Identification of the Clinical Candidate (R)-(1-(4-Fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone (CORT125134): A Selective Glucocorticoid Receptor

Article abstract of DOI:10.1021/acs.jmedchem.7b00162

The nonselective glucocorticoid receptor (GR) antagonist mifepristone has been approved in the U.S. for the treatment of selected patients with Cushing’s syndrome. While this drug is highly effective, lack of selectivity for GR leads to unwanted side effe

Full text of DOI:10.1021/acs.jmedchem.7b00162

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Article
The Identification of the Clinical Candidate (R)-(1-(4-fluorophenyl)-6-
(1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-
pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone
(
(CORT125134):– A Selective Glucocorticoid Receptor (GR) Antagonist.
Hazel J. Hunt, Joseph K. Belanoff, Iain Walters, Benoit Gourdet, Jennifer thomas,
Naomi Barton, John Unitt, Timothy Phillips, Denise Swift, and Emily Eaton
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00162 • Publication Date (Web): 03 Apr 2017
Downloaded from http://pubs.acs.org on April 3, 2017
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The Identification of the Clinical Candidate (R)-(1-(4-fluorophenyl)-6-((1-methyl-1H-
pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-
(trifluoromethyl)pyridin-2-yl)methanone (CORT125134):– A Selective Glucocorticoid
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Receptor (GR) Antagonist.
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Hazel J. Hunt *, Joseph K. Belanoff , Iain Walters , Benoit Gourdet , Jennifer Thomas , Naomi
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Barton , John Unitt , Timothy Phillips , Denise Swift , Emily Eaton
1
Corcept Therapeutics, 149 Commonwealth Drive, Menlo Park, California, 94025
Sygnature Discovery, BioCity, Pennyfoot Street, Nottingham, NG1 1GF
2
* Author to whom correspondence should be addressed
Abstract
The nonꢀselective glucocorticoid receptor (GR) antagonist, mifepristone, has been approved in
the USA for the treatment of selected patients with Cushing’s syndrome. Whilst this drug is
highly effective, lack of selectivity for GR leads to unwanted side effects in some patients.
Optimization of the previously described fused azadecalin series of selective GR antagonists led
to the identification of CORT125134, which is currently being evaluated in a Phase 2 clinical
study in patients with Cushing’s syndrome.
Introduction
The glucocorticoid receptor (GR) is a ligandꢀactivated transcription factor and a member of the
nuclear receptor superfamily. The GR is widely expressed, and is present in nearly all cell types
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and tissues. The essential role played by the GR is confirmed by the observation that mice with
1
targeted disruption of the GR do not survive postpartum due to a number of defects . The GR
mediates a breadth of biological processes, including inflammation, gluconeogenesis, immunity,
cardiovascular function, bone metabolism, brain function and homeostasis/development,
predominantly through transcriptional mechanisms. In the absence of ligand, the GR resides in
the cytoplasm and associates with chaperone proteins, including hsp90 and hsp70. Receptor
ligand binding (cortisol in humans and higher mammals, corticosterone in rodents) causes a
conformational change in the GR, leading to its dissociation from the chaperones and
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translocation of the ligandꢀbound receptor to the nucleus . Once in the nucleus, the GR
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regulates gene transcription by both activating and repressing mechanisms . Analogous to other
nuclear hormone receptors, the GR serves as an assembly point for transcription coregulators that
can directly modify chromatin structure and/or impact the activity of the gene transcription
apparatus. The GR can function either as a monomer or as a homoꢀdimer. Mechanisms by which
the GR modulates gene expression include direct binding of the dimer to glucocorticoid response
elements (GREs) in the promoter or enhancer region of genes, and binding of the monomer to
other transcription factors such as nuclear factorꢀkappa B (NFꢀkB) and activator protein 1 (APꢀ
1), through proteinꢀprotein interactions. Ligand structure, deoxyribonucleic acid (DNA)
sequence, cellular transcription factor composition and regulatory inputs all contribute to geneꢀ
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specific regulation .
In healthy individuals, cortisol, the natural ligand for GR, is secreted from the cortical cells of
the adrenal glands under the control of adrenocorticotropic hormone (ACTH). Endogenous
Cushing’s syndrome is a rare multisystem disorder that results from overproduction of cortisol.
In both adults and children, Cushing’s syndrome is most commonly caused by an ACTHꢀ
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secreting pituitary tumor (Cushing’s disease). Other forms of Cushing’s syndrome result from
autonomous production of cortisol from adrenal cortical tumors or overproduction of ACTH
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from nonꢀpituitary tumors
.
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The only curative treatment for Cushing’s Syndrome is resection of the tumor that is the source
of the excess ACTH or cortisol. Pharmacological treatment is not curative in Cushing’s
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syndrome, but it serves to control the disease after unsuccessful surgery . It may also be used to
lower cortisol activity to improve a patient’s condition prior to surgery. Pharmacological
treatment may be employed as an interim therapy under specific circumstances, such as in
1
2
patients who are waiting for their radiotherapy to be effective .
Currently, there are two United States (US) Food and Drug Administration (FDA)ꢀapproved
medical therapies for the treatment of endogenous Cushing’s syndrome. The first is mifepristone
(Figure 1), a nonꢀselective GR antagonist, which has been approved for use in selected adult
patients with endogenous Cushing’s syndrome who have type 2 diabetes mellitus or glucose
intolerance and have failed surgery or are not candidates for surgery. The second is pasireotide, a
somatostatin receptor agonist, which has been approved for the treatment of adult patients with
Cushing’s disease (a subꢀset of Cushing’s syndrome) for whom pituitary surgery is not an option
or has not been curative.
As noted above, Cushing’s syndrome is caused by excessive cortisol activity. Excess cortisol
activity leads to a plethora of severe symptoms, including the typical body habitus of patients
with Cushing’s syndrome, as well as diabetes mellitus, hypertension, dermatologic
manifestations and psychiatric disturbances. Mifepristone is effective in reducing the clinical
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impact of excessive cortisol activity, and improving the patients’ overall medical condition . Its
role in endogenous Cushing´s syndrome includes adjunct therapy following radiation treatment,
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and in cases where a patient cannot safely undergo surgery or has experienced failure of surgery.
Mifepristone does not decrease cortisol production; it acts by blocking the interaction of cortisol
with the GR.
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Although mifepristone is very effective, the lack of selectivity for GR has certain disadvantages.
Due to its high affinity for the progesterone receptor (PR), mifepristone causes termination of
pregnancy, and endometrial thickening or irregular vaginal bleeding in some patients. Our
objective was to identify a selective GR antagonist that would provide all the beneficial effects
of mifepristone, but would not cause the side effects associated with PR antagonism.
We have previously described the discovery and optimization of a series of selective GR
1
4,15
15
antagonists, based on a fused azadecalin template
. In our most recent publication , we
reported the identification of compounds such as CORT113176 1, which possess a heteroaryl
ketone substituent at the ring junction position, Figure 1. Compound 1 demonstrated modest
efficacy in a rat model of olanzapine induced weight gain and also in a rat model of exogenous
16
Cushing’s syndrome. In addition, Vendruscolo et al.
have reported that 1 reduced alcohol
17
intake in rats made dependent on alcohol, and Pineau et al. have reported the efficacy of 1 in a
rat model of Alzheimer’s disease. Further investigation of the heteroaryl ketone series provided
1
5
compounds such as the previously described compound 33 , designated compound 2 herein,
with reduced activity in a manual patch clamp hERG assay, good bioavailability in rats, and
efficacy in our rat model of exogenous Cushing’s syndrome. Although both compounds 1 and 2
demonstrated efficacy in our rat model of exogenous Cushing’s syndrome, neither were as
effective as mifepristone and we hoped to improve on their activity by the design and synthesis
of additional analogues. The structures of these compounds, and the nonꢀselective GR antagonist
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mifepristone are shown in Figure 1. We now wish to report further optimization of this series of
compounds, and the identification of two clinical candidates, 7 and 12.
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Figure 1. Structures of compounds 1, 2 and mifepristone
Chemistry
1
4
All the compounds described herein were synthesised from the enantiopure azadecalin ester 3
by two principal routes, described in Schemes 1ꢀ2. The first route is exemplified in Scheme 1 by
the conversion of 3 to the ketoꢀsulphonamide 5 via the Bocꢀprotected ketone intermediate 4. It
was found that low temperature addition of aryllithium reagents to ester 3 generally proceeded
only as far as the ketone (eg. 4, Scheme 1), perhaps due to the steric hindrance afforded by the
adjacent quaternary carbon centre. Two additional Bocꢀketone intermediates 4a,b were similarly
prepared and readily converted to several different sulphonamides described in Tables 1 and 2. A
complication arose with the addition of the aryllithium reagent derived from 2ꢀbromoꢀ4ꢀ
trifluoromethyl pyridine, which gave rise to the production of an inseparable byꢀproduct thought
to be derived from loss of the entire ketone substituent at the ring junction. Since the synthesis of
the paraꢀtrifluoromethyl substituted pyridyl ketone intermediate 6 was important to this
programme, improved conditions were developed involving aryl Grignard addition to the ester 3,
which afforded a mixture of ketone and hemiketal products, easily driven to clean ketone product
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by in situ acidic hydrolysis. Isolation of the hemiketal intermediate suggests that double
addition of the Grignard in this case is thwarted by stabilization of the initial tetrahedral
intermediate, perhaps due to magnesium ion chelation to the adjacent pyridyl nitrogen atom.
Again, ketone intermediate 6 was progressed to give a number of different sulphonamides such
as the methyl pyrazole analogue 7 shown in Scheme 1.
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Scheme 1. Synthesis of ketoꢀsulphonamides
a
Reagents and conditions: (a) 2ꢀbromopyridine, nꢀBuLi, THF, ꢀ78 °C, 45 min, 53%; (b) (i) TFA,
DCM, rt, 1 h; (ii) 3ꢀmethylbenzeneꢀ1ꢀsulfonyl chloride, Hunig’s base, DCM, rt, 0.5 h, 38%; (c)
(i) isopropylmagnesium chloride, 2ꢀbromoꢀ4ꢀ(trifluoromethyl)pyridine, ether/THF, 0 °C → rt, 2
h; (ii) aq HCl, MeCN, rt, 30 min, 84%; (d) (i) TFA, DCM, rt, 1.5 h; (ii) 1ꢀmethylꢀ1Hꢀpyrazoleꢀ4ꢀ
sulfonyl chloride, K CO , DCM/water, rt, 20 min, 73%.
2
3
In the second preparative route, the order of steps was reversed, as illustrated in Scheme 2, by the
synthesis of ketoꢀsulphonamide 9 via sulphonamide ester 8, easily prepared from Bocꢀester 3.
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Again, monoꢀaddition of aryllithium reagents to give the desired ketones generally occurred with
negligible overꢀaddition to tertiary alcohol byꢀproducts. Three alternative sulphonamides 8a-c
were also used to prepare some of the ketoꢀsulphonamides listed in Tables 1 and 2.
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a
Scheme 2. Alternative synthesis of ketoꢀsulphonamides
a
Reagents and conditions: (a) (i) HCl, dioxane, rt, 45 min; (ii) 3,4ꢀdifluorobenzeneꢀ1ꢀsulfonyl
chloride, Hunig’s base, DCM, rt, 15 min, 92%; (b) 2ꢀbromothiazole, nꢀBuLi, diethyl ether, ꢀ78
°
C, 45 min, 36%.
The coreꢀreduced analogues of Table 3 were synthesized by a variant of the route shown in
Scheme 1. Hydrogenation of ester 3 occurred with essentially complete diastereoselectivity to
afford the trans azadecalin intermediate 10 shown in Scheme 3. This ester reacted with
aryllithium or aryl Grignard reagents to give ketones such as 11 or 11a, which were deprotected
and treated with a variety of sulphonyl chlorides to give ketoꢀsulphonamides such as 12, as
shown in Scheme 3. The sense of diastereoselection shown by hydrogen addition to 3 was
determined by evaluation of the Xꢀray crystal structure of ketoꢀsulphonamide 12, which clearly
shows the trans azadecalin ring geometry (Figure 2).
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Scheme 3. Synthesis of reduced core analogues
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Reagents and conditions: (a) H , Pd/C, 55 °C, 45 min, Hꢀcube, 1 mL/min, 99%; (b) 2ꢀbromoꢀ4ꢀ
2
(trifluoromethyl)pyridine, isopropylmagnesium bromide, toluene, 0 °C → rt, 18 h, 45%; (i) TFA,
DCM, rt, 45 min; (ii) 2ꢀmethylꢀ2Hꢀ1,2,3ꢀtriazoleꢀ4ꢀsulfonyl chloride, Hunig’s base, DCM, rt, 2
h, 72%.
Figure 2. Crystal structure of 12
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Most of the sulphonyl chlorides required to make the sulphonamides described in Tables 1, 2 and
5
were commercially available, with the exception of the triazoles 18-20. These were synthesized
by the route shown in Scheme 4. Benzylation of triazole thiol 13 followed by methylation
afforded a mixture of methyl triazole regioisomers 15-17 which were readily separable by
column chromatography. Oxidative debenzylation with Nꢀchlorosuccinimide or chlorine then
gave the three sulphonyl chlorides 18-20. The regiochemistry of the 2,4ꢀisomer 19 was readily
assigned with the aid of the crystal structure of the derived ketoꢀsulphonamide product 12 shown
in Figure 2. The 1,4ꢀisomer 18 and the 1,5ꢀisomer 20 were assigned through comparison of the
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H NMR spectra of the precursor sulphides 15 and 17 with those previously reported
.
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Scheme 4. Synthesis of triazole sulphonyl chlorides
a
Reagents and conditions: (a) BnBr, EtOH, 0 °C → rt, 20 min, 87%; (b) MeI, K CO , DMF, 0 °C
2
3
→
rt, 1 h, 22% (15) / 40% (16) / 20% (17); (c) NCS, AcOH/water, rt, 2 h, 64% (18) / 82% (19);
(
d) Cl , DCM/water, 0 °C, 5 min, 90%.
2
Results and Discussion
All compounds were tested for their affinity to GR using a fluorescence polarization (FP) assay.
Functional activity was assessed in the human HepG2 cell line. The GR agonist, dexamethasone,
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induces the activity of the enzyme tyrosine amino transferase (TAT). GR antagonism is readily
determined by measuring the ability of the test compounds to inhibit the effect of
dexamethasone. The minimum significant ratio in this assay was 2.2 at the 95% confidence level,
so potency differences of 2.2 fold can be detected. For key compounds, competitive antagonism
was confirmed by Schild analysis. GR agonism of the test compounds is assessed by testing the
compounds in the absence of dexamethasone and measuring any induction in TAT activity. Full
details of both assays are provided in the Experimental section.
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The next step in our investigation of this very promising series of compounds was the
incorporation of a substituent on the heteroaryl ketone. Although our compounds have excellent
potency in our binding assay and standard functional GR antagonist assay, most of them are not
quite as potent as mifepristone. As can been seen in Figure 3, the heteroaryl ketone substituent in
1
does not fully occupy the space filled by the dimethylanilinophenyl group in mifepristone. We
wanted to determine whether we could obtain additional functional potency by the inclusion of a
larger heteroaryl ketone group, by adding a substituent to the heteroaryl ring. An overlay of 1
(blue) and mifepristone (pink) is shown in Figure 3, which depicts the GR active site (green
except for helix 12 in red). The GR and mifepristone structure comes from PDB 3H52, chain c.
A minimised conformer of 1 was placed onto mifepristone using an RMS fitting procedure. The
conformer of 1 was generated with Spartan software using the conformer distribution option and
then minimised using MMFF94. The inclusion of a methyl substituent on the pyridyl or thiazolyl
ring was investigated. We were encouraged to find that a methyl substituent para to the nitrogen
often provided a modest increase in potency (see for example compound 5 compared to
compound 21; 26 vs 27; 28 vs 29; 32 vs 33). A comparison of some pairs of compounds, with
and without a methyl substituent, is provided in Table 1. However, the inclusion of the methyl
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group meta to the pyridyl nitrogen was not advantageous (for example see compound 22). In the
thiazole series, the position of the methyl group also made a significant difference, with 5ꢀmethyl
providing an enhancement in potency (compound 37), but a 3ꢀmethyl being detrimental
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(
compound 38). In the pyridyl series we also investigated ethyl substitution (compound 34), and
found that this was beneficial.
Figure 3. Overlay of 1 and mifepristone
Table 1. Effect of Substitution on the Heteroaryl Ketone Group
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2
2
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
R
Ar
X
FP
Ki (nM)
0.09
HepG2 TAT Cmax
AUC
(ng.h/mL)
nt
Ki (nM)
(ng/mL)
mifepristone NA
NA
NA
3.0
nt
1
5
2
2
2
2
2
pꢀCF
3
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
H
0.28
0.14
0.08
0.14
0.20
0.22
0.14
0.12
0.14
0.1
12
nt
nt
mꢀCH
H
6.8
3.3
7.5
14
31
11
nt
83
3
3
3
3
1
2
3
4
5
mꢀCH
mꢀCH
mꢀCH
4ꢀCH
5ꢀCH
6ꢀCH
H
3
3
3
44
nt
nt
nt
m,pꢀdi F
m,pꢀdi F
m,mꢀdi F
m,mꢀdi F
7.5
5.7
9.2
4.3
14
215
113
72
nt
974
421
143
nt
4ꢀCH
H
3
3
3
3
26
27
4ꢀCH
H
2
2
3
3
3
8
9
0
1
2
mꢀCF
3
3
nt
nt
mꢀCF
4ꢀCH
H
0.21
0.29
0.42
0.14
5.1
11
nt
nt
mꢀF,pꢀCF
mꢀF,pꢀCF
mꢀCN
125
48
140
726
173
239
3
3
4ꢀCH
H
7.5
15
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3
3
3
4
mꢀCN
mꢀCN
mꢀCN
2ꢀpyridyl
2ꢀpyridyl
2ꢀpyridyl
2ꢀthiazole
2ꢀthiazole
2ꢀthiazole
2ꢀthiazole
2ꢀthiazole
2ꢀthiazole
2ꢀthiazole
4ꢀCH
4ꢀCH
3
2
0.15
0.19
0.32
0.48
0.29
0.88
0.23
1.3
4.5
1.7
4.8
17
70
nt
217
nt
CH
3
35
36
4ꢀCF
H
3
20
103
nt
49
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mꢀCF
mꢀCF
mꢀCF
446
nt
3
3
3
9
3
4
4
7
8
3
3
5ꢀCH
3ꢀCH
H
3
3
10
63
nt
nt
m,pꢀdi F
m,pꢀdi F
m,pꢀdi Cl
m,pꢀdi F
37
nt
nt
9
0
1
5ꢀCH
H
3
7.4
16
30
197
30
135
1,222
135
0.59
0.32
5ꢀCH
7.4
3
NA = not applicable; nt = not tested
Encouraged by the improved potency of several of the methylated analogues, we conducted
some cassette PK studies in rats. We included three test compounds and a reference compound in
each cassette, and administered a total oral dose of 12mg/kg (3mg/kg for each compound) by
oral gavage. The additional methyl substituent appeared to have a detrimental effect on plasma
concentrations, since several compounds provided low Cmax and AUC when compared to the
corresponding compounds lacking the methyl group, see Table 1. For example, the methyl
analogues 25, 31 and 33 all provided lower Cmax and AUC than the corresponding unsubstituted
compounds 24, 30 and 32. We considered that the additional methyl group may have been
subject to metabolism, thus explaining the reduced plasma concentrations in analogues
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Journal of Medicinal Chemistry
Page 14 of 62
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
incorporating this group. On this basis, replacement of methyl by trifluoromethyl was expected
to provide higher plasma concentrations, but we were concerned by the already higher than ideal
lipophilicity and molecular weight of our compounds. This concern was confirmed by the low
Cmax and very low AUC observed with compound 35. The corresponding unsubstituted pyridyl
analogue, compound 32, exhibited considerably better exposure, see Table 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In an effort to reduce lipophilicity and molecular weight, we investigated the replacement of the
substituted phenyl sulphonamide by a heteroaryl sulphonamide, but we found that these
compounds were typically not sufficiently potent in the HepG2 TAT assay. A representative
compound, 42, is included in Table 2. It occurred to us that the combination of a heteroaryl
sulphonamide (to reduce lipophilicity and molecular weight) with a trifluoromethyl substituted
pyridyl ketone (to provide improved potency compared with the unsubstituted pyridyl) at the
ring junction might provide an acceptable balance of physicochemical properties and potency.
As illustrated in Table 2, this expectation proved to be justified, and we identified a number of
compounds with acceptable potency (for example compounds 7, 43, 44, 45, 46, 48, 50 and 51).
Various substituted pyrazole and triazole sulphonamides provided good GR antagonist potency,
although some interesting SAR emerged related to the position of methyl groups on the pyrazole
sulphonamides. For example, whereas Nꢀalkylation (compare compound 7 with compound 47)
was well tolerated, it appeared that Cꢀalkylation was often detrimental (compare compound 49
with compound 47). We were also very gratified to discover that the incorporation of the
trifluoromethyl substituent on the pyridyl ketone did not have any negative consequences from a
PK perspective. In contrast, the addition of this group appeared to be beneficial, compare
compounds 42 and 7.
Table 2. Heteroaryl sulphonamides
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Page 15 of 62
Journal of Medicinal Chemistry
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32
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35
36
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound R
Het
HepG2 TAT Cmax
AUC
Ki nM
ng/mL
ng.h/mL
4
7
4
4
4
4
2
H
28
150
262
126
291
239
110
780
1219
701
863
967
283
CF
CF
CF
CF
CF
7.2
4.6
6.6
11
3
3
3
3
3
3
4
5
6
12
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Journal of Medicinal Chemistry
Page 16 of 62
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
7
CF
3
3
7.6
10
nt
nt
nt
nt
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
48
CF
4
9
0
CF
3
69
nt
nt
5
CF
CF
CF
3
3
3
3.1
9.7
31
255
91.5
nt
860
450
nt
51
52
Having identified a number of compounds with the required potency in the HepG2 TAT assay
and encouraging plasma levels in the rat cassette PK studies, we selected several compounds for
additional pharmacokinetic profiling. Good plasma levels in the cassette PK studies generally
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Page 17 of 62
Journal of Medicinal Chemistry
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7
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correlated with good plasma levels and high bioavailability in single compound i.v./p.o. rat PK
studies, using an oral dose of 5mg/kg and an i.v. dose of 1mg/kg. We also evaluated the PK
profile of several of the most promising compounds in i.v./p.o. PK studies in monkeys, using an
oral dose of 20mg/kg and an i.v. dose of 1mg/kg. Unfortunately, good bioavailability in rats did
not always translate into good bioavailability in monkeys, as shown in Table 3. Compound 7 was
the only compound with a good PK profile in monkeys. Even minor changes to the structure of
the compound, such as moving the position of a nitrogen in the pyrazole ring, had a detrimental
effect (compare compound 7 with compound 45).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Oral PK data for selected compounds
Compound Rat
Monkey
Cmax
AUC
%F
Cmax
AUC
%F
ng/mL
ng.h/mL
ng/mL
ng.h/mL
7
297
1550
986
89
68
62
52
1140
nt
11663
nt
88
nt
44
510
528
511
45
1561
1527
16
217.4
193
~1
1.5
48
20.8
For i.v. dosing in both species the compounds were formulated in 10% DMSO, 45% PEG 400
and 45% propylene glycol.
For oral dosing in rats, the compounds were formulated in 10% DMSO and 0.5%
methylcellulose in water.
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Journal of Medicinal Chemistry
Page 18 of 62
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For oral dosing in monkeys, the compounds were formulated in 10% DMSO, 0.1% Tween 80,
0.5% hydroxypropylmethylcellulose in water.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 7 was the clearly the most promising compound in this series, and it was selected for
full profiling. Selectivity for GR over the progesterone receptor, androgen receptor and estrogen
receptor was assessed in radioligand binding assays, and no affinity (< 10% inhibition of
radioligand binding) for the other receptors was detected at a concentration of 1ꢀM whereas
100% inhibition was measured in an analogous GR binding assay. GR antagonism was measured
in primary hepatocytes from several species, including human, rat, dog and monkey.
Dexamethasone increases the activity of TAT in primary hepatocytes, and the ability of
compound 7 to prevent this effect was determined. In addition, GR agonism was assessed by
testing the compound in the absence of dexamethasone and measuring TAT activity. Whereas
Compound 7 demonstrated full antagonism and no agonism in human and monkey hepatocytes,
the compound acted predominantly as a GR agonist in dog hepatocytes, with only modest
antagonism observed. There was also evidence for incomplete antagonism, and partial agonism
at high (10ꢀM) concentrations in rat hepatocytes. These data are provided in Table 4. GR
antagonism was also assessed in human peripheral blood mononuclear cells by testing the ability
of compound 7 to prevent the dexamethasoneꢀinduced inhibition of LPS stimulated production of
tumour necrosis factor alpha (TNFα) (data not shown). Compound 7 was selective for GR over a
standard panel of diverse receptors, enzymes and channels (data not shown).
Table 4. TAT assay data for compound 7 in primary hepatocytes from several species
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Parameter
Human
81.2
100
Monkey
210
99
Rat
12
Dog
>10,000
31%
Antagonist K (nM)
i
Maximum % antagonism
Agonist EC (nM)
54.2
10
11
12
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25
26
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30
31
32
33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N/A
0
N/A
0
>10,000
22
>10,000
80
50
Maximum % agonism
In vivo efficacy was assessed in a rat model of exogenous Cushing’s syndrome as described
1
5
previously . A dose of 30mg/kg compound 7 administered orally twice a day significantly
inhibited effects on plasma insulin and completely prevented the cortisone induced increase in
plasma glucose, as shown in Figure 4. Similar effects were achieved with a lower dose, 7.5mg/kg
twice a day. Mifepristone at a dose of 30mg/kg was included for comparison. The results
15
obtained with compound 7 were superior to those reported previously with compound 1 , and
similar to results obtained with mifepristone in the same experiment.
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Journal of Medicinal Chemistry
Page 20 of 62
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1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 4. Effects of Compound 7 in the cortisone model of exogenous Cushing’s syndrome
*
#
** denotes p < 0.001 compared with vehicle treated mice.
## denotes p < 0.001 compared with cortisone treated mice.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 7 was found to have high plasma protein binding in rats, monkeys and humans, with
9.7, 98.9 and 99.5% binding respectively. Significant inhibition of CYP3A4 and CYP2C8 was
9
observed, with modest inhibition of other CYPs as shown in Table 5. This CYP inhibition was
not time dependent. CYP induction studies carried out in cryopreserved human hepatocytes
indicated no induction of CYP1A2, CYP2B6 or CYP3A4.
Table 5. CYP Inhibition of Compound 7
CYP
1A2
2B6
2C8
2C9
2C19
2D6
3A4
3A5
IC50 (µM)
>10
>10
0.21
2
8
9
1.3
4.9
2
0
Compound 7 (CORT125134 , see Figure 5 for chemical structure) was selected for preꢀclinical
development and subjected to the usual panel of toxicology and safety pharmacology studies. We
have completed a Phase I study in healthy human volunteers. In addition to the usual assessment
of safety, tolerability and pharmacokinetics, we also included an assessment of pharmacological
effect. This was achieved by the administration of the GR agonist, prednisone, and assessing the
ability of 7 to counteract the effects of prednisone on a variety of GR mediated parameters. The
21
results obtained in this Phase I clinical study will be published elsewhere . A Phase 2 study in
patients with endogenous Cushing’s syndrome is currently in progress in the USA and Europe.
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Page 21 of 62
Journal of Medicinal Chemistry
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5 Chemical structure of 7
Having identified compound 7 as our first clinical candidate, we next turned our attention to the
identification of a backꢀup compound. A potential minor concern with compound 7 was the
presence of the carbonꢀcarbon double bond in the central ring of the fused azadecalin scaffold,
since we observed some propensity for this double bond to migrate to the adjacent six membered
ring under certain conditions. In order to avoid this possibility, we opted to prepare compounds
in which the double bond had been reduced. In general, reduction of the double bond resulted in
decreased potency in the HepG2 TAT assay. For example, the reduced analogue of compound 7
(compound 54) had a Ki of 34 nM, compared with 7.2 nM for compound 7. As shown in Figure
6, reduction of the double bond affected the conformation of the tricyclic ring system, and
altered the position of the key substituents, relative to the unsaturated analogues. Numerous other
pairs of compounds (e.g. compound 28 compared to compound 53; 44 vs 55 and 50 vs 12) show
the same trend, as shown in Table 6.
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Journal of Medicinal Chemistry
Page 22 of 62
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Overlay of Compounds 7 and 12
Table 6. Reduced Core Compounds
A
B
Compound A or B
R
Ar
HepG2 TAT
Ki (nM)
14
28
A
H
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Page 23 of 62
Journal of Medicinal Chemistry
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7
5
3
B
A
B
A
B
A
B
A
B
H
44
10
11
12
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22
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25
26
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28
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30
31
32
33
34
35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CF
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
3
7.2
34
4
44
6.6
24
55
50
3.1
12
12
51
9.7
56
60
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Journal of Medicinal Chemistry
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
4
2
7
8
A
B
A
CF
CF
CF
3
3
3
31
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
120
10
58
B
CF
3
68
Compound 12 was one of the most potent compounds identified in the reduced core series, so we
decided to obtain additional information to determine whether this subꢀseries provided any
benefit over the original compounds. We were very gratified to discover that compound 12
provided excellent plasma levels in rats after oral dosing, higher than any other compound we
tested. Following the administration of a 5mg/kg oral dose, the Cmax was 1255 ng/mL and the
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Page 25 of 62
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AUC was 9418 ng.h/mL. Bioavailability was 100%. Compound 12 (CORT125281 ) was
selected for further profiling and will be the subject of a later publication.
Conclusions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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By further optimization of our fused azadecalin series of selective GR antagonists, we have
identified the clinical candidate compound 7. This compound combines excellent potency,
selectivity and oral bioavailability in several species with acceptable physicochemical properties.
In vivo efficacy has been demonstrated in a relevant animal model. We have obtained proof of
GR antagonism in human subjects in a Phase I study in healthy subjects, and the evaluation of
the compound in patients with Cushing’s syndrome is currently in progress. Although
mifepristone is a very effective in the treatment of selected patients with Cushing’s syndrome,
the use of this compound is limited by side effects which result from its affinity for the
progesterone receptor. There are also political issues in some countries concerning the use of the
“abortion pill” which will be avoided by the development of a compound lacking affinity for the
progesterone receptor. By removing affinity for the progesterone receptor, compound 7 provides
a significant benefit over mifepristone.
Experimental Section
Chemistry All starting materials and reagents were either commercially available or their
synthesis had been previously described in the literature. All purchased chemicals and solvents
were used without further purification. All reactions involving air or moisture sensitive reagents
were performed under an inert atmosphere. Hydrogenations were performed on a Thales Hꢀcube
flow reactor under the conditions stated. Silica gel chromatography was done using the
appropriate size prepacked silica filled cartridges (230ꢀ400 mesh, 40ꢀ63 µm). NMR spectra were
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acquired on a Bruker Avance III spectrometer at 400 MHz with chemical shifts reported in ppm
relative to residual undeuterated solvent as reference. Reactions were monitored by silica gel F254
TLC plates with UV visualisation at 254 nm or by LCMS. LCMS analyses to determine purities
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and associated mass ions were performed using an Agilent Infinity 1260 LC 6120 quadrupole
mass spectrometer with positive and negative ion electrospray and ELS / UV @ 254nm detection
using an Agilent Zorbax Extend C18, Rapid Resolution HT 1.8 micron C18 30 x 4.6 mm column
and a 2.5 mL / minute flow rate with a 4 minute run time. High resolution masses (HRMS) were
determined on a Waters Xevo G2ꢀXS QTOF mass spectrometer. Preparative reverse phase
HPLC was performed using UV detection at 215 and 254 nm with a Waters XꢀSelect PrepꢀC18,
5
µm, 19x50 mm column eluting with a H OꢀMeCN gradient containing 0.1% v/v formic acid
2
over 10 min. All final compounds had an HPLC purity of 95% or better. All chemical names
have been generated using CambridgeSoft ENotebook 12.0.
(R)-tert-butyl
1-(4-fluorophenyl)-4a-picolinoyl-4a,5,7,8-tetrahydro-1H-pyrazolo[3,4-
g]isoquinoline-6(4H)-carboxylate (4). 2ꢀbromopyridine (23.6 ml, 243 mmol) in dry THF (180
mL) was added to nꢀbutyllithium (99 ml, 249 mmol) in dry THF (100 mL) at ꢀ78 °C. The
reaction mixture was stirred at ꢀ78 °C for 45 min. A solution of (R)ꢀ6ꢀtertꢀbutyl 4aꢀmethyl 1ꢀ(4ꢀ
fluorophenyl)ꢀ4a,5,7,8ꢀtetrahydroꢀ1Hꢀpyrazolo[3,4ꢀg]isoquinolineꢀ4a,6(4H)ꢀdicarboxylate
15
3
(34.0 g, 80 mmol) in dry THF (280 mL) was added dropwise over 30 min, and the reaction
mixture was stirred for 45 min at ꢀ78 °C. Water (400 mL) was added and the reaction mixture
was stirred at RT for 10 min. The aqueous phase was extracted with ethyl acetate (2 x 400 mL).
The combined organic phases were washed with brine (400 mL), dried (MgSO ), and solvent
4
removed to give an orange oil. The crude product was purified by chromatography on silica (330
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g column, 10ꢀ60% ethyl acetate/isohexane) to afford 20.5 g (53%) of 4 as a pale yellow solid.
+
LC/MS m/z 475 (M+H) .
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(
R)-tert-butyl
1-(4-fluorophenyl)-4a-(4-methylpicolinoyl)-4a,5,7,8-tetrahydro-1H-
pyrazolo[3,4-g]isoquinoline-6(4H)-carboxylate (4a). Compound 4a was prepared using the
same procedure as described for the synthesis of 4 by substituting 2ꢀbromoꢀ4ꢀmethylpyridine for
+
2
ꢀbromopyridine. LC/MS m/z 489 (M+H) .
(R)-tert-butyl
4a-(4-ethylpicolinoyl)-1-(4-fluorophenyl)-4a,5,7,8-tetrahydro-1H-
pyrazolo[3,4-g]isoquinoline-6(4H)-carboxylate (4b). Compound 4b was prepared using the
same procedure as described for the synthesis of 4 by substituting 2ꢀbromoꢀ4ꢀethylpyridine for 2ꢀ
+
bromopyridine. LC/MS m/z 503 (M+H) .
(R)-(1-(4-fluorophenyl)-6-(m-tolylsulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-
g]isoquinolin-4a-yl)(pyridin-2-yl)methanone (5). A solution of (R)ꢀtertꢀbutyl 1ꢀ(4ꢀ
fluorophenyl)ꢀ4aꢀpicolinoylꢀ4a,5,7,8ꢀtetrahydroꢀ1Hꢀpyrazolo[3,4ꢀg]isoquinolineꢀ6(4H)ꢀ
carboxylate 4 (0.2 g, 0.42 mmol) in 20% TFA/DCM (6 mL) was stirred at RT for 1 h. Solvent
was removed in vacuo, azeotroping twice with toluene, to give a dark orange oil. This was
dissolved in DCM (6 mL) and 3ꢀmethylbenzeneꢀ1ꢀsulfonyl chloride (0.073 mL, 0.504 mmol)
and diꢀisopropylethylamine (0.367 ml, 2.10 mmol) were added. The reaction mixture was stirred
at RT for 0.5 h, before solvent was removed in vacuo. The crude product was purified by
chromatography on silica (12 g column, 0ꢀ40% Ethyl acetate/isohexane) and by preparative
+
1
HPLC to afford 88 mg (38%) of 5 as a pale yellow solid. LC/MS m/z 529 (M+H) ; H NMR
(CDCl3):
δ
8.66 (1H, ddd, J = 4.7, 1.7, 0.8 Hz), 7.91ꢀ7.89 (1H, m), 7.84 (1H, dt, J = 7.4, 1.7 Hz),
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.50ꢀ7.40 (5H, m), 7.36ꢀ7.34 (2H, m), 7.29 (1H, s), 7.19ꢀ7.13 (2H, m), 6.47 (1H, d, J = 2.1 Hz),
.51 (1H, dd, J = 12.4, 2.1 Hz), 4.31 (1H, d, J = 16.9 Hz), 3.84ꢀ3.80 (1H, m), 2.92ꢀ2.78 (2H, m),
.68 (1H, d, J = 12.4 Hz), 2.49ꢀ2.40 (5H, m).
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(R)-tert-butyl 1-(4-fluorophenyl)-4a-(4-(trifluoromethyl)picolinoyl)-4a,5,7,8-tetrahydro-1H-
pyrazolo[3,4-g]isoquinoline-6(4H)-carboxylate (6). Isopropylmagnesium chloride (2M in
THF, 35.1 ml, 70.2 mmol) was added to dry ether (100 mL) cooled to 0 °C. A solution of 2ꢀ
bromoꢀ4ꢀ(trifluoromethyl)pyridine (15.9 g, 70.2 mmol) in dry ether (50 mL) was then added over
20 min. The resulting dark coloured suspension was stirred at 0°C for 50 min, then (R)ꢀ6ꢀtertꢀ
butyl 4aꢀmethyl 1ꢀ(4ꢀfluorophenyl)ꢀ4a,5,7,8ꢀtetrahydroꢀ1Hꢀpyrazolo[3,4ꢀg]isoquinolineꢀ
2
3
4a,6(4H)ꢀdicarboxylate 3 (10.0 g, 23.4 mmol) was added as a solution in dry ether:THF (4:1,
total volume 100 mL) dropwise over 30 min. The reaction was stirred at 0°C for 15 min, then at
RT for 2 h. The reaction was reꢀcooled to 0°C, and quenched by the dropwise addition of iceꢀ
cold water (40 mL) and then diluted further with water (120 mL). The layers were then
separated, and the aqueous layer extracted with Ethyl acetate (3 x 150 mL). The combined
organic extractions were washed with brine (150 mL) and dried over MgSO , and solvent
4
removed in vacuo to give a brown oil. This was diluted with acetonitrile (100 mL) and aqueous
HCl (1M, 23.4 mL, 23.4 mmol) and the resulting solution stirred at RT for 30 min. The reaction
mixture was diluted with ethyl acetate (300 mL) and washed sequentially with brine (100 mL),
saturated aqueous NaHCO (100 mL) and brine again (100 mL). The organic layer was then
3
dried over MgSO and solvent removed in vacuo. The crude product was purified by
4
chromatography on silica (220 g column, 0ꢀ60% ethyl acetate/isohexane) to afford 10.9 g (84%)
+
of 6 as a pale yellow solid. LC/MS m/z 543 (M+H) .
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(
R)-(1-(4-fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-
pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone (7). To
a
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solution of (R)ꢀtertꢀbutyl 1ꢀ(4ꢀfluorophenyl)ꢀ4aꢀ(4ꢀ(trifluoromethyl)picolinoyl)ꢀ4a,5,7,8ꢀ
tetrahydroꢀ1Hꢀpyrazolo[3,4ꢀg]isoquinolineꢀ6(4H)ꢀcarboxylate 6 (9.59 g, 17.7 mmol) in DCM
(80 mL) was added TFA (27.2 mL, 354 mmol) dropwise over 20 min, and the resulting dark
coloured solution stirred at RT for 1.5 h. The reaction mixture was diluted with toluene (30 mL)
and solvent removed in vacuo. The dark brown residue was then azeotroped with toluene (3 x 30
mL). The resulting brown gum was diluted with DCM (80 mL) and water (80 mL) and
potassium carbonate (12.2 g, 88 mmol) added to the biphasic mixture portionwise over 10 min.
1ꢀmethylꢀ1Hꢀpyrazoleꢀ4ꢀsulfonyl chloride (3.51 g, 19.4 mmol) was then added portionwise to
the reaction mixture, and the resulting biphasic solution was stirred at RT for 20 min. The
reaction mixture was diluted with water (200 mL) and the layers separated. The aqueous layer
was extracted with DCM (2 x 100 mL) and the combined organic extractions washed with brine
(
200 mL) and dried over MgSO . The solvent was removed to give a thick brown oil, which was
4
purified by chromatography on silica (220 g column, load in DCM, 0ꢀ70% ethyl
acetate/isohexane) to afford 7.66 g (73%) of 7 as a pale orange solid. HRMS calculated for
+
1
C H F N O S [(M+H) ], 587.1483; found, 587.1483; H NMR (CDCl ):
δ
8.87 (1H, d, J = 5.0
Hz), 8.16ꢀ8.15 (1H, m), 7.72ꢀ7.70 (1H, m), 7.69ꢀ7.66 (2H, m), 7.47ꢀ7.42 (2H, m), 7.30 (1H, s),
.21ꢀ7.14 (2H, m), 6.51 (1H, d, J = 2.1 Hz), 5.44 (1H, dd, J = 12.0, 2.1 Hz), 4.21 (1H, d, J = 16.9
27 22
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Hz), 3.93 (3H, s), 3.80ꢀ3.76 (1H, m), 2.94 (1H, J = 16.9 Hz), 2.88ꢀ2.79 (1H, m), 2.66 (1H, d, J =
13
1
2.1 Hz), 2.53ꢀ2.48 (1H, m), 2.43 (1H, ddd, J = 12.6, 10.5, 3.5 Hz); C NMR (CDCl ):
δ
197.65
3
(s), 161.75 (d, J = 247 Hz), 153.91 (s), 148.80 (s), 139.88 (q, J = 35 Hz), 138.86 (s), 138.22 (s),
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