Discovery of a novel, orally active, small molecule gonadotropin-releasing hormone (GnRH) receptor antagonist

Article abstract of DOI:10.1021/jm060012g

Gonadotropin releasing hormone (GnRH) plays an important role in the biology of reproduction. The use of GnRH receptor antagonists has been reported in the literature for the treatment of breast, ovarian, and prostate cancers. In this article, we report the synthesis, in vitro characterization, pharmacokinetics, and pharmacodynamics of an orally bioavailable, potent, small molecule GnRH receptor antagonist N-{4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl) amino]pyrimidin-5-yl}-5-[3,3,6-trimthyl-2,3-dihydro-1H-inden-5-yl)oxy] -2-furamide (compound 1).

Full text of DOI:10.1021/jm060012g

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3362
J. Med. Chem. 2006, 49, 3362-3367
Discovery of a Novel, Orally Active, Small Molecule Gonadotropin-Releasing Hormone (GnRH)
Receptor Antagonist
Haitao Li,* Kenna L. Anderes, Eugenia A. Kraynov, David R. Luthin, Quyen-Quyen Do, Yufeng Hong, Eileen Tompkins,
Eric T. Sun, Ranjan Rajapakse, Ved P. Pathak, Lance C. Christie, Haresh Vazir, Rosemary Castillo, Margaret L. Gregory,
Mary Castro, Karen Nared-Hood, Genevieve Paderes, and Mark B. Anderson
Department of Medicinal Chemistry, Research Pharmacology, and Pharmacokenetics, Dynamics & Metabolism, Pfizer Global Research &
DeVelopment, 10770 Science Center DriVe, San Diego, California 92121
ReceiVed January 5, 2006
Gonadotropin releasing hormone (GnRH) plays an important role in the biology of reproduction. The use
of GnRH receptor antagonists has been reported in the literature for the treatment of breast, ovarian, and
prostate cancers. In this article, we report the synthesis, in vitro characterization, pharmacokinetics, and
pharmacodynamics of an orally bioavailable, potent, small molecule GnRH receptor antagonist N-{4,6-
dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-yl}-5-[3,3,6-trimthyl-2,3-dihydro-1H-inden-5-
yl)oxy]-2-furamide (compound 1).
Introduction
Results and Discussion
Gonadotropin-releasing hormone (GnRH) plays an important
role in the biology of reproduction.1 The GnRH decapeptide
(pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is pro-
duced in the hypothalamus. The decapeptide is released into
the pituitary, where GnRH interacts with high-affinity receptors
(7-transmembrane G-Protein coupled receptors). In the pituitary,
GnRH triggers the release of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH).2 GnRH, as the primary
regulator of LH, is consequently the primary regulator of the
sex hormones testosterone and estrogen.3 GnRH and its
analogues have stimulated much interest because of their
potential therapeutic benefit in treating sex-hormone-dependent
diseases, such as prostate, ovarian, and breast cancer as well as
endometriosis, uterine fibroids, benign prostate hyperplasia,
fertility disorders, and precocious puberty.4-7
GnRH receptor agonists have been clinically used in the
treatment of sex-steroid-dependent diseases. The agonists mimic
GnRH to stimulate receptors on the pituitary gland, mediating
the release of LH and FSH. After a transient rise in hormone
production or the flare effect, the administration of GnRH
receptor agonists results in a down-regulation of GnRH recep-
tors. GnRH receptor down-regulation leads to a decrease in
circulating levels of LH, testosterone, and estrogen.
In recent years, peptide GnRH receptor antagonists have been
available for clinical evaluation. They directly lower the
hormones levels without the observed flare effect associated
with the agonists.8 Several laboratories have reported active
nonpeptide GnRH receptor antagonists;9-17 few have shown oral
activity in animal models.18-21 In this article, we report the
synthesis, in vitro characterization, pharmacokinetics, and
pharmacodynamics of an orally bioavailable, potent, small
molecule GnRH receptor antagonist (compound 1). Our effort
toward the discovery of small molecule GnRH receptor antago-
nists started with the screening of our exploratory library.
Compound 2 was obtained as an initial lead.22,23 Following this
discovery, an efficient combination of targeted library syntheses
and medicinal chemistry approaches resulted in compound 1
as a potent, orally active GnRH receptor antagonist.
Chemistry. The synthesis of compound 1 is outlined in
Scheme 1. Friedel-Crafts alkylation of o-cresol 3 with 3,3-
dimethylacrylic acid provided indanone 4, which was reduced
by hydrogenation to yield indanol 5. Alkylation of phenol 5
with methyl 5-bromo-2-furoate afforded 6. Saponification of 6
provided furoic acid 7, which was acylated with 4,6-dimethoxy-
N-(3-morpholin-4-yl-propyl)-pyrimidine-2,5-diamine (11) to
furnish compound 1. The synthesis of diamine 11 is shown in
Scheme 2. Nitration of 2-chloro-4,6-dimethoxy-pyrimidine (8)
provided nitro-pyrimidine 9. Nucleophilic aromatic substitution
of 9 with 3-morpholin-4-yl-propylamine afforded compound 10.
The nitro group was then reduced by hydrogenation to provide
compound 11.
In Vitro Characterization. Compound 1 was characterized
in competition radioligand-binding assays utilizing 125I-GnRH-A
as previously described.24 Compound 1 bound to rat pituitary
and recombinant human GnRH receptors with identical binding
Ki values of 0.4 ( 0.1 nM. In cell-based functional assays,
compound 1 was an antagonist of GnRH-stimulated total inositol
phosphate accumulation with IC50 values of 3.3 ( 0.9 nM (Hill
coefficient of 0.96 ( 0.08; 1 nM GnRH in assay) and 8.8 (
1.5 nM (Hill coefficient of 0.83 ( 0.15; 100 nM GnRH in assay)
for recombinant human and rat receptors, respectively. GnRH
stimulated increases in total inositol phosphates with EC50 values
of 53 ( 16 nM (N ) 4; Hill coefficient of 1.2 ( 0.2) and 1.0
( 0.2 (N ) 4; Hill coefficient of 0.72 ( 0.2) for recombinant
rat and human receptors, respectively. In selectivity assays,
compound 1 was examined in 54 different assays including ion
channels, GPCRs, and monoamine transporters. The Ki values
were determined for hits at 10 µM. These are depicted in Table
1. With the exceptions of the Ca2+ channel assays, GnRH
selectivity of compound 1 was more than 1000-fold for all 54
assays examined. Compound 1 was subsequently profiled for
effects on basal (agonist) and KCl-stimulated Ca2+ channel
activity. Compound 1 produced no effect on basal Ca2+ channel
activity and inhibited KCl-stimulated Ca2+ flux with an IC50
value of 6.3 µM (nH 1.6), suggesting that although compound
1 readily binds to these channels, it has only modest effects as
a Ca2+ channel antagonist. Compound 1 has low potential for
drug-drug interactions mediated by CYP 1A2, 2C19, 2D6, and
* To whom correspondence should be addressed. Tel: 858-526-4659.
Fax: 858-526-4508. E-mail: haitao.li@pfizer.com.
10.1021/jm060012g CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/26/2006

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Gonadotropin-Releasing Hormone Receptor Antagonist
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3363
Chart 1
Scheme 1a
a
Reagents and conditions: (a) 3,3-dimethylacrylic acid, polyphosphoric acid, 105 °C; (b) H2, Pd/C, H2SO4, MeOH; (c) methyl 5-bromo-2-furoate,
Cs2CO3, DMF; (d) NaOH, MeOH; (e) compound 11, HATU, DIEA, DMF.
Scheme 2a
Conclusion
We have reported the discovery of a novel, small molecule
GnRH receptor antagonist, N-{4,6-dimethoxy-2-[(3-morpholin-
4-ylpropyl)amino]pyrimidin-5-yl}-5-[3,3,6-trimthyl-2,3-dihydro-
1H-inden-5-yl)oxy]-2-furamide (compound 1). Compound 1
exhibited picomolar affinities to human and rat GnRH receptors
and was more than 1000-fold selective for GnRH receptors
versus 54 G-protein-coupled receptors, other enzymes, and ion
channels. In gonad intact male rats, compound 1 dose-
dependently suppressed testosterone to below castrate levels for
12 h following a single oral dose. These data suggest that
compound 1 is a potent and selective, orally active GnRH
receptor antagonist that may have potential in treating sex-
hormone dependent diseases, such as prostrate cancer and
endometriosis.
a Reagents and conditions: (a) tetramethylammonium nitrate, triflic
anhydride, DCM; (b) 3-morpholin-4-yl-propylamine, DMF; (c) H2, Pd/C,
MeOH
Experimental Section
3A4 (IC50 > 30 µM). The IC50 value for CYP2C9 inhibition
was 3 µM (in-house data).
Chemistry. 5-Hydroxy-3,3,6-trimethyl-indan-1-one (4). To a
three-necked round-bottom flask assembled with a condenser,
thermometer, and mechanic stirrer under nitrogen, o-cresol 3 (1.197
mol, 124 mL) and 3,3-dimethylacrylic acid (1.52 mol, 154 g) were
added. The mixture was gently stirred and heated at 40 °C while
polyphosphoric acid (3.9 L) was added. After the addition of
polyphosphoric acid was completed, the contents were rapidly
heated to 105 °C and then promptly removed from the heating
mantle. The reaction was quenched by pouring the hot mixture into
a large bucket of ice water with constant stirring. The aqueous layer
was extracted with ethyl acetate. The organic layer was dried over
sodium sulfate and filtered, and the solvent was removed in vacuo.
The crude product was crystallized with ethyl acetate to obtain 21.0
g of compound 4 (9%). 1HNMR (300 MHz, CDCl3) δ 1.36 (s, 6H),
2.27 (s, 3H), 2.55 (s, 2H), 6.06 (bs, 1H), 7.50 (s, 1H), 7.83 (s, 1H).
APCI-MS: calcd for C12H14O2 190; found, 191, [M + H]+.
3,3,6-Trimethyl-indan-5-ol (5). To a solution of 5-hydroxy-
3,3,6-trimethyl-indan-1-one (21.5 mmol, 4.1 g) and sulfuric acid
(290 µL) in methanol (150 mL) was added Pd/C (4.3 mmol, 0.63
g). The ketone was reduced overnight under 40 psi of H2. The
reaction mixture was filtered through Celite. The filtrate was taken
to dryness to give a brown oil that was redissolved in ethyl acetate
and washed with water and brine. The organic layer was dried over
sodium sulfate and filtered, and the solvent was removed in vacuo
to yield a light-yellow oil. The crude product was purified by flash
column chromatography (ethyl acetate/hexane 1:3) to afford 3.6 g
Pharmacokinetics. When compound 1 was given to intact
male rats, it showed long T1/2 (5.1 h), low CLa (0.33 L/hr/kg),
and medium Vss (1.1 L/kg). The compound had an oral
bioavailability of 26% (Figure 1).
In Vivo Efficacy. In gonad intact rats, compound 1 dose-
dependently suppressed testosterone to below castrate levels for
12 h following a single oral dose (Figure 2). In gonad intact
rats, g2 µM plasma concentration of compound 1 was required
to sustain the suppression of testosterone at or below castrate
levels. In castrated rats, compound 1 dose-dependently sup-
pressed luteinizing hormone levels to below the detection limit
(0.12 mIU/mL) (Figure 3). Complete suppression of LH was
obtained in castrated rats at g1.2 µM concentration. The plasma
concentration was different between intact and castrated rats
because of the hormone-dependent metabolism of compound 1
as described in Iatsimirskaia et al.25 Following seven days of
oral treatment with compound 1 in intact rats, testosterone
suppression was sustained at or below castrate levels, and the
plasma concentration was maintained at 2 µM. Testosterone
levels returned to normal 24 h after the final dose (Figure 4).
a Abbreviations: bid, twice a day dosing; p.o., oral dosing; i.v.,
intravenous dosing; CL, clearance; Vss, volume of distribution.

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3364 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Table 1. Selectivity of Compound 1 in Broad Ligand Profilea
Li et al.
ref comp
Ki
(nM)
GnRH
fold
selectivity
compd 1
Ki (nM)
compd 1
nH
ref compd
assay
ref compd
IB-MECA
protriptyline
saralasin
SCH 23390
(+)butaclamol
clozapine
GBR12909
cimetidine
pirenzepine
4-DAMP
[Sar9,Met(O2)11]-SP
U50488
DAMGO
WEB 2086
ketanserin
mesulergine
serotonin
nH
A3b
628
2.0
2.8
1.2
2.1
1.8
2.4
2.7
1.0
2.0
2.3
1.3
1.7
1.4
1.2
1.5
1.3
1.7
1.9
1.2
0.8
11
0.76
0.53
3.8
35
6.2
877
14
1.2
0.9
1.5
1.0
1.0
1.3
1.1
2.0
0.8
1.0
1.4
0.9
1.3
1.0
0.7
1.1
1.1
1.1
1.0
1.4
1570
12725
9275
4050
2950
6275
4500
3050
10300
9200
3125
4900
3500
2650
1458
2410
4375
3575
363
NE transporterb
AT1b
5090
3710
1620
1180
2510
1800
1220
4120
3680
1250
1960
1400
1060
583
D1b
D3b
D4.4b
DA transporterb
H2
M1b
M4b
NK1b
1.1
Kappa opiate
Mu opiateb
PAF
0.28
0.42
1.2
0.67
0.65
0.43
1.9
5HT2Ab
5HT2Cb
964
5HT7b
1750
1430
145
5HT transporterb
L-type Ca2+
DHP site
L-type Ca2+
diltiazem site
L-type Ca2+
verapamil site
N-type Ca2+
imipramine
nitrendipine
0.57
370
2.0
1.0
1.7
diltiazem
15
1.1
0.9
1.3
925
166
D600
4.2
415
5240
ω-conotoxin GVIA
0.003
13100
a Assays all performed at Cerep. b human isoform utilized for assay.
Figure 1. Plasma concentrations of compound 1 in intact rats after
i.v. (5 mg/kg) and p.o. (20 mg/kg) administration. Data are means (
SD for n ) 3.
of compound 5 as a light-yellow solid (93%). 1HNMR (300 MHz,
CDCl3) δ 1.21 (s, 6H), 1.89 (t, 2H), 2.21 (s, 3H), 2.78 (t, 2H),
4.58 (s, 1H), 6.57 (s, 1H), 6.94 (s, 1H). Anal. (C12H16O) C, H.
5-(3,3,6-Trimethyl-indan-5-yloxy]-furan-2-carboxylic Acid
Methyl Ester (6). In a round-bottom flask assembled with a
condenser and gas outlet, a solution of compound 5 (22.73 mmol,
4.0 g), methyl 5-bromo-2-furoate (17.6 mmol, 3.6 g) and cesium
carbonate (22.8 mmol, 7.4 g) in DMF (45 mL) was heated to 100
°C for 7 h under a N2 atomosphere. The mixture was cooled to
room temperature and neutralized with 1 M HCl. The content was
extracted with ethyl acetate. The organic layer was dried over
sodium sulfate and filtered, and the solvent was removed in vacuo.
The crude product was purified by column chromatography (ethyl
acetate/hexane 1:5) to afford 5.8 g of compound 6 as a yellow oil
(84%). Compound 6 was partially pure and used in the next reaction
Figure 2. (a) Effect of a single oral dose of compound 1 (5.0, 25, 50,
and 100 mg/kg) on testosterone time course in intact male rats. The
testosterone level below 0.5 ng/mL is equivalent to the castrated levels
denoted by CX. (b) Plasma concentration (µM) of compound 1 (5.0,
25, 50, and 100 mg/kg) following a single oral dose to intact male
rats.
1
step without any further purification. HNMR (300 MHz, CDCl3)
δ 1.21 (s, 6H), 1.89 (t, 2H), 2.20 (s, 3H), 2.77 (t, 2H), 3.86 (s,
3H), 5.22 (d, 2H), 6.56 (s, 1H), 6.93 (s, 1H), 7.12 (d, 1H). APCI-
MS: calcd for C18H20O4 300; found 301 [M + H]+.
5-(3,3,6-Trimethyl-indan-5-yloxy)-furan-2-carboxylic Acid (7).
To a solution of compound 6 (19.3 mmol, 5.8 g) in methanol (10
mL) was added 4 M NaOH solution (20 mL). The reaction mixture
was stirred at room-temperature overnight. The clear brown solution
was acidified with 2 M HCl and extracted with ethyl acetate. The
combined organic layer was washed with brine, dried over sodium

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Gonadotropin-Releasing Hormone Receptor Antagonist
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3365
Figure 3. (a) Effect of a single oral dose of compound 1 (5.0, 20, 50, and 100 mg/kg) on luteinizing hormone time course in castrated male rats.
(b) Plasma concentration of compound 1 (5.0, 20, 50, and 100 mg/kg) in castrated male rats following a single oral dose.
Figure 4. Effect of seven-day pretreatment with compound 1 (100 mg/kg, p.o., bid) on testosterone time course in intact rats. The plasma samples
were collected following the last dose. The testosterone level below 0.5 ng/mL is equivalent to the castrated levels denoted by CX.
sulfate, filtered and the solvent was removed in vacuo. The crude
product was crystallized with CH3CN to afford 3.18 g compound
7 as a light yellow solid (57%). 1HNMR (300 MHz, CDCl3) δ 1.21
(s, 6H), 1.93 (t, 2H), 2.20 (s, 3H), 2.84 (t, 2H), 6.85 (s, 1H), 7.05
(s, 1H), 7.26 (d, 1H). APCI-MS: calcd for C17H18O4 286; found,
287 [M + H]+.
5-(3,3,6-Trimethyl-indan-5-yloxy)-furan-2-carboxylic Acid [4,6-
dimethoxy-2-(3-morpholin-4-yl-propylamino)-pyrimidine-5-yl]-
amide (1).To a solution of compound 7 (9.74 mmol, 2.8 g), 11
(8.0 mmol, 2.4 g), and HATU (10.2 mmol, 3.4 g) in DMF (18
mL), diisopropylethylamine (33.5 mmol, 6 mL) was slowly added
via a syringe under a N2 atomosphere. The reaction mixture was
stirred at room-temperature overnight. The solvent was removed
in vacuo. The crude mixture was purified by HPLC (32-95% in
70 min, CH3CN in water with 0.1M NH4OAc) to give 2.84 g of
compound 1 as a white solid (52%). It was dissolved in methylene
chloride and the solvent was removed in vacuo to give an
amorphous solid. 1HNMR (300 MHz, CDCl3) δ 1.21 (s, 6H), 1.81
(t, 4H), 1.93 (t, 2H), 2.23 (s, 3H), 2.49 (m, 6H), 2.84 (t, 2H), 3.48
(bt, 2H), 3.75 (t, 4H), 3.87 (s, 6H), 5.29 (d, 1H), 5.72 (bs, 1H),
6.82 (s, 1H), 6.95 (s, 1H), 7.05 (s, 1H), 7.10 (d, 1H). Anal.
(C30H39N5O6‚1.0H2O) C, H, N, O.
2-Chloro-4,6-dimethoxy-5-nitro-pyrimidine (9). In a 12 L flask
assembled with an overhead stirrer, thermometer, N2 inlet, and an
additional funnel were added tetramethylammonium nitrate (587
g, 4.31 mol) and dichloromethane (4 L). The contents were stirred
under a N2 atomosphere for 1 h at room temperature (20 °C). Triflic
anhydride (1216 g, 4.31 mol, 725 mL) was added dropwise in 45
min so that the temperature was not raised above 25 °C. The
contents were stirred at room temperature for 2 h. The reaction
mixture was then cooled to -78 °C in a dry ice/acetone bath.
Compound 8 (500 g, 2.87 mol) was dissolved in minimal amount
of DCM (3 L) and added dropwise over a 1.5 h time period. The
flow rate was critical in controlling the temperature and preventing
it from rising more than 5 °C. After the addition of compound 8,
the ice bath was removed, and the reaction was stirred over the
weekend under a N2 atomosphere. The reaction was quenched by
pouring the reaction mixture into ∼2 kg of ice. The contents were