Identification and optimization of a novel series of [2.2.1]-oxabicyclo imide-based androgen receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.02.006

A novel series of [2.2.1]-oxabicyclo imide-based compounds were identified as potent antagonists of the androgen receptor. Molecular modeling and iterative drug design were applied to optimize this series. The lead compound [3aS-(3aα,4β,5β,7β,7aα)]-4-(octahydro-5-hydroxy-4,7-dimethyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl)-2-iodobenzonitrile was shown to have potent in vivo efficacy after oral dosing in the CWR22 human prostate tumor xenograph model.

Full text of DOI:10.1016/j.bmcl.2008.02.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1910–1915
Identification and optimization of a novel series
of [2.2.1]-oxabicyclo imide-based androgen receptor antagonists
Mark E. Salvati,a,* Aaron Balog,a Weifang Shan,a Richard Rampulla,a Soren Giese,a
Tom Mitt,a Joseph A. Furch,a Gregory D. Vite,a Ricardo M. Attar,a Maria Jure-Kunkel,b
Jieping Geng,b Cheryl A. Rizzo,b Marco M. Gottardis,b Stanley R. Krystek,c
Jack Gougoutas,c Michael A. Galella,c Mary Obermeier,d
Aberra Furad and Gamini Chandrasenad
aDepartment of Oncology Chemistry, Bristol-Myers Squibb Pharmaceutical Research and Development,
PO Box 4000, Princeton, NJ 08543-4000, USA
bDepartment of Discovery Biology, Bristol-Myers Squibb Pharmaceutical Research and Development,
PO Box 4000, Princeton, NJ 08543-4000, USA
cDepartment of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research and Development,
PO Box 4000, Princeton, NJ 08543-4000, USA
dPharmaceutical Candidate Optimization Department, Bristol-Myers Squibb Pharmaceutical Research and Development,
PO Box 4000, Princeton, NJ 08543-4000, USA
Received 25 December 2007; accepted 1 February 2008
Available online 8 February 2008
Abstract—A novel series of [2.2.1]-oxabicyclo imide-based compounds were identified as potent antagonists of the androgen recep-
tor. Molecular modeling and iterative drug design were applied to optimize this series. The lead compound [3aS-(3aa,4b,5b,7b,7aa)]-
4-(octahydro-5-hydroxy-4,7-dimethyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl)-2-iodobenzonitrile was shown to have potent in vivo
efficacy after oral dosing in the CWR22 human prostate tumor xenograph model.
Published by Elsevier Ltd.
Carcinoma of the prostate (CaP) is the 2nd leading
cause of cancer related death in men in the United
States.1 The androgen receptor (AR) is a ligand binding
transcription factor in the nuclear hormone receptor
super family and is a key molecular target in the etiology
and progression of prostate cancer. Androgen ablation
via surgical castration or by chemical castration with a
luteinizing hormone releasing hormone agonist, in com-
bination with an anti-androgen,2 is currently the treat-
ment of choice for advanced CaP. Although this
therapy initially shows an 80–90% response rate,3
approximately 50% of patients progress to fatal andro-
gen independent CaP (AI-CaP) after about 18 months
of treatment.4 Recent data have shown that cytotoxic
agents such as Docetaxel5 can be effective in the treat-
ment of AI-CaP, however the survival benefit is mini-
mal. Recent advances in the field have shown that
reactivation of the AR signaling pathway is the root
cause for the development of AI-CaP.6 The identifica-
tion of the role of the AR in AI-CaP suggests that
new agents which act at the level of the AR may be effec-
tive in the treatment of this disease. For this reason, we
are interested in identifying novel small molecule antag-
onists of the AR that are more effective than the current
AR antagonists at targeting the AR in AI-CaP.
Previous work from our laboratories has described a
series of bicyclic imide7 and hydantoin-based8 AR
antagonists (1, 2 & 3, Table 1). As found with the clin-
ically used anti-androgens bicalutamide3 (4) and
hydroxyflutamide3 (5), our series of AR antagonists
demonstrated potent binding (Ki) to, and functional
antagonism (IC50) of the wild type (WT) AR as found
in the MDA-453 cell line.9 Subsequent efforts led to
Keywords: Androgen receptor; Prostate cancer; AR antagonist;
CWR22R; [2.2.1]-Oxabicyclo.
*
Corresponding author. Tel.: +1 609 818 5259; fax: +1 609 818
5880; e-mail: mark.salvati@bms.co
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.02.006

M. E. Salvati et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1910–1915
1911
Table 1. Initial AR antagonist leads
O
H
H
H
O
O
O
O
N
N
N
N
CF3
N
CF3
N
CF3
CN
H
O
O
3
O
1
2
OH
OH
O
O2N
O
O
H
N
H
O
S
CF3
CN
N
CF3
O
N
O
F
NO2
O
H
bicalutamide (4)
6
hydroxyflutamide (5)
H
b
MDA-453 IC50 (nM)
Compound #
MDA-453 Kia (nM)
1
2
3
4
5
6
360
21
152
130
774
173
26
40
64
43
3.0
>5000
a Binding (Ki) determined through direct displacement with [3H]-DHT in the MDA-453 cell line.9
b Functional antagonist activity (IC50) determined through a transiently transfected reporter assay system utilizing the secreted alkaline phosphatase
reporter gene driven by the AR-dependent, PSA promoter.9
the discovery of a new series of [2.2.1]-oxabicyclo imide-
based analogs (6)9,10 which demonstrated potent bind-
ing to, but weak antagonism of the WT AR. Mouse oral
PK studies of key analogs from our imide, hydantoin
and oxabicyclo series of AR antagonists, as well as bica-
lutamide (Table 2), revealed that of our three series, the
oxabicyclo series possessed the most optimal oral PK
profile. As our goal was to identify a new orally active
anti-androgen for the treatment of CaP, we focused on
improving the in vitro profile of our new [2.2.1]-oxabicy-
clo imide-based AR antagonists.
For our first approach to improve upon the lead com-
pound 6, we explored replacement of the 4-nitronaph-
thyl group with a series of substituted aniline and
naphthyl groups which had previously been shown to
improve antagonist activity in our earlier series.7,8,10,12
As shown in Table 3, all attempts to replace the 4-nitro-
naphthyl group led to a dramatic drop in binding to the
AR (Ki), but improved overall antagonist activity (IC50).
Table 2. Mouse oral PK profiles11
Compound #
Dose
(mmol/kg)
Cmax
Tmax
AUC (0–6 h)
lM h
(lM)
(h)
Bicalutamide (4)
0.1
0.1
0.1
0.1
110
0.5
0.5
0.5
0.5
768
1
2
6
0.023
1.74
100
0.056
11.4
11.4
To aid us in our efforts to improve the potency of our
[2.2.1]-oxabicyclo imide series, we turned to a modeling
Table 3. SAR around aniline and naphthyl portiona
O
H
O
R
N
O
H
R
Ki (nm)
MDA 453 IC50 (nm)
Compound #
R
Ki (nM)
MDA 453 IC50
500
Me
Cl
53.0
670
350
480
2290
11
77.0
Br
I
Cl
Cl
Cl
NO2
Cl
221
377
486
12
13
14
692
1150
3310
2860
CF3
Cl
1110
1170
Cl
CF3
CN
Me
a Synthesis previously described.10,12 See Scheme 1.

1912
M. E. Salvati et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1910–1915
Table 4. SAR around aniline portion for 4,7-dimethyl analogsa
approach. Previous efforts from this lab have led to the
first crystal structures of the AR with DHT as well as
small molecule analogs from our earlier bicyclic imide
series.13,7 Using our available structural information,
we constructed a model of compound 6 bound into
the AR ligand binding domain (LBD).14 Utilizing the
minimized model shown in Figure 1, we explored poten-
tial sites where we could engineer interactions between
compound 6 and the AR LBD protein backbone. As
seen in Figure 1, we noted that adjacent to the C-4
and C-7 positions of the oxabicyclo ring system are
two methionine residues (M780 and M895). M780 is lo-
cated on the linker region between helix 6 and 7, on one
side of the ligand binding pocket, while M895 resides on
the hinge region of helix 12, on the opposite side of the
ligand binding pocket. Extending a methyl group from
the C-4 and C-7 position of the oxabicyclo core should
result in positive lipophilic interactions with each of
the methionine residues, generating improved binding
of the ligand to the LBD. The crystal structure of AR
O
O
R
N
O
H
H
Compound #
R
Kia (nM)
MDA 453b
IC50 (nM)
NC
18
17
7.0
7.0
19
F3C
NC
I
19
20
21
1.0
3.0
3.0
NC
S
Cl
16
Cl
O2N
22
23
24
25
26
27
28
17
4.0
12
17
27
3.0
18
11
38
F3C
NC
Cl
N
O
O
S
N
N
20
120
70
Figure 1. (A) Docking of a probe compound into the AR LBD. View
of AR LBD binding site with a portion of the backbone ribbon (red)
removed for clarity. Key side chains are displayed and colored by atom
type (C white, N blue, O red). Residues M780 and M895 are in gold
CPK rendering. Probe compound is shown in stick form colored by
atom type (C yellow, N blue, O red) with the C-4 and C-7 methyl
groups in green.
NH2
NH2
N
N
S
N
N
N
NC
NC
16
O
O
F3C
N
O
Compound 15
H
aKi = 4.1 μM
H
N
88
bMDA 453 IC50 = 11.5 μM
O
O
O
H
O
F3C
N
F3C
N
O2N
Br
O
Compound 16
aKi = 410 nM
Compound 17
aKi = 41 nM
O
H
29
17
40
H
H
bMDA 453 IC50 = 343 nM
bMDA 453 IC50 = 36 nM
a Synthesis previously described.12 See Scheme 1.
Figure 2. Effects of methyl groups at C-4 and C7 positions.

M. E. Salvati et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1910–1915
1913
NC
with DHT shows a similar interaction between M780
NC
I
O
O
and the C15-C16 atoms of DHT.13 Additional AR co-
crystal structures demonstrate interactions between
M895 and residues from several non-steroidal AR
agonists.15
O
H
O
H
OH
H
N
I
N
OH
H
O
O
H
H
Compound 34b:
Compound 34a:
a,cKi = 2.41 0.18 nM
a,cKi = 7.18 1.21 nM
To validate this model, we designed a series of [2.2.1]-
oxabicyclo imide analogs having a methyl at the C-4
or both the C-4 and C-7 positions of the molecule.10,12
Figure 2 shows the results of one set of analogs synthe-
sized. Compound 15 demonstrates weak binding to and
antagonism of the AR. Addition of a C-4 methyl group
(compound 16), demonstrates a 10-fold improvement in
binding and antagonist activity. The opposite antipode
of compound 16 showed a similar increase in binding
to the AR (data not shown). Addition of a second
methyl at the C-7 position yields compound 17, which
again results in a 10 fold increase in both binding and
potency as compared to the mono-methyl analog 16.
To expand on this observation, we once again explored
an array of substitutions at the aniline portion of the
molecule. As seen in Table 4, addition of a methyl group
at the C-4 and C-7 position of the [2.2.1]-oxabicyclo
imide core consistently resulted in improved binding to
and antagonism of the AR though a wide variety of ani-
line replacements. Through this exercise, we were able to
identify compound 19 as the most potent analog in our
series. Compound 19 demonstrates a 64-fold improve-
ment in binding affinity and a 25-fold improvement in
antagonist activity as compared to bicalutamide (4). De-
spite the potent activity of compound 19, this compound
was found to be rapidly metabolized by liver micro-
somes (human liver microsome rate: 0.8 nmol/min/mg
protein),16 and thus was not suitable for progression
as an oral agent.
bMDA 453 IC50 = 19 nM
bMDA 453 IC50 =10 nM
Human liver microsome rate:
0.001 nmol/min/mg protein
Human liver microsome rate:
0.04 nmol/min/mg protein
cValue calculated as average of 5 test occasions
Figure 3. Lead compounds 34a and 34b.
2-iodo-4-nitroaniline. 2-Iodo-4-nitroaniline was treated
with NaNO2 to form the diazonium salt, which was then
treated with a mixture of CuCN and KCN to yield 2-
iodo-4-nitrobenzonitrile. Reduction of the nitro group
was achieved with iron powder to give 4-amino-2-
iodobenzonitrile (30). 4-Amino-2-iodobenzonitrile and
furan-2,5-dione (31) were heated together in the pres-
ence of acetic acid to yield 4-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-yl)-2-iodobenzonitrile (32), which was car-
ried forward in a Diels–Alder reaction with 2,5-dimeth-
ylfuran to yield intermediate 33. Hydroboration of
intermediate 33 is achieved through treatment with bor-
ane-dimethylsulfide to yield the racemic mixture of com-
pounds 34a and 34b, which were separated by chiral
HPLC using standard conditions. The crystal structure
of compound 34a was determined, establishing the abso-
lute stereochemistry of both enantiomers.18
As seen in Figure 3, enantiomers 34a and 34b, demon-
strated high metabolic stability in liver microsomes
and potent binding to, and antagonism of, the AR.
Enantiomer 34b demonstrated an 3-fold increase in
binding relative to 34a, even though this compound
showed a slight decrease in antagonist activity. With this
data, we returned to our original model to try to better
understand the difference in binding found between the
two enantiomers. Figure 4A and B shows the binding
models for both enantiomers 34a and 34b in the AR
LBD structure. Interestingly, the models suggest that
for both enantiomers 34a and 34b a H-bond can exist
between the bridgehead oxygen as well as the C-5 hydro-
Past work in our group and literature reports10,12,17 had
shown that functionalization at the C-5 position of the
oxo-bicyclo ring system resulted in improved metabolic
stability. Based on this observation, we synthesized the
C-5 hydroxyl analog of compound 19. Scheme 1 shows
the general pathway used to make the two enantiomers
of the C-5 hydroxylated analog of compound 19, details
of which have previously been published.12 The synthe-
sis begins with commercially available 4-nitroaniline
which is treated with I2 in the presence of AgSO4 to yield
NH2
CN
O
O
N
I
I
a, b, c, d
e
O
CN
+
O
O
NH2
NO2
29
32
30
31
O
I
O
I
g, h
f
O
N
CN
O
N
CN
HO
O
O
33
34a(S) and 34b(R)
Scheme 1. Synthesis of C-5 hydroxy compounds 34a and 34b. Reagents and conditions: (a) I2, AgSO4, EtOH, 22 °C (95%); (b) NaNO2, HCl, H2O,
0 °C, then NaBF4, 0 °C; (c) CuCN (0.1 equiv), KCN (1.0 equiv), H2O, 22 °C (44% over b and c); (d) iron powder (325 mesh), NH4Cl (aq), THF,
EtOH, 60 °C (97%); (e) AcOH, 110 °C (90%); (f) neat 2,5-dimethylfuran, 60 °C (94%); (g) borane-dimethylsulfide, 0–25 °C, then Na2HPO4/KH2PO4
(pH 7), H2O2, 0 °C (25%); (h) chiral HPLC.

1914
M. E. Salvati et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1910–1915
Table 5. CWR22R xenograft study: mice were dosed orally in a vehicle
of PEG-400/Tween 80 (80:20) for 62 consecutive days with compound
34a or bicalutamide (4)
xyl group and N-705 of helix 3, in the AR LBD binding
pocket. In previous AR co-crystal structures, the N705
residue has been shown to make critical hydrogen bond
interactions with the C-17 hydroxyl group of DHT13 as
well as key hydroxyl residues of other non-steroidal AR
agonist ligands.15 In the case of enantiomer 34b, an
additional H-bond may occur from the C-5 hydroxyl
group to T877 of the AR LBD, which completely reca-
pitulates the bifurcated hydrogen bond network seen
with the C-17 hydroxyl of DHT to T877 of the AR
LBD in the co-crystal structure.13 Thus, the model ap-
pears to predict the observed binding data and suggests
that compound 34b should have improved binding rela-
tive to compound 34a, due to the additional formation
of an H-bond to T-877. As is often noted for ligands
of nuclear hormone receptors, the binding affinities of
compounds 34a and 34b do not correlate with their
functional antagonist activities.
1250
1000
Control vehicle
q1dx60;po;25
750
bicalutamide,
50 mg/kg,
q1dx60; po
34a,
500
10 mg/kg,
q1dx60;po
34a,
90 mg/kg,
250
0
q1dx60;po
20
30
40
50
60
70
80
90 100 110
Days post Implant
Drug
Dose (mpk)
Mean exposure @
1 and 24 h ( M)
34a
10
34a
90
bicalutamide (4)
50
Due to the improved metabolic stability16 and suitable
antagonist activity found with compound 34a, we ad-
vanced this compound forward into efficacy studies in
the CW22R human tumor xenograft model.19 Compound
26, 8
55.5, 23.6
4.6, 15.6
48.5, 86.0
19.4, 11.0
μ
Stdev (
3, 1.7
μM)
Plasma samples were taken 1 and 24 h post-dose on day 60 and drug
concentrations were measured.21 Tumor volumes were measured twice
weekly.21
34a was tested along side bicalutamide (4) in this model,
with daily oral dosing. As can been seen in Table 5, com-
pound 34a demonstrated superior efficacy in this model
despite achieving significantly lower plasma levels than
that of bicalutamide. It should be noted that the dose of
bicalutamide used in these studies generates plasma levels
consistent with levels clinically found in humans.20
In summary, we have identified a novel series of [2.2.1]-
oxabicycloimide-based AR antagonists. Through molec-
ular modeling approaches and an iterative drug design
process we identified several unique contact points in
the AR LBD binding pocket which we were able to ex-
ploit to increase the affinity of our early [2.2.1]-oxabicy-
clo imide-based leads. Ultimately, we were able to
identify the highly potent compounds 34a and 34b.
Compound 34a demonstrated significantly improved
efficacy relative to the clinically used anti-androgen bica-
lutamide (4) in the CWR22R human prostate xenograft
model. This work serves to demonstrate pre-clinical
proof of concept that novel AR antagonists can be
developed that may demonstrate improved efficacy for
the treatment of advanced CaP. The unique interactions
identified through our application of modeling have
since been exploited in the design of additional novel
AR antagonists for the treatment of advanced CaP,
the subject of which will be described elsewhere.
Figure 4. (A) Docking of compound 34a into the AR LBD. (B)
Docking of compound 34b into the AR LBD. View of AR LBD
binding site with a portion of the backbone ribbon (red) removed for
clarity. Key side chains are displayed and colored by atom type (C
white, N blue, O red). Dotted lines signify potential hydrogen bonds
from key amino acid residues (N705 and T877) to specific atoms on
ligands with the distance of each potential hydrogen bond listed in
angstroms. Compound 34a and 34b are shown in stick form colored by
atom type (C yellow, N blue, O red).
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16. In vitro metabolism was determined using pooled human
liver microsomes. The liver microsomal incubation mix-
tures contain the substrate compound (10 lM in DMSO),
microsomal protein (1 mg/mL), NADPH (1 mM), and
phosphate buffer (56 mM, pH 7.4). The reaction, was
conducted in triplicate, was initiated by the addition of
NADPH followed by incubation at 37 °C for 120 min.
Aliquots of samples (0.2 mL) were taken at various time
points over 120 min, and the reaction was quenched by the
addition of an equal volume of acetonitrile. Samples were
analyzed by standard HPLC method by comparing the
peak area ratio of the samples at each time point to the
peak area ratios at 0 h. The rate of metabolism was
determined from the amount of the parent compound
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18. Structure submitted into the Cambridge Structural Data-
base, Reg# CCDC 664455.
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11. The pharmacokinetics of test compounds were investi-
gated in male Balb/C mice. Groups of three male mice
(20 2 g) received a single oral dose of test compound at
10 mg/kg in PEG 400/water (80:20, v/v). The animals were
fasted overnight prior to dosing. After dosing, serial blood
samples were collected, by retro-orbital bleeding, in
microcentrifuge tubes at predose, 10, 30 min and 1, 2, 4,
6, 8, 10, and 24 h post-dose. Three blood samples were
obtained from each mouse. Samples from three mice were
obtained for each blood collection time point for a
composite pharmacokinetic profile. The blood volume
collected at each time point was 0.2 mL. Blood samples
were immediately centrifuged at 4 °C and the separated
serum was frozen on dry ice and stored at À20 °C until
LC/MS/MS analysis under standard conditions.
21. Blood samples were collected, by retro-orbital bleeding, in
microcentrifuge tubes at 1 and 24 h post-dose. Sample
work-up and analysis were as described in Ref. 10. Details
for the experimental procedure used for the CWR22
Human Prostate Xenograft study can be found in US-
7,141,578 B2.