M.; Eggert, M. Curr. Pharma. Des. 2004, 10, 2817.
7. Almawi, W. Y.; Melemedijian, O. K. J. Leukocyte Biol.
2002, 71, 9.
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Inflamm. Allergy 2002, 1, 127.
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Vujic, M.; Weih, F.; Angel, P.; Herrlich, P.; Schutz, G.
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Metab. 1997, 8, 152.
In summary, a series of GR ligands containing a 6,5-
A,B-ring system with a p-fluorophenyl pyrazole append-
age and an aromatic or heteroaromatic substituent at
the C1 hydroxyl center have been synthesized using
newly developed chemistry. These compounds were
evaluated in functional in vitro assays and compared
to analogous compounds containing a 6,6-A,B-ring sys-
tem. It was found that monocyclic aromatic or heteroa-
romatic B-ring substituents lacked potency in our TR
and TA assays, particularly in mouse cell lines. Bicyclic
ring systems displayed strong activity in both mouse and
human cell lines, although the SAR was much tighter
than in the 6,6-series, with the point of attachment to
the aryl or heteroaryl substituent of critical importance.
Molecular modeling was employed to better understand
this divergence in activity between the 6,6- and 6,5-sys-
tems. Modeling explained the in vitro data nicely with
the lowest energy conformation of active compounds
displaying the aromatic or heteroaromatic substituent
in one region of space and inactive compounds display-
ing their substituents in another. Interestingly, as in the
6,6-series, a benzothiophene paired with an R configura-
tion at the hydroxyl center (compound 18) gave the best
profile, with TR activity more than double that of TA in
both mouse and human cell lines. Although this par-
tially dissociated compound was able to suppress inflam-
mation in a mouse PD model, several lines of evidence
point to a poorly dissociated metabolite that may ac-
count for the strong in vivo activity. This is a cautionary
tale for other investigators working in the field. As in the
6,6-series, dissociative properties dropped off in the most
potent compounds (i.e., 19 and 23), and although com-
pounds with similar properties were identified, the 6,5-
series does not appear to offer any advantage over the
previously explored 6,6-series in terms of a dissociative
window.
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22. For details of receptor binding and cell based assays, see
Ref. 17a.
23. Preliminary experiments showed that dissociation in
mouse cell lines generally correlated well with dissocia-
tion in human cell lines; rat cell lines did not correlate as
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pendant aromatic 420° about the dihedral torsion already
described and determining the energy of the rotamer at
each 10° increment using MMFF with a distance-depen-
dent dielectric of 2r.
20. For a similar homologation approach on a related
molecule, see: (a) Sevillano, L. G.; Melero, C. P.;
´
´
`
Caballero, E.; Tome, F.; Lelievre, L. G.; Geering, K.;
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21. A crystal structure of compound 19 was obtained, which
allowed assignment of the absolute stereochemistry of
this compound. The stereochemistry of other compounds
was assigned by analogy using characteristic NMR
shifts. The chemical shifts for the protons on the
methylene group adjacent to the pyrazole ring were
particularly diagnostic. The signals for these protons
were generally observed close to 2 ppm for the diaste-
reomers with an R configuration at the hydroxyl center
and close to 3 ppm for compounds with an S configu-
ration at the hydroxyl center.
31. Incubation of compound 18 (10 lM) with mouse liver
microsomes (1 mg/mL) in the presence of NADPH
showed no parent and primarily
a metabolite of
MW = parent + 32 after 30 min. In contrast, compound
19 showed primarily the parent compound and very little
metabolism under the same conditions.
32. Data for compound 26: GR IC50 = 2.8 nM, hIL-6
EC50 = 2 nM (92% dex), hTAT EC50 = 24 nM (77% dex),
mIL-6 EC50 = 4.8 nM (88% dex), and mGS EC50
3.3 nM (67% dex). 26 was not prepared in sufficient
=
quantity to be tested in vivo.