Synthesis and biological evaluation of piperazinyl heterocyclic antagonists of the gonadotropin releasing hormone (GnRH) receptor

Article abstract of DOI:10.1016/j.bmcl.2010.02.099

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional antagonism in vitro. Additional structure-activity relationships within this series are reported along with a pharmacokinetic comparison to the benzimidazole-based lead molecule.

Full text of DOI:10.1016/j.bmcl.2010.02.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2512–2515
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of piperazinyl heterocyclic antagonists
of the gonadotropin releasing hormone (GnRH) receptor
Matthew D. Vera a, Joseph T. Lundquist IV a, Murty V. Chengalvala b, Joshua E. Cottom b, Irene B. Feingold c,
Lloyd M. Garrick a, Daniel M. Green a, Diane B. Hauze a, Charles W. Mann a, John F. Mehlmann a,
John F. Rogers a, Linda Shanno c, Jay E. Wrobel a, Jeffrey C. Pelletier a,
*
a Departments of Chemical & Screening Sciences, Wyeth Research, 500 Arcola Rd., Collegeville, PA 19426, USA
b Musculoskeletal Biology, Wyeth Research, 500 Arcola Rd., Collegeville, PA 19426, USA
c Drug Safety and Metabolism, Wyeth Research, 500 Arcola Rd., Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical
results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent
discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought
to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the
synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and
benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was
shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional
antagonism in vitro. Additional structure–activity relationships within this series are reported along with
a pharmacokinetic comparison to the benzimidazole-based lead molecule.
Received 22 January 2010
Accepted 26 February 2010
Available online 3 March 2010
Keywords:
GnRH
GnRH antagonist
Gonadotropin releasing hormone
Gonadotropin releasing hormone antagonist
GPCR
Ó 2010 Elsevier Ltd. All rights reserved.
Gonadotropin releasing hormone (GnRH) is a decapeptide syn-
thesized in the hypothalamus and released into the hypophyseal
circulation in a pulsating fashion, where it travels to the anterior
pituitary gland.1–3 GnRH acts to stimulate the G-protein coupled
GnRH receptor leading to the downstream release of follicle stim-
ulating hormone (FSH) and leuteinizing hormone (LH). The release
of these hormones leads to gametogenesis and the synthesis of sex
steroids. Consequently, antagonism of the GnRH receptor and the
resulting inhibition of sex steroid synthesis have shown clinical
promise in the treatment of hormone-dependent disorders such
as endometriosis and prostate cancer.4,5
the benzimidazole core to ensure reliable behavior in screening as-
says and consistency across templates in data analysis.
The heterocyclic replacements (3–10) for the benzimidazole
core template are shown in Scheme 1. Each template adds, changes
or repositions heteroatoms across the fused ring system. Templates
3 and 4 incorporate an additional nitrogen atom, giving imidazo-
pyridines. Imidazopyridines 5 and 6 formally migrate one of the
benzimidazole nitrogens of 1 into the six-membered ring. Tem-
plates 7 and 8 replace the benzimidazole system with regioisomer-
ic benzoxazoles, while
benzothiazoles.
9 and 10 are the corresponding
We have recently reported the elaboration and structure–activ-
ity relationships of piperazinyl benzimidazoles6–8 including com-
pound 1 (Table 1), which has demonstrated oral pharmacological
activity by lowering plasma levels of LH in rats.9 In order to further
explore structural novelty, binding potency and pharmaceutical
properties, we sought to compare several heterocyclic replace-
ments for the benzimidazole core template of 1. The substituted
imidazole derivative 2 had better solubility10 in aqueous buffer
than 1 and showed comparable binding potency at both the human
and rat GnRH receptors. Hence, the substituted imidazole side
chain was kept constant in our comparison of replacements for
Schemes showing the preparation of 3–10 are available as Sup-
plementary data. Additional details describing the preparation of 3
and 4,11 5 and 6,12 and 7–1013 have been reported elsewhere. Com-
pounds 9 and 10 are also available by a tandem benzothiazole ring
closure/Buchwald coupling reaction.14
Benzimidazoles 1 and 2 were prepared as described previously
by reductive amination of the appropriate aldehyde with the corre-
sponding piperazine derivative.8,15 As shown in Scheme 1, imidaz-
ole derivatives 3a through 10a were prepared using the analogous
reaction with 2-ethyl-5-methyl-imidazole-4-carboxaldehyde and
piperazinyl derivatives 3 through 10 (Scheme 1). Yields were gen-
erally high.
Compounds 3a through 10a were assayed for receptor binding
potency against the human GnRH receptor as shown in Table 2.
Binding data for 3a and 4a indicates the formal incorporation of
* Corresponding author. Tel.: +1 484 865 2912; fax: +1 484 865 9399.
E-mail addresses: Pelletj2@wyeth.com, pelletj@comcast.net (J.C. Pelletier).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.099

M. D. Vera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2512–2515
2513
Table 1
Table 2
Structures, GnRH receptor (GnRH-R) binding data and solubility for lead benzimid-
azole derivatives
GnRH receptor binding activity of 2-methyl-5-ethyl-imidazolyl derivatives of core
templates 3–10 (Scheme 1)
N
Compound
hGnRH receptor
rGnRH receptor
binding IC50
a,b,c
a,b,c
1
R =
R =
binding IC50
(nM)
(nM)
N
3a
4a
5a
6a
7a
8a
9a
10a
(32%)d
58
290
37
>10,000
9.8
N
N
R
N
2
(61%)d
>10,000
(70%)d
>10,000
HN
N
NH
Compound IC50 (nm) hGnRH-R
bindinga,b,c
IC50 (nm) rGnRH-R
bindinga,b,c
Solubilityd
g/mL)
a
Binding to overexpressed human or rat GnRH receptors in competition with
(
l
125I-(
D
-Trp6)-GnRH (see Ref. 8 for details).
b
1
2
12
10
72
34
1
20
Results are reported as an average of two independent experiments run in
triplicate.
c
Standard deviations for assay results were within 50% of the average.
Values in parentheses indicate percent inhibition at 500 nM.
a
Binding to overexpressed human or rat GnRH receptors in competition with
d
125I-(
D
-Trp6)-GnRH (see Ref. 8).
b
Results are reported as an average of two independent experiments run in
triplicate.
c
Standard deviations for assay results were within 50% of the average.
Concentration is measured after dilution of a DMSO stock solution into pH 7.4
effect on receptor binding. Benzoxazoles 7a and 8a are both signif-
icantly less active than 2, as are benzothiazoles 9a and 10a. How-
ever, when the oxygen or sulfur of both analogue pairs is proximal
to the piperazine ring (i.e., 8a and 10a) the analogues are signifi-
cantly less active than their regioisomeric counterparts (7a and
9a). Overall, these observations indicate that a nitrogen atom in
the five-membered ring which is situated proximal to the piperaz-
inyl substituent is an important determinant of GnRH receptor
binding activity. Moreover the receptor binding data for this series
suggested that the imidazopyridine template 6 would be a good
surrogate for the benzimidazole core of compounds 1 and 2.
We recently reported the beneficial effects on potency using the
appended imidazoles, uracils and quinoxalines shown in Figure
1.7–9 To investigate further the properties of 6 as a template for
d
buffer followed by overnight equilibration. See Ref. 10 for details.
a pyridine-type nitrogen into the six-membered ring of the benz-
imidazole core is tolerated only in the 4-position (4a) while the
5-analogue is much less potent (3a).
The imidazopyridines 5a and 6a also show disparate binding
potencies with 6a displaying low nanomolar binding affinity at
both the human and rat GnRH receptors. This level of binding po-
tency is comparable to that of benzimidazole 2. Compound 5a,
however, is significantly less potent, indicating that transposing
the benzimidazole nitrogen distal to the piperazine ring has little
O
H
N
N
H
N
N
NH
N
N
R
R
N
H
NaBH(OAc)3, NMP or DCE
3-10
3a-10a
R =
N
N
Y
HN
N
X
N
N
X
Y
7: X = O, Y = N
8: X = N, Y = O
9: X = S, Y = N
10: X = N, Y = S
3: X = N, Y = CH
4: X = CH, Y = N
5
6
Scheme 1. Reductive amination with templates 3–10 and 2-ethyl-4-methylimidazol-5-carboxaldehyde provided final products 3a through 10a. See Supplementary data for
synthetic schemes leading to compounds 3–10.

2514
M. D. Vera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2512–2515
R =
O
N
N
NH
N
N
N
H
O
H
6a
6b
6c
N
R
N
O
N
N
N
N
NH
N
N
N
O
6d
6e
6f
Figure 1. Derivatives of imidazopyridine template 6.
GnRH antagonists, several additional analogs were synthesized and
assayed for both receptor binding and functional antagonist activ-
ity. Hence compounds 6b–6f (Fig. 1) were prepared by reductive
amination of the corresponding aldehydes using template 6. Along
with 6a, these compounds were assayed for receptor binding to the
human and rat GnRH receptors. Additionally, all six compounds
were tested for functional antagonist activity by measuring the
in vitro inhibition of inositol phosphate in recombinant cells over-
expressing the human GnRH receptor. As an additional measure of
functional activity, the vitro inhibition of LH release in primary cul-
tures of rat anterior pituitary cells was assessed.
As shown in Table 3 6a–6f demonstrated nanomolar binding
potency at both the rat and human GnRH receptors, indicating that
template 6 is a general mimic of the benzimidazole core in terms of
receptor binding potency. Moreover, 6a–6f also showed good lev-
els of in vitro functional antagonist activity against the GnRH
Table 3
Receptor binding and functional antagonist potency for analogs derived from template 6
a,b,c
a,b,c
d
e
Compound
hGHRH binding IC50
(nM)
rGHRH binding IC50
(nM)
Human IP inhibition IC50 (nM)
Rat LH release IC50 (lM)
6a
6b
6c
6d
6e
6f
1b (Fig. 2)
1
2
9.8
6.7
4.3
2.3
44
47
5.8
12
37
12
95
5.7
29
81
24
72
34
60
41
20
5.0
39
90
15
28
18
0.92
0.42
4.4
0.26
1.5
3.4
0.20
0.36
0.16
10
a
b
c
Binding to overexpressed human or rat GnRH receptors in competition with 125I-( -Trp6)-GnRH (see Ref. 8 for details).
D
Results are reported as an average of two independent experiments run in triplicate.
Standard deviations for assay results were within 50% of the average.
d
e
Compound driven IP reduction in whole cells following stimulation with (D
-Trp6)-GnRH (see Ref. 8 for details).
Compound driven reduction in LH release from primary rat pituitary cells stimulated with (
D
-Trp6)-GnRH (see Ref. 8 for details).
N
N
N
N
N
N
HN
N
N
N
N
N
1b
6b
Figure 2. Benzimidazole and imidazopyridine derivatives chosen for pharmacokinetic evaluation.
Table 4
Pharmaceutical properties of selected imidazopyridine analogsa
Compound
PAMPA (Pe  10À6 cm/s)
Solubility (
l
g/mL)
Rat liver microsome t1/2 (min)
Cyp450 2C9% inhib.
Cyp450 2D6% inhib.
Cyp450 3A4% inhib.
6a
6b
6d
1b
0.2
1.1
1.2
2.7
3
9
6
3
21
11
6
6
10
0
8
9
0
78
54
54
73
21
27
14
a
See Ref. 10 for experimental details.

M. D. Vera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2512–2515
2515
Table 5
compared to 1b, do not support in vivo efficacy studies. As these
results suggest, the program continues to focus on benzimi-
dazoles.
Pharmacokinetic parameters for 6b and 1b
Parameter
Compound (route of administration)a,b,c
In conclusion, eight alternative heterocyclic templates were
prepared and derivatized to yield a set of analogs (3a–10a) which
were evaluated for GnRH receptor binding potency. Imidazopyri-
dine analog 6a showed binding potency comparable to that of
the benzimidazole leads 1 and 2. Several additional imidazopyri-
dine derivatives (6b–6f) demonstrated both binding and functional
GnRH antagonist activity in vitro. A comparative pharmacokinetic
study of an imidazopyridine analog and the corresponding benz-
imidazole showed the latter having better oral pharmacokinetics
as measured by oral exposure and bioavailability. The benzimid-
azole series continues to show superior in vitro and in vivo proper-
ties when compared to derivatives of templates 3–10.
6b (iv)
6b (po)
1b (iv)
1b (po)
Dose (mg/kg)
Tmax (h)
Cmax or C0 (ng/mL)
T1/2 (h)
AUC0–inf (ng * h/mL)
Cl (mL/min/kg)
Vss (L/kg)
3
—
30
1
—
30
0.8 0.4
788 146
1.1 0.2
1946 394
—
3.7 2.5
533 78
2.2 0.9
3444 933
—
1764 353
1.4 0.1
685 149.3
75.1 14.8
3.5 0.7
—
458 75
0.4 0.0
124 5.9
134 6.5
2.7 0.4
—
—
28
—
93
F (%)
a
Single-dose pharmacokinetic studies using orchidectomized Sprague-Dawley
rats (n = 3).
b
Dosing vehicle: iv = DMSO/PEG200, po = PEG400.
Values represent the mean standard deviation.
c
Acknowledgments
receptor as measured by inhibition of inositol phosphate and LH
release. Compound 6b and its direct comparator from the benz-
imidazole series (1b,Fig. 2) have very comparable levels of receptor
binding and in vitro functional activity. Three analogs (6a, 6b and
6d) showed sub-micromolar potency in the in vitro inhibition of
rat LH release, comparable to the level of activity of lead benzimi-
dazoles 1 and 2. These results demonstrate that the surrogacy of
the imidazopyridine template 6 extends beyond binding affinity
to in vitro functional antagonism, heightening our interest in pur-
suing compounds of this type.
We thank Magid Abou-Gharbia, Ron Magolda, Richard Lyttle
and Len Freedman for their support of this work. We also thank
our colleagues in the Wyeth Discovery Analytical Chemistry and
Compound Profiling groups for their expertise in compound
characterization.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.099.
In order to assess further the differences between the imidazo-
pyridine and benzimidazole derivatives, we sought to compare the
pharamocokinetics of the two templates with the side chain moi-
ety being held constant. In selecting a compound for further eval-
uation, we examined the pharmaceutical properties of the most
active members of the imidazopyridine series, as shown in Table
4. While 6d showed the highest level of potency (Table 3) in all
four in vitro assays, it suffered from a short half-life in rat liver
microsomes. Compound 6a, which had the longest microsomal
half-life, showed reduced cell permeability and a relatively high le-
vel of Cyp450 3A4 isozyme inhibition. While all compounds shown
in Table 3 exhibited relatively low solubility in pH 7.4 buffer, 6b
was modestly more soluble than the others. Consistent with our
previous efforts to balance structure–property relationships with
structure–activity relationships to obtain an orally active mole-
cule,9 6b appeared to have the best overall combination of
in vitro activity and pharmaceutical properties.
References and notes
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Imidazopyridine 6b and its benzimidazole comparator 1b were
dosed in orchidectomized Sprague-Dawley rats at 1 or 3 mg/kg iv
and 30 mg/kg orally. Pharmacokinetic parameters are shown in
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and 93%, respectively. Greater oral bioavailability and exposure
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1b has better permeability properties than 6b, making it a better
candidate for intestinal absorption following oral administration.
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