3-[(2R)-amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3- methoxyphenyl)-6-methylpyrimidin-2,4-dione (NBI 42902) as a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor. Design, synthesis, and in vitro and in vivo characterization

Article abstract of DOI:10.1021/jm049218c

Further structure-activity relationship studies of a series of substituted uracils at the 1, 3, and 5 positions resulted in the discovery of several potent antagonists of the human gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylglycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6- difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-di-one (R-13b, NBI 42902) displayed subnanomolar binding affinity (K_i = 0.56 nM) and was a potent functional antagonist (IC₅₀ = 3.0 nM in Ca ²⁺ flux assay) at the human GnRH receptor. It also bound to the monkey GnRH receptor with high affinity (K_i = 3.9 nM). In addition, R-13b had good plasma exposure in cynomolgus monkeys after oral administration, with a C_max of 737 ng/mL and an AUC of 2392 ng/mL·h at a 10 mg/kg dose. Moreover, oral administration of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum levels of luteinizing hormone. These results demonstrate that compounds from this series of uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman primates.

Full text of DOI:10.1021/jm049218c

J. Med. Chem. 2005, 48, 1169-1178
1169
3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-
6-methylpyrimidin-2,4-dione (NBI 42902) as a Potent and Orally Active
Antagonist of the Human Gonadotropin-Releasing Hormone Receptor. Design,
Synthesis, and in Vitro and in Vivo Characterization
Fabio C. Tucci,† Yun-Fei Zhu,† R. Scott Struthers,‡ Zhiqiang Guo,† Timothy D. Gross,† Martin W. Rowbottom,†
Oscar Acevedo,† Yinghong Gao,† John Saunders,† Qiu Xie,‡ Greg J. Reinhart,‡ Xin-Jun Liu,‡ Nicholas Ling,†
Anne K. L. Bonneville,§ Takung Chen,§ Haig Bozigian,§ and Chen Chen*,†
Departments of Medicinal Chemistry, Endocrinology, and Preclinical Development, Neurocrine Biosciences, Inc.,
12790 El Camino Real, San Diego, California 92130
Received September 24, 2004
Further structure-activity relationship studies of a series of substituted uracils at the 1, 3,
and 5 positions resulted in the discovery of several potent antagonists of the human
gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylg-
lycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-
phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-di-
one (R-13b, NBI 42902) displayed subnanomolar binding affinity (Ki ) 0.56 nM) and was a
potent functional antagonist (IC50 ) 3.0 nM in Ca2+ flux assay) at the human GnRH receptor.
It also bound to the monkey GnRH receptor with high affinity (Ki ) 3.9 nM). In addition, R-13b
had good plasma exposure in cynomolgus monkeys after oral administration, with a Cmax of
737 ng/mL and an AUC of 2392 ng/mL‚h at a 10 mg/kg dose. Moreover, oral administration
of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum
levels of luteinizing hormone. These results demonstrate that compounds from this series of
uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman
primates.
Introduction
Since the first nonpeptidic antagonist of the human
gonadotropin-releasing hormone receptor (hGnRH-R)
T-98475 (1), which possesses high binding affinity (IC50
) 0.2 nM),7 was reported in 1998, several small mol-
ecules from different chemical classes have appeared in
the literature.8 3-Arylquinolones, represented by com-
pound 2, have been discovered as potent antagonists of
the hGnRH-R (IC50 ) 0.44 nM). Interestingly, unlike 1
which binds to the rat GnRH receptor (rGnRH-R) with
only moderate affinity (IC50 ) 60 nM), 2 also possesses
high binding affinity (IC50 ) 4 nM) at the rat receptor.9
Tryptamine compounds such as 3 are reported to be
potent GnRH antagonists, and several derivatives from
this class have showed oral bioavailability in rats and
dogs.10 Very recently, a potent antagonist of the hGnRH-
R, thieno[2,3-b]pyrimidin-2,4-dione TAK-013 (4), has
been reported to be clinically efficacious in humans in
suppressing luteinizing hormone when given orally.11
Previously, we had reported on a series of pyrazolo-
pyrimidones12 and imidazolopyrimidones13 such as 5
and 6 as potent antagonists of the hGnRH-R. In our
efforts to optimize small molecules toward oral bioavail-
ability, we focused on reducing the molecular weight of
these early compounds. On the basis of the successful
optimization toward high potency of imidazolopyrimi-
done derivatives, we designed and synthesized a series
of uracils (pyrimidin-2,4-ones) bearing a 3-(N-alkyl-
aminoethyl) group (7-9, Figure 1).14 While compounds
from this series such as 8 (Ki ) 0.5 nM) exhibit high
binding affinity,15 many of them, especially tertiary
amines such as 8, suffer from poor oral bioavailability
Gonadotropin-releasing hormone (GnRH), also known
as luteinizing hormone-releasing hormone (LH-RH), is
a linear decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-
Leu-Arg-Pro-Gly-NH2, originally isolated and character-
ized from porcine and ovine hypothalami.1 GnRH exerts
its action at the level of the pituitary by activation of
its cell surface receptor, a member of the class A
G-protein-coupled receptor superfamily,2 to stimulate
the secretion of the gonadotropins-luteinizing hormone
(LH) and follicle-stimulating hormone (FSH). These
hormones, in turn, act on the reproductive organs where
they participate in the regulation of steroid production,
gametogenesis, and ovulation.3 Several disease condi-
tions, such as endometriosis and prostate cancer, can
be treated by suppression of the pituitary-gonadal axis.
GnRH peptide superagonists, represented by leuprore-
lin,4 which activate and subsequently down-regulate the
receptor, are currently used in the treatment of these
conditions.5 Clinical evidence shows that peptidic GnRH
antagonists directly lower gonadal sex hormone levels,
alleviating disease symptoms without the concomitant
flare effect caused by superagonists.6 An orally bioavail-
able small molecule will have the advantage of flexible
dosing and titration of drug concentrations, which may
provide novel clinical management options.
* To whom correspondence should be addressed. Phone: 858-617-
7634. Fax: 858-617-7967. E-mail: cchen@neurocrine.com.
† Department of Medicinal Chemistry.
‡ Department of Endocrinology.
§ Department of Preclinical Development.
10.1021/jm049218c CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

1170 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Tucci et al.
Figure 1. Some small-molecule GnRH antagonists.
Scheme 1. Synthesis of
because of high clearance. One of the major metabolites
from the in vitro liver microsomes assay is identified
as the N-dealkylation product, which possesses low
binding affinity. Because an N-alkyl group such as a
benzyl moiety at the basic nitrogen is required for high
affinity in 7-9, we designed analogues by moving this
small lipophilic group from the nitrogen to the R-carbon
of the 2-aminoethyl side chain to eliminate one of the
nitrogen-carbon bonds that are easily cleaved through
oxidation by enzymes. In a previous communication, we
reported on our initial success by using this strategy
and synthesized potent compounds with improved meta-
bolic stability.16 In this paper we describe the details of
this structure-activity relationship study on compounds
bearing a 2-aminoalkyl side chain at the 3-position of
the uracils (13-16), as well as substitutions at the 5
and 1 positions (19, 20, and 24).17 Finally, we report on
the pharmacokinetic profiles and oral activity of repre-
sentative compounds from this series.
5-(2-Fluoro-3-methoxypheyl)uracils Bearing a
3-Aminoalkyl Side Chaina
a Reagents and conditions: (a) R- or S-R1CH(NR2Boc)CH2OH,
PPh3, DEAD, THF, room temp, 55-70%; (b) (i) 2-F-3-MeOPh-
B(OH)2, Pd(PPh3)4, Na2CO3, benzene, EtOH, DME, reflux; (ii) TFA,
CH2Cl2, room temp, 35-90%; (c) for 13, CH2O or cyclobutanone,
NaBH(OAc)3, CH2Cl2, room temp, ∼50%.
Chemistry
To examine the aminoalkyl side chain at the 3-posi-
tion of the uracil, 13 and 14 were synthesized according
to Scheme 1 starting from 1-(2,6-difluorobenzyl)-5-
bromo-6-methyluracil 10, which was obtained in three
steps from readily available chemicals as previously
reported.14 Thus, coupling reactions of 10 with either
R- or S-substituted N-Boc-aminoethanols, or their N-
methyl analogues, under Mitsunobu conditions (DEAD/
Ph3P in THF) gave the corresponding uracils 11 and 12
in 55-70% yields. These were then subjected to a
Suzuki coupling reaction with 2-fluoro-3-methoxyphen-
ylboronic acid catalyzed by palladium, followed by TFA
treatment, to afford the 5-aryluracils 13 and 14, respec-
tively, in 35-90% yields. Compounds 13 were N-
dimethylated with formaldehyde in the presence of
sodium triacetoxyborohydride to give 15 in about 50%
yield. Compound 13 could also be monoalkylated with
cyclobutanone to give the secondary amine 16a. To
explore the SAR at the 5-position, 5-bromouracils 17 or
18 bearing the R-2-aminophenethyl side chain was
subjected to Suzuki coupling reactions with various
Scheme 2. Synthesis of Uracils with Various
Substituted Phenyl Group at the 5-Positiona
a Reagents and conditions: (a) (i) ArB(OH)2, Pd(PPh3)4, Na2CO3,
benzene, EtOH, DME, reflux; (ii) TFA, CH2Cl2, 30-93%.
substituted phenylboronic acids to afford compounds 19
and 20 after deprotection (Scheme 2).
To quickly survey the effect of substitution at the
1-position of the 6-methyluracil, a new synthetic route
was developed and is shown in Scheme 3.17 Thus,
reaction of (2-fluorophenyl)acetone with chlorosulfonyl

Dione as Antagonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1171
Table 1. SAR of (R)- and (S)-3-Aminoalkyluracils 13-16 at the
hGnRH-R
Scheme 3. Synthesis of Uracils with Various
Substituents at the 1-Positiona
compd
R1
R2
R3
Ki (nM)
R-13a
S-13a
R-13b
S-13b
R-13c
S-13c
R-13d
S-13d
R-13e
S-13e
R-13f
S-13f
S-14a
R-14b
R-14c
S-15a
R-15b
S-15b
S-15c
R-15e
S-15e
S-16a
PhCH2
PhCH2
Ph
H
H
66 ( 11
H
H
16 ( 1.1
0.56 ( 0.03
22 ( 3.1
6.4 ( 1.0
39 ( 13
H
H
Ph
H
H
a Reagents and conditions: (a) ClSO2NCO, Et2O, room temp,
33%; (b) (R)-PhCH(NHBoc)CH2OH, PPh3, DEAD, THF, room
temp, 81%; (c) R4NH2, heat, 5-60%; (d) TFA, CH2Cl2, quantitative.
i-Bu
i-Bu
H
H
H
H
cyclohexyl
cyclohexyl
i-Pr
H
H
6.5 ( 1.0
46 ( 12
51 ( 18
970 ( 226
600 ( 87
>10000
8.1 ( 0.5
1.8 ( 0.3
1.2 ( 0.3
7.9 ( 1.5
6.5 ( 0.3
15 ( 1.3
7.5 ( 2.0
27 ( 11
H
H
Scheme 4. Acetylation of Amino Compound 19ba
H
H
i-Pr
H
H
Me
H
H
Me
H
H
PhCH2
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
i-Bu
H
PhCH2
Ph
Me
Me
Me
Me
Me
Me
a Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2, room temp,
93%.
Ph
i-Bu
i-Pr
i-Pr
1100 ( 460
3.6 ( 0.3
isocyanate, according to the general method previously
described by Hassner and co-workers,18 gave 5-(2-
fluorophenyl)-6-methyl-1,3-oxazine-2,4-(3H)-dione 21 in
33% yield after separation by crystallization. Alkylation
at the 3-position of 21 with R-N-Boc-2-phenylglycinol,
via a Mitsunobu protocol, gave 1,3-oxazine-2,4-dione 22
in 81% yield. Reaction of 22 with various primary
amines at 100 °C, followed by N-Boc deprotection, gave
the desired products 24 in 5-60% isolated yield. Anilines
failed to give the corresponding products under these
conditions.
Compound 19b was acetylated with acetic anhydride
in the presence of triethylamine to afford 25 in 93% yield
in order to examine the importance of the basic amine
in the binding mode of uracil-based GnRH antagonists
(Scheme 4).
Me
cyclobutyl
Table 2. SAR of 5-Phenyluracils 19 and 20 at the hGnRH-R
compd
R2
X
Ki (nM)
R-13b
19a
19b
19c
19d
19e
19f
19g
19h
20a
20b
20c
H
2-F, 3-MeO
3-MeO
0.56 ( 0.03
2.3 ( 0.1
3.4 ( 0.3
5.1 ( 0.4
13 ( 2.3
8.5 ( 0.9
2.8 ( 0.9
5.6 ( 0.7
3.1 ( 0.2
6.4 ( 0.5
5.6 ( 0.7
13 ( 2.5
H
H
3,4-OCH2O
3,4-OCH2CH2O
4-MeS
H
H
H
4-PhO
Pharmacology
H
2-Cl
The compounds herein synthesized were evaluated for
their ability to inhibit [125I-Tyr5,DLeu6,NMeLeu7,Pro9-
NEt]GnRH agonist binding to the cloned human, mon-
key, and rat GnRH-Rs as previously described,19 and
the data are summarized in Tables 1-3. Selected
compounds were tested in the inhibition of GnRH-
stimulated calcium flux in RBL cells stably expressing
the hGnRH-R to determine functional antagonism.
Table 4 summarizes the binding affinities of selected
compounds at the human, monkey, and rat GnRH
receptors, as well as functional antagonism of these
compounds at the human receptor.
Previously, we reported on the structure-activity
relationships of a series of uracils bearing a N-alkyl-
aminoethyl side chain at the 3-position as potent
antagonists of hGnRH-R.13c For example, by incorpora-
tion of a methyl group at the â- or R-position of the
3-side-chain in the initial lead 7, potent antagonists
exemplified by 8 and 9 were identified. However,
H
2-F
H
2-F, 3-Me
3-MeO
Me
Me
Me
3,4-OCH2O
3,4-OCH2CH2O
N-dealkylation by liver enzymes in the in vitro assay
generates a 3-(aminopropyl)uracil metabolite, which,
without a small N-alkyl group, is much less potent in
binding. For example, the primary amine S-13f was
inactive, while the corresponding N-cyclobutyl analogue
S-16a had a Ki value of 3.6 nM. We anticipated that
compounds bearing a 3-side-chain derived from an
amino acid, in which a small lipophilic group required
for high receptor binding is attached through a carbon-
carbon bond, should be metabolically more stable. The
successful Mitsunobu reaction of uracil 10 at the 3-posi-
tion with an alcohol allowed us to quickly examine a
series of chiral N-Boc-amino alcohols, readily available
from commercial sources or from the corresponding

1172 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Tucci et al.
Table 3. SAR of 1-Substitutions of Uracils 24a-t at the
phenylglycinol S-13b (Ki ) 22 nM). More importantly,
it was dramatically more active than the S-alaninol
analogue S-13f (Ki > 10 µM). All these results may
suggest that, in addition to a key interaction with the
basic amine, the receptor possesses a relatively large
pocket lined by lipophilic amino acids to accommodate
a group such as a phenyl or cyclohexyl moiety from the
R-isomeric ligands, or a benzyl moiety from the S-
isomer.
hGnRH-R
compd
R4
Ki (nM)
23
>10000
1200 ( 210
880 ( 83
570 ( 69
140 ( 24
880 ( 100
64 ( 9
Interestingly, addition of a small methyl group to the
amino nitrogen of the R-isomers of these aminoethyl
derivatives impacted their binding affinity in an un-
predicted fashion. For example, mono- and dimethyl-
ation of the phenylglycinol derivative R-13b reduced its
binding affinity about 3- and 11-fold, respectively (R-
14b and R-15b). On the other hand, methylation of the
leucinol derivative R-13c increased potency over 5-fold
(R-14c, Ki ) 1.2 nM), and this was also observed for
the S-isomers (S-13a, S-14a, and S-15a). One possible
explanation for these changes is that the methylation
causes the lipophilic side chain to adopt alternative
conformations and interact with the receptor in a more
or less favorable way. However, alkylation of the much
smaller alaninol side chain of S-13f (Ki g 10 µM) with
the small cyclobutyl group led to a dramatic increase
in binding affinity (S-16a, Ki ) 3.6 nM), and this
improvement in potency most likely is caused by the
additional lipophilic interaction (Table 1).
The SAR of the 5-aromatic group of uracil was also
briefly examined with the 3-side-chain kept constant as
either (R)-2-aminophenethyl or (R)-2-(N-methyl)amino-
phenethyl, and the results are shown in Table 2. While
the 3-methoxy-, 3,4-methylenedioxy-, and 3,4-ethylene-
dioxyphenyl derivatives (19a-c) exhibited good binding
affinities (Ki ) 2.3-5.1 nM), their N-methylation (20a-
c) resulted in about 2-fold reduction (Ki ) 5.6-13 nM).
The large and lipophilic 4-phenoxyphenyl derivative
(19e) possessed a Ki value of 8.5 nM, similar to the
4-methylthiophenyl analogue (19d, Ki ) 13 nM). The
2-chloro-, 2-fluoro-, and 2-fluoro-3-methylphenyl com-
pounds (19f-h) had Ki values of 2.8-5.6, and the
2-fluoro-3-methoxyphenyl analogue R-13b (Ki ) 0.56
nM) was significantly more potent than the 3-methoxy-
phenyl 19a and 2-fluorophenyl 19g. The increase in
binding affinity of R-13b over 19a is most likely a
consequence of the enforcement of an orthogonal con-
formation between the 5-(2-fluoro-3-methoxyphenyl)
ring and the uracil core. This phenomenon was observed
in the X-ray crystal structure of R-13b (Figure 2).
24a
24b
24c
24d
24e
24f
MeOCH2CH2
c-PrCH2
i-PrCH2
c-HxCH2
PhCH2CH2
2-PyCH2
24g
24h
24i
24j
24k
24l
24m
24n
24o
24p
24q
24r
19g
24s
24t
3-PyCH2
980 ( 120
53 ( 4
PhCH2
4-FC6H4CH2
3-FC6H4CH2
2-FC6H4CH2
2-ClC6H4CH2
2-BrC6H4CH2
2-MeC6H4CH2
2-MeOPhCH2
2-CF3SC6H4CH2
2-CF3C6H4CH2
2-CF3-5-FC6H4CH2
2,6-F2C6H4CH2
2-Cl-6-FC6H4CH2
2-Cl-4-FC6H4CH2
63 ( 5
61 ( 4
20 ( 2
14 ( 4
4.6 ( 0.9
20 ( 5
100 ( 37
180 ( 19
3.1 ( 0.3
3.9 ( 0.3
5.6 ( 0.7
0.61 ( 0.1
21 ( 6
Table 4. Species Selectivity and Antagonistic Activity of
Uracils at the GnRH-Rs
GnRH-R binding Ki (nM)
IC50 (nM)a
compd
human
monkey
rat
Ca2+ fluxb
R-13b
R-13d 6.5 ( 1.0
S-14a
R-14b
R-15b
19a
0.56 ( 0.03 3.5 ( 0.3
3000 ( 850
3200c
3.0
6.5
20
120 ( 11
200 ( 17
15 ( 9.0
8.1 ( 0.5
1.8 ( 0.3
6.5 ( 0.3
2.3 ( 0.1
3.4 ( 0.3
5.1 ( 0.4
6.4 ( 0.3
5.6 ( 0.7
0.61 ( 0.1
12000 ( 4700
120 ( 3.7 11000 ( 2300
49
5.0
6.2
10
34
45 ( 6.1
42 ( 4.5
74 ( 12
65 ( 12
100 ( 7
6.4 ( 1.8
7600 ( 1100
9900 ( 2400
18000 ( 4700
7700 ( 1300
19b
19c
20a
20b
24s
2900 ( 110
2.8
a Average of two or more independent determinations. b Func-
tion at the hGnRH-R. c Single determination.
amino acids by a simple reduction. We examined both
R- and S-isomers to determine the chiral selectivity for
high affinity to the receptor.
Among the primary amines we examined, the com-
pound derived from R-phenylglycinol possessed the best
binding affinity (R-13b, NBI-42902, Ki ) 0.56 nM),
followed by R-leucinol and R-cyclohexylglycinol deriva-
tives (R-13c and R-13d, Ki ) 6.4 and 6.5 nM, respec-
tively). Smaller lipophilic side chains showed further
reductions in potency (R-13e and R-13f, Ki ) 51 and
600 nM, respectively) On the other hand, the larger
R-phenylalaninol R-13a only exhibited a Ki value of 66
nM, suggesting that a lipophilic group is important for
binding, but there is a size limit for the interaction with
the hGnRH-R for the R-isomers.
While the S-configured compounds 13 generally pos-
sessed lower binding affinity than the corresponding
R-isomers, the S-phenylalaninol derivative (S-13a, Ki
) 16 nM) was about 4-fold more potent than the
corresponding R-isomer, about equivalent to the S-
A survey of the three-dimensional homology models
for the hGnRH-R, built from the human receptor protein
by using the X-ray structure of bovine rhodopsin as a
template,2b,20 suggests that two amino acids, Tyr-283
and Tyr-284 in the middle of the transmembrane helix
6, are possible residues to interact with the electron-
deficient 1-(2,6-difluorobenzyl) group of R-13b and its
close analogues. A cavity formed by these two residues
and Tyr-290 around the Gly-267 could be perfect to host
the 2,6-difluorobenzyl group. These two amino acids
have been implicated in the binding and function of the
peptide GnRH ligand, and thus, either Y283A or Y284A
mutant cannot be activated by GnRH.20 In addition,
these residues align with the highly conserved aromatic
amino acids of many class A aminergic G-protein-

Dione as Antagonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1173
and a bulky chlorine at the two ortho positions resulted
in compound 24s with subnanomolar binding affinity
(Ki ) 0.61 nM). An additional fluorine at the para
position of the 1-(2-chlorobenzyl) group of 24l or at the
meta position of the 1-(2-trifluoromethylbenzyl) group
of 24q had no effect on binding affinity (24t and 24r,
Ki ) 21 and 3.9 nM, respectively).
These results may indicate that a bulky ortho sub-
stituent such as chlorine at the benzyl group favors a
certain conformation that interacts well with the recep-
tor and could be similar to that observed in the X-ray
structure of R-13b. The SAR results also suggest that
the 1-benzyl group interacts with an electron-rich
aromatic ring such as tyrosine. Thus, an electron-
deficient aromatic ring with a proper dipolar moment
would be optimal to have a strong edge-to-face σ-π
interaction22 or offset face-to-face π-π stacking with the
tyrosine residues.23
Figure 2. X-ray crystal structure of R-13b indicates an
orthogonal relationship between the 5-(2-fluoro-3-methoxyphe-
nyl) group and the uracil ring.
Acetylation of 19b at the basic nitrogen afforded the
amide 25, which exhibited almost 500-fold reduction in
binding affinity (Ki ) 1.5 µM). These data suggest that
the basic nitrogen is very important for high binding
affinity, possibly through charge-charge attraction.
However, since the acetyl group is quite large, it may
cause a steric clash with the receptor and consequently
a conformational change of the 3-side-chain of 19b. A
hint of this hypothesis is the phenomenon observed from
the methylation of 19b, which led to a modest reduction
in binding affinity in 20b. The role of this basic amine
in receptor binding is not very clear.
Characterization of Atropisomeric 5-(2-Fluoro-
phenyl)-6-methyluracils. As seen in the X-ray crystal
structure of R-13b, the 5-(2-fluoro-3-methoxy)phenyl
ring lay in an orthogonal orientation to the uracil ring.
While this feature contributed to binding affinity at the
hGnRH-R, it also created a unique property for these
compoundssan interconvertible mixture of atropisomer-
ers. Thus, when examined by NMR, all compounds
containing a 5-(2-fluorophenyl) or 5-(2-fluoro-3-meth-
oxyphenyl) group displayed two distinct sets of signals
in proton, fluorine, and carbon spectra (see Experimen-
tal Section for details). The ratio of the mixture was
typically 1:1. This observation was somewhat surpris-
ing, since the des-fluoro analogue 19a did not show
evidence of isomers, at least by NMR spectroscopy. The
van der Waals radius of a fluorine is comparable to that
of hydrogen (1.47 versus 1.20 Å), so it is difficult to infer
that steric hindrance plays a major role in the slow
rotation around the carbon-carbon bond between the
5-phenyl ring and the uracil. Although variable tem-
perature NMR experiments did not result in coalescence
of the two sets of signals at 90 °C in D2O, this finding
was confirmed by HPLC analysis. On a chiral column,
these two rotameric diastereoisomers were easily sepa-
rated at room temperature. The atropisomeric proper-
ties and their significance will be further discussed
separately.24
coupled receptors, which are known to face the putative
binding pocket and to be involved in the interaction with
small-molecule ligands.21 We investigated the structure-
activity relationships of the substituent at the 1-position
to explore this potential interaction further. By use of
the chemistry shown in Scheme 3, several analogues
were quickly synthesized and tested, and the results are
summarized in Table 3. The oxazine 23, which lacks the
2,6-difluorobenzyl moiety of R-13, had no measurable
binding affinity to the hGnRH-R. Methoxyethyl at this
position resulted in a compound with poor affinity (24a,
Ki ) 1.2 µM). Increasing lipophilicity such as isobutyl
afforded analogues with moderate binding affinity (i.e.,
24c, Ki ) 570 nM), which was improved further in
cyclohexylmethyl analogue 24d (Ki ) 140 nM). 24d was
the most potent analogue without an aromatic func-
tionality at this position. In comparison with the phen-
ethyl compound 24e (Ki ) 880 nM), the benzyl analogue
24h exhibited much better binding affinity (Ki ) 53 nM).
Interestingly, the 2-pyridylmethyl compound (24f, Ki )
64 nM) possessed much higher potency than the 3-
pyridyl analogue (24g, Ki ) 980 nM). These results
indicate that an aromatic ring connected by a methylene
is favored at this site.
To further explore the SAR at this site and to improve
the affinity of 1-benzyluracils as antagonists of the
hGnRH-R, we carefully examined a series of substitu-
tions at the benzyl group. Among the monofluorinated
analogues, the 2-fluoro derivative (24k, Ki ) 20 nM)
exhibited a binding affinity about 3-fold better than that
of the parent 24h, while 3- or 4-fluorination (24i and
24j) had little effect. For the 2-substituent, it is apparent
that the binding affinity increased along with the size.
Thus, the Ki values for the 2-fluoro 24k, 2-methyl 24n,
2-chloro 24l, and 2-bromo compound 24m were 20, 20,
14, and 4.6 nM, respectively. The electron-donating
2-methoxy substitution resulted in a 2-fold reduction in
binding (24o, Ki ) 100 nM), whereas the electron-
deficient 2-trifluoromethyl improved binding affinity
(24q, Ki ) 3.1 nM).
Species Selectivity and Functional Antagonism
of Selected Compounds. Compounds from this series
also bound to the monkey GnRH receptor with good
affinity, although typically a 10-fold or more reduction
was observed (Table 4). However, none of the com-
pounds in this study bound well to rGnRH-R, and the
best affinity was about 3 µM (R-13b, R-13d, and 24s).
These observations prompted us to investigate disub-
stituted benzyl groups. While a second fluorine at the
6-position of the 2-fluorophenyl ring (19g, Ki ) 5.6 nM,
Table 2) increased binding affinity by about 4-fold, the
combination of a strongly electron-withdrawing fluorine

1174 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Tucci et al.
Table 5. Pharmacokinetic Characterization of Uracils in
Cynomolgus Monkeysa
CL
Vd
t1/2 Tmax
Cmax
AUC
F
compd (ml/(min‚kg)) (L/kg) (h) (h) (ng/mL) (ng/(mL‚kg)) (%)
R-13b
S-14a
19a
12.6
13.5
16.9
15.5
3.4 3.1 3.0
3.6 3.1 5.5
4.9 3.3 2.3
4.6 3.5 4.0
737
98
380
420
2392
912
1126
3280
16
22
9.9
32
19b
a Dosed at 10 mg/kg for both po and iv administrations.
For example, R-13b exhibited Ki values of 0.56, 3.5, and
3000 nM at the human, monkey, and rat GnRH recep-
tors, respectively.
Compounds R-13b, 19a, 19b, and 24s possessed good
functional antagonism at the hGnRH-R. The IC50 values
for these compounds ranged from 2.8 to 6.2 nM in
inhibition of GnRH-stimulated calcium flux in RBL cells
expressing the hGnRH-R.
Figure 3. Suppression of plasma LH concentrations in
castrated male cynomolgus macaques after administration of
R-13b at 40 mg/kg orally (b), at 10 mg/kg intravenously (1),
or with intravenous vehicle (0). Values shown are the mean
( SEM of bioactive LH levels expressed as a percentage of
pretreatment LH levels for each of three individual animals.
Pharmacokinetics. The pharmacokinetic profiles of
selected compounds were determined in cynomolgus
monkeys to study their oral exposure. Compound 19b
gave, after a 10 mg/kg dose intravenously, a plasma
clearance (CL) of 15.5 mL/(min‚kg), a volume distribu-
tion (Vd) of 4.6 L/kg, and a terminal half-life (t1/2) of 3.5
h. After oral administration of 19b at a dose of 10 mg/
kg, the plasma concentration reached maximal 420 ng/
mL at 4 h, the plasma level area under the curve (AUC)
was 3280 ng/mL‚h. The oral bioavailability calculated
from these data was 32%. These results confirmed that
adequate exposure of compounds of this class could be
obtained in the cynomolgus monkeys after oral admin-
istration. Similarly, both compounds 19a and R-13b had
good exposure after an oral dose of 10 mg/kg. Thus, the
Cmax values for 19a and R-13b were 380 and 737 ng/
mL, respectively, and the oral AUC values were 1126
and 2392 ng/mL‚h, respectively (Table 5).
series of compounds have lower molecular weight (R-
13b, MW ) 495) than most known GnRH antagonists.
Compounds from this class also exhibited good oral
bioavailability in cynomolgus monkeys (32% for 19b).
Compound R-13b was characterized to possess good
binding affinity with a Ki of 0.56 nM and potent
functional antagonism with an IC50 of 3.0 nM in
inhibition of GnRH-stimulated calcium flux at the
hGnRH-R. This compound was moderately lipophilic
with a log D value of 2.4. Its hydrochloride salt was
readily soluble in water (>10 mg/mL). Compound R-13b
also possessed a Ki value of 3.5 nM at the monkey
receptor, and it significantly suppressed the plasma
luteinizing hormone level by oral administration in this
species. The ability of this compound to suppress
circulating LH concentrations following oral adminis-
tration to postmenopausal women will be reported
elsewhere.
In Vivo Studies with Compound R-13b. Because
all uracil derivatives synthesized herein lack the neces-
sary high binding affinity to the rat receptor, in vivo
pharmacology from selected compounds was character-
ized in castrated male cynomolgus macaques. Three
monkeys each received various doses of R-13b, vehicle,
or other GnRH antagonists as part of the evaluation of
multiple advanced compounds, and circulating lutein-
izing hormone (LH) concentrations were measured.
Data from the R-13b (at 40 mg/kg po, at 10 mg/kg iv,
and with vehicle iv) are presented in Figure 3. Sup-
pression of circulating LH was observed with nadirs
ranging from 33% to 43% of the individual predose
concentrations at 4-8 h after administration. Suppres-
sion by the 40 mg/kg oral dose was roughly equivalent
to the 10 mg/kg intravenous dose. After 24 h, LH levels
returned to baseline, demonstrating that the suppres-
sion was reversible.
Experimental Section
Chemistry. Proton, carbon, and fluorine NMR spectra were
recorded on Varian spectrometer (Mercury) using TMS as the
internal standard and CDCl3 as the solvent except where
indicated. LC-MS analyses were performed on a Perkin-Elmer
Sciex API-100 mass spectrometer using the electrospray
ionization technique or on a SpectraSystem P4000 HPLC
system coupled with a Finnigan LCD/Deca mass spectrometer
using the electrospray ionization technique. HRMS ESI was
carried out on a Bruker 4.7T BIOAPEX FTMS mass spectrom-
eter. All compounds were characterized by LC-MS and proton
NMR (300 MHz). All commercially available reagents were
used without further purification.
3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-6-
methyl-5-(2-fluoro-3-methoxyphenyl)pyrimidin-2,4-di-
one Hydrochloride (R-13b). To a solution of triphenylphos-
phine (104.1 g, 397 mmol), 1-(2,6-difluorobenzyl)-5-bromo-6-
methyluracil (10, 87.7 g, 265 mmol) and Boc-R-phenylglycinol
(66.0 g, 278 mmol) in THF (3.5L) under nitrogen atmosphere,
was added diethyl azodicarboxylate (69.2 g, 397 mmol). The
reaction mixture was stirred at room temperature for 16 h.
The solvent was removed under reduced pressure, and the
crude was purified by column chromatography on silica gel
(hexane/EtOAc ) 2:1) to afford 3-[(2R)-(tert-butoxycarbonyl-
amino)-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-bromo-6-meth-
yluracil 11b as a white solid (80.0 g, 55%).
Conclusion
We have reported on the design and synthesis of a
series of uracil compounds bearing a 2-aminoalkyl group
at the 3-position as potent antagonists of the hGnRH-
R. Significant improvements in antagonist activity, as
well as pharmacokinetic properties, for this class have
been achieved by incorporation of a phenyl group at the
3-side-chain of the uracil core. SAR study at the 1-posi-
tion also results in identification of electron-deficient
benzyl groups as the most favored at this position. This
In a pressure vessel, the bromide (11b, 2.20 g, 4.0 mmol)
was dissolved in a 45:5:50 v/v/v mixture of benzene, EtOH,
and 1,2-dimethoxyethane (80 mL). To that, 2-fluoro-3-meth-
oxyphenylboronic acid (816 mg, 4.8 mmol) was added, followed
by an aqueous saturated solution of Ba(OH)2 (28.6 mL). The
resulting heterogeneous mixture was degassed by N2 bubbling

Dione as Antagonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1175
for 30 min. Tetrakis(triphenylphosphine)palladium (462 mg,
0.4 mmol) was added, and the vessel was tightly capped and
immersed in an oil bath with the mixture being stirred at 80
°C. The reaction mixture was maintained at that temperature
for 15 h, after which time the reaction was checked by TLC
and LC-MS. Only a small amount of starting material
remained. The reaction mixture was cooled, diluted with
EtOAc, and washed with H2O. The organic layer was washed
with brine, dried over anhydrous MgSO4, filtered, and evapo-
rated. The residue was purified by column chromatography
on silica gel, eluting with a 4:1 v/v mixture of hexanes and
ethyl acetate. The R-13b free base was obtained as a white
foam (1.32 g, 2.2 mmol, 55%). The corresponding hydrochloride
salt was obtained as a white solid by dissolving the free base
in CH2Cl2 and treating with HCl (4.0 M solution in dioxane, 1
the mixture was further stirred for 10 h and then diluted with
DCM, washed with water, dried (sodium sulfate), and evapo-
rated. The crude product was purified by flash chromatography
(silica, 20-50% EtOAc/hexanes).
The above free base was converted to the hydrochloride salt
by dissolution in dichloromethane and addition of 1 N HCl
solution in ether, followed by removal of the solvent under
vacuum to provide the HCl salt as an amorphous solid. 1H
NMR (DMSO-d6): δ 1.12 (d, J ) 5.1 Hz, 3H), 1.75 (m, 2H),
2.14 (m, 2H), 2.18 (s, 3H), 2.22 (m, 2H), 3.38 (m, 1H), 3.82 (m,
1H), 3.86 (s, 3H), 4.05 (m, 2H), 5.27 (s, 2H), 6.77 (m, 1H), 7.08-
7.22 (m, 4H), 7.42 (m, 1H)9.10 (brs, 2H). 19F NMR: δ -115.0
(t, J ) 7.7 Hz, 2F), -135.3 and -135.4 (t, J ) 4.5 Hz, 1F). 13
C
NMR: δ 14.3, 14.7 and 14.8, 17.5, 26.3 and 26.4, 26.5, 38.8,
42.6, 48.9, 49.9, 56.0, 106.9, 111.9 (m, 2C), 112.1 (t, J ) 17
Hz), 113.5, 122.2 (d, J ) 13.6 Hz), 123.6, 124.0, 130.1 (t, J )
10.7 Hz), 147.4 and 147.5, 149.5 (d, J ) 242 Hz), 151.1 and
151.2, 151.2 and 151.3, 106.6 (dd, J ) 7.6, 245.8 Hz, 2C), 160.6.
MS, m/z: 488 (MH+). Anal. (C25H28F3N3O3‚HCl‚0.5H2O) C, H,
N.
1
equiv), followed by ether. H NMR: δ 2.04 and 2.06 (s, 3H),
3.81 and 3.84 (s, 3H), 3.89 and 4.42 (d, J ) 12.0 Hz, 1H), 4.13
and 4.41 (m, 1H), 4.46 and 4.57 (dd, J ) 10.0, 14.0 Hz, 1H),
4.79 and 5.10 (d, J ) 16.4 Hz, 1H), 5.23 and 5.37 (d, J ) 16.4
Hz, 1H), 6.80 and 6.86 (t, J ) 8.0 Hz, 2H), 6.91 (m, 1H), 7.03
and 7.13 (t, J ) 7.2 Hz, 1H), 7.22 (m, 2H), 7.15-7.26 (m, 3H),
7.28 (t, J ) 7.6 Hz, 2H), 7.44 7.49 (d, J ) 7.2 Hz, 2H). 13C
NMR: δ 17.58 and 17.65, 39.13 and 39.26, 45.6 and 46.1, 54.1
and 54.3, 56.1 and 56.2, 107.97 and 108.03, 111.7 and 111.9
(AA′XX′, 2C), 112.0 and 112.2 (t, J ) 6.7 Hz), 113.2 and 113.4,
122.2 and 122.4 (d, J ) 12.8 Hz), 123.8 and 124.8 (d, J ) 3.8
Hz), 124.2 and 124.6, 127.1 and 127.2 (2C), 128.7 and 128.8
(2C), 129.5 and 129.6, 129.7 and 129.8, 134.1 and 134.3, 147.6
and 147.8 (d, J ) 10.6 Hz), 149.9 and 150.3 (d, J ) 244 Hz),
151.3 and 151.5, 151.6 and 152.3, 161.1 and 161.2 (dd, J )
7.6, 248 Hz, 2C), 161.4 and 162.9. 19F NMR: δ -113.3 and -
113.5 (t, J ) 7.7 Hz, 2F), -134.0 and -1134.7 (m). MS, m/z:
496 (MH+). Anal. (C27H24F3N3O3‚HCl‚H2O) C, H, N.
3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-6-
methyl-5-(3,4-methylenedioxyphenyl)pyrimidin-2,4-di-
one Hydrochloride (19a). 3-[(2R)-(tert-Butoxycarbonylamino)-
2-phenylethyl]-1-(2,6-difluorobenzyl)-5-bromo-6-methyluracil
11b (22.6 g, 41 mmol), 3-methoxyphenylboronic acid (7.5 g,
49 mmol), and saturated barium hydroxide solution (280 mL)
were added to a mixture solvent (benzene/EtOH/DME ) 10:
1:11, 800 mL). The reaction mixture was degassed with
nitrogen, and tetrakis(triphenylphosphine)palladium (4.7 g, 4
mmol) was added. The reaction mixture was then heated at
80 °C for 18 h, cooled to room temperature, and filtered
through a pad of Celite. The organic layer was separated and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/EtOAc ) 3:1) to yield
3-[(2R)-(tert-butoxycarbonylamino)-2-phenylethyl]-1-(2,6-dif-
luorobenzyl)-5-(3-methoxyphenyl)-6-methyluracil (22.0 g, 93%).
The above compound was treated with 1 equiv of 4.0 M HCl
in dioxane (100 mL) for 30 min. The solvent was removed in
vacuo and the residue was treated with ether to precipitate
the title compound as a white solid (14.7 g, 75%). 1H NMR: δ
2.10 (s, 3H), 3.99 (m, 1H), 4.52 (m, 2H), 5.18 (s, 2H), 5.91 (s,
1H), 5.96 (s, 1H), 6.60 (m, 2H), 6.86 (m, 3H), 7.23 (m, 2H),
7.33 (m, 2H), 7.59 (d, J ) 7.5 Hz, 2H), 8.70 (brs, 3H). 19F NMR
(CFCl3): δ -115.0 (s). MS, m/z: 478 (MH+). Anal. (C27H23-
F2N3O4‚HCl‚1.4H2O) C, H, N.
3-[(2R)-Methylamino-2-phenylethyl]-1-(2,6-difluoro-
benzyl)-6-methyl-5-(3,4-methylenedioxyphenyl)pyrimi-
din-2,4-dione Hydrochloride (20b). A solution of BH3/THF
(20 mL of a 1.5 M solution in THF, 29.4 mmol, 2 equiv) was
added dropwise in neat form to d-(N-Boc-N-methyl)phenyl-
glycine (3.90 g, 14.7 mmol) at 0 °C under an inert atmosphere
of N2. After 15 min, the ice/water bath was removed and the
mixture was allowed to reach room temperature. After 1 h,
the reaction mixture was cooled to 0 °C and the reaction was
carefully quenched by addition of H2O (20 mL). The resulting
mixture was extracted with EtOAc, and the organic layer was
separated and washed with a 10% aqueous citric acid solution
and brine. The organics were dried (MgSO4), filtered, and
evaporated. The crude (2R)-(N-Boc-N-methyl)phenylglycinol
was isolated as a white solid, and it was used without any
further purification, 2.67 g (72% yield). 1H NMR: δ 1.43 (s,
9H), 2.69 (br s, 3H), 4.09-4.02 (m, 2H), 5.30 (br, 1H), 7.38-
7.22 (m, 5H).
To a suspension of 1-(2,6-difluorobenzyl)-5-bromo-6-methyl-
uracil (10, 3.35 g, 10.1 mmol), the above alcohol (2.67 g, 10.6
mmol), and triphenylphosphine (4.0 g, 15.2 mmol) in THF (150
mL) under N2 at room temperature, was added DEAD (2.40
mL, 15.2 mmol) dropwise. The initial yellow color was dis-
charged, and a clear, colorless solution resulted. This was
stirred at room temperature for 15 h, and then the solvent
was removed in vacuo. The crude residue was purified by
column chromatography on silica gel, eluting with a 3:1 v/v
mixture of hexanes and ethyl acetate. 3-[(2R)-[N-(tert-Butoxy-
carbonyl)-N-methylamino)-2-phenylethyl]-1-(2,6-difluoroben-
Sample for X-ray Crystal Structure. R-13b (free base,
1 g) was dissolved in a mixture of methanol (5 mL) and water
(12 mL) with heating. Upon cooling to room temperature, the
sample turned milky and was further treated with a few drops
of methanol until clear. The sample vial was capped loosely
and placed in a hood to evaporate slowly.
3-[(2R)-Dimethylamino-2-phenylethyl]-1-(2,6-di-
fluorobenzyl)-6-methyl-5-(2-fluoro-3-methoxyphenyl)-
pyrimidin-2,4-dione Hydrochloride (R-15b). 3-[(2R)-Amino-
2-phenylethyl]-1-(2,6-difluorobenzyl)-6-methyl-5-(2-fluoro-3-
methoxyphenyl)pyrimidin-2,4-dione (R-13b, 450 mg, 0.91 mmol)
was dissolved in CH2Cl2 (20 mL) and treated with formalde-
hyde (200 µL of a 37% solution in H2O). The resulting mixture
was stirred at room temperature for 10 min. Sodium tri-
acetoxyborohydride (1.33 g, 6.3 mmol) was added portionwise,
and the reaction mixture was stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo,
diluted with EtOAc, and washed with H2O, aqueous saturated
NaHCO3, and brine. The organic layer was dried over anhy-
drous MgSO4 and filtered. Evaporation gave a residue that
was purified by column on silica gel, eluting with a 1:1 v/v
mixture of hexanes and EtOAc. The free base was isolated as
a white foam (282 mg, 0.54 mmol, 59%). This was dissolved
in CH2Cl2 (5 mL) and treated with HCl (1 mL of a 4.0 M
solution in dioxane). After 1 h, the mixture was evaporated to
dryness and triturated with Et2O to give the hydrochloride
salt as a white solid (256 mg, 0.46 mmol). 1H NMR: δ 2.15
and 2.16 (s, 3H), 2.68-2.79 (m, 6H), 3.89 (s, 3H), 4.94 and 4.52
(m, 1H), 4.82-5.50 (m, 4H), 6.86-7.49 (m, 11H), 12.33 and
12.40 (brs, 1H). 19F NMR: δ - 114.2 and - 114.1 (s, 2F),
-136.2 and -136.0 (s, 1F). 13C NMR: δ 17.9, 38.3 and 38.9,
39.4 and 39.7, 41.4 and 41.9, 43.8 and 43.9, 56.4, 66.8 and 67.0,
108.4 and 108.5, 111.9 (m, 2C), 112.1 (d, J ) 16.7 Hz), 113.5,
122.4 and 122.5 (d, J ) 11.1 Hz). MS, m/z: 524 (MH+). Anal.
(C29H28F3N3O3‚HCl‚1.3H2O) C, H, N.
3-[(2S)-Cyclobutylaminopropyl]-1-(2,6-difluorobenzyl)-
6-methyl-5-(2-fluoro-3-methoxyphenyl)pyrimidin-2,4-di-
one Hydrochloride (S-16a). To S-13f14 (216 mg, 0.5 mmol)
in dichloroethane (10 mL) was added cyclobutanone (0.6
mmol), and the mixture was stirred at room temperature for
10 min. Then NaBH(OAc)3 (255 mg, 1.2 mmol) was added, and

1176 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Tucci et al.
zyl)-5-bromo-6-methyluracil R-12b was isolated as a white
solid (2.91 g, 51%). 1H NMR: δ 1.39 (s, 9H), 2.51 (br, 6H),
4.33-4.21 (m, 1H), 4.86 (q, J ) 12.3 Hz, 1H), 5.29 (q, J ) 14.1
Hz, 1H), 5.33 (q, J ) 15.7 Hz, 1H), 5.85 (m, 1H), 6.93-6.89
(m, 2H), 7.43-7.27 (m, 6H). MS, m/z: 464 (MH+ - Boc).
4.62 (m, 1H), 7.15 (m, 3H), 7.40 (m, 6H), 8.20 (brs, 3H). MS,
m/z: 341 (MH+).
3-[2(R)-Amino-2-phenylethyl]-6-methyl-5-(2-fluoro-
phenyl)-1-(2-methoxybenzyl)uracil Trifluoroacetate (24o).
A mixture of 22 (29 mg) and 2-methoxybenzylamine (0.15 mL)
was heated in a sealed reaction vial at 100 °C for 1 h.
Chromatography on silica gel with 1:2 ethyl acetate/hexanes
gave 3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-6-meth-
yl-5-(2-fluorophenyl)-1-(2-methoxybenzyl)uracil as a colorless
oil. 1H NMR: δ 1.40 (s, 9H), 2.04 (s, 3H), 3.87 (s, 3H), 4.18 (m,
1H), 4.44 (m, 1H), 5.22 (m, 2H), 5.65 (brs, 1H), 5.78 (m, 1H),
6.85-7.42 (m, 13H). MS, m/z: 460 (MH+ - BuOCO).
In a pressure vessel, 3-[(2R)-(tert-butoxycarbonylamino)-2-
phenylethyl]-1-(2,6-difluorobenzyl)-5-bromo-6-methyluracil (226
mg, 0.40 mmol) was dissolved in a 45:5:50 v/v mixture of
benzene, EtOH, and 1,2-dimethoxyethane (8 mL). To that, 3,4-
methylenedioxyphenylboronic acid (80 mg, 0.48 mmol) was
added followed by an aqueous saturated solution of Ba(OH)2
(3 mL). The resulting heterogeneous mixture was degassed
by N2 bubbling for 30 min. Tetrakis(triphenylphosphine)-
palladium (46 mg, 0.04 mmol) was added, and the vessel was
tightly capped and immersed in an oil bath at 100 °C. The
reaction mixture was maintained at that temperature for 22
h, after which time the reaction was deemed complete by TLC
and LC-MS. The reaction mixture was cooled, diluted with
EtOAc, and washed with H2O. The organic layer was washed
with brine, dried over anhydrous MgSO4, filtered, and evapo-
rated. The residue was purified by column chromatography
on silica gel, eluting with a 3:1 v/v mixture of hexanes and
ethyl acetate. The product was obtained as a yellow foam (85
mg, 0.14 mmol, 35%).
The above compound (20 mg) was treated with trifluoro-
acetic acid (1 mL) at room temperature for 30 min. Concentra-
tion in vacuo gave the title compound as a colorless oil in
1
quantitative yield. H NMR: δ 2.04 (s, 3H), 3.82 and 3.85 (s,
3H), 4.20 (m, 1H), 4.62 (m, 2H), 5.10 (m, 2H), 6.82-7.40 (m,
13H), 8.05 (brs, 3H). MS, m/z: 460 (MH+). HRMS (CI - CH4)
calcd for C27H26FN3O3 (MH+): 460.203 65. Observed:
460.202 10.
3-[(2R)-Acetamido-2-phenylethyl]-1-(2,6-difluoro-
benzyl)-6-methyl-5-(3,4-methylenedioxyphenyl)pyrimi-
din-2,4-dione (25). A solution of 3-[(2R)-amino-2-phenyl-
ethyl]-1-(2,6-difluorobenzyl)-6-methyl-5-(3,4-methylenedioxy-
phenyl)pyrimidin-2,4-dione (19b, 400 mg, 0.81 mmol) in acetic
anhydride (3 mL) was stirred at room temperature overnight.
Volatiles were evaporated, and the residue was dried under
vacuum overnight at 60 °C. The crude material was then
dissolved in dichloromethane (5 mL) and the product was
precipitated with ether/hexane (1/1 volume) to obtain the title
The above compound (85 mg, 0.14 mmol) was dissolved in
CH2Cl2 (1.5 mL) and treated with trifluoroacetic acid (0.75
mL). The resulting mixture was stirred for 1.5 h at room
temperature and then concentrated in vacuo. The residue was
partitioned between EtOAc and an aqueous NaHCO3 saturated
solution. The organics were separated, washed with brine,
dried (MgSO4), filtered, and evaporated. The residue was
purified by column chromatography on silica gel, eluting with
EtOAc. The free base was obtained as a white foam (52 mg,
0.10 mmol, 71%). This was dissolved in CH2Cl2 (1 mL) and
treated with HCl (3 mL of a 1.0 M solution in Et2O). After 1
h, the volatiles were removed in vacuo and the residue was
triturated with Et2O (10 mL) to give the hydrochloride salt as
1
compound as a white solid (406 mg, 93%). H NMR (DMSO-
d6): δ 1.72 (s, 3H), 2.17 (s, 1H), 4.00 (dd, J ) 5.6, 12.8 Hz,
1H), 4.09 (dd, J ) 9.2, 12.8 Hz, 1H), 5.16 (d, J ) 16.4 Hz, 1H),
5.23 (d, J ) 16.4 Hz, 1H), 5.27 (m, 1H), 6.04 (s, 2H), 6.54 (d,
J ) 7.2 Hz, 1H), 6.59 (s, 1H), 6.92 (d, J ) 8.4 Hz, 1H), 7.11 (t,
J ) 8.4 Hz, 2H), 7.26 (m, 5H), 7.42 (m, 1H), 8.21 (d, J ) 9.2
Hz, 1H). 13C NMR: δ 17.6, 22.4, 45.5, 50.2, 101.0, 108.0, 111.0,
111.8 (m, 2C), 112.4 (t, J ) 16.7 Hz), 112.7, 124.2, 126.6 (2C),
127.0, 127.9, 128.1, 129.8 (t, J ) 9.9 Hz), 140.1, 146.5, 146.9,
149.4, 150.9, 160.6 (dd, J ) 7.6, 245.7 Hz, 2C), 161.2, 168.7.
19F NMR: δ -114.8 (t, J ) 7.0 Hz). MS, m/z: 534 (MH+). Anal.
(C29H25F2N3O5) C, H, N.
1
a white solid (50 mg). H NMR: δ 2.13 (s, 3H), 2.44 (s, 3H),
4.25 (d, J ) 13.2 Hz, 1H), 4.60 (m, 1H), 4.78 (d, J ) 10.4 Hz,
1H), 5.25 (brs, 2H), 5.89 (s, 2H), 6.71 (m, 2H), 6.85 (t, J ) 6.3
Hz, 2H), 7.23 (m, 1H), 7.35 (m, 3H), 7.53 (m, 2H), 9.16 (brs,
1H), 10.3 (brs, 1H). 19F NMR: δ -115.1. MS, m/z: 506 (MH+).
Anal. (C28H25F2N3O4‚HCl‚1.5H2O) C, H, N.
Pharmacokinetics of Uracil Compounds. A total of 10
mg/kg test articles were administrated to postsurgery monkeys
via oral (po) and intravenous (iv) routes. For iv dosing, a
formulated (water or 5% chromophore) dosing solution was
administered as an intravenous infusion over a period of 15
min. Blood was collected from an accessible vein at predose,
0.25, 0.33, 0.5, 1, 1.5, 4, 8, and 24 h after dosing. For oral
dosing, a sterile water solution was administered with a
nasogastric gavage. Blood was collected at predose, 0.25, 0.5,
1, 1.5, 2, 4, 8, and 24 h after dosing. Plasma sample was
harvested from blood by centrifugation within 30 min after
collection. All samples were stored at -70 °C or below until
analysis.
The bioanalytical method applied for the measurement of
test article in plasma along with added internal standard
consisted of protein precipitation from 50 µL of plasma with
200 µL of acetonitrile. The processed samples were then
centrifuged to recover the supernatant. The supernatant was
dried in a vacuum and then reconstituted in acetonitrile/water
solutions before being introduced into an LC-MS/MS system
for analysis. The analytical system consisted of a Waters 2790
HPLC module coupled to a Micromass Quattro-LC system. An
Agilent Zorbax SB-C18 (5 µm, 4.6 mm × 5.0 mm) column
provided separations for sample analysis. The lower limit of
quantification (LLOQ) for the analytical method was 5 ng/mL
of test article in plasma. All pharmacokinetic parameters were
calculated from noncompartmental models using the WinNon-
lin program, version 3.2. Oral bioavailability was calculated
from the dose-adjusted area under the curve (AUC) from the
oral route over the dose-adjusted AUC from the iv route.
3-[(2R)-Amino-2-phenylethyl]-6-methyl-5-(2-fluoro-
phenyl)oxazine-2,4-dione Trifluoroacetate (23). To a
stirred solution of 2′-fluorophenylacetone (7.6 g, 50 mmol) in
ether (50 mL) was added dropwise chlorosulfonyl isocyanate
(16.2 g, 115 mmol) at room temperature. The yellowish solution
was stirred overnight, poured onto ice (100 g), and basified
with sodium carbonate. The product was extracted with ethyl
acetate (2 × 200 mL), and the extract was washed with water
and brine, dried over magnesium sulfate, and concentrated
in vacuo to give a yellow residue (9.5 g, proton NMR, about
70% product). The crude product was crystallized from ether/
hexanes to give 6-methyl-5-(2-fluorophenyl)oxazine-2,4-dione
1
(21) as a yellowish solid (3.6 g, 33% yield). H NMR: δ 2.14
(s, 3H), 7.16 (t, J ) 9.0 Hz, 1H), 7.24 (m, 2H), 7.41 (m, 1H),
9.20 (brs, 1H).
DEAD (348 mg, 1.2 mmol) was added to a solution of the
above oxazine 21 (221 mg, 1.0 mmol), triphenylphosphine (314
mg, 1.2 mmol), and (R)-N-Boc-phenylglycinol (249 mg, 1.05
mmol) in dry THF (5 mL). The mixture was stirred at room
temperature for 2 h, concentrated, and purified by chroma-
tography on silica gel with 1:3 ethyl acetate/hexanes to give
3-[2(R)-tert-butoxycarbonylamino-2-phenylethyl]-6-methyl-5-
(2-fluorophenyl)oxazine-2,4-dione (22) (380 mg, 87%) as a
white solid. 1H NMR: δ 1.39 (s, 9H), 2.14 (s, 3H), 4.02 (m,
1H), 4.28 (m, 1H), 5.21 (brs, 1H), 5.30 (m, 1H), 7.38 (m, 9H).
MS, m/z: 341 (MH+ - BuOCO).
Compound 22 (30 mg) was treated with trifluoroacetic acid
(1 mL) at room temperature for 30 min. Concentration in vacuo
gave the title compound as a colorless oil in quantitative yield.
1H NMR: δ 2.05 and 2.08 (s, 3H), 4.10 (m, 1H), 4.45 (m, 1H),
In Vivo Efficacy in Cynomolgus Monkeys. Complete
orchiectomy was performed on six male cynomolgus macaques

Dione as Antagonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1177
(10) Ashton, W. T.; Sisco, R. M.; Kieczykowski, G. R.; Yang, Y. T.;
Yudkovitz, Cui, J.; Mount, G. R.; Ren, R. N.; Wu, T.-J.; Shen,
X.; Lyons, K. A.; Mao, A.-H.; Carlin, J. R.; Karanam, B. V.;
Vincent, S. H.; Chang, K.; Goulet, M. T. Orally bioavailable,
indole-based nonpeptide GnRH receptor antagonists with high
potency and functional activity. Bioorg. Med. Chem. Lett. 2001,
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(11) Clark, E.; Boyce, M.; Warrington, S.; Johnston, A.; Suzuki, N.;
Cho, N.; Dote, N.; Nishizawa, A.; Furuya, S. Effects of single
doses of TAK-013, a new non-peptide orally active gonadotropin-
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(12) (a) Zhu, Y.-F.; Struthers, R. S.; Connors, P. J., Jr.; Gao, Y.; Gross,
T. D.; Saunders, J.; Wilcoxen, K.; Reinhart, G. J.; Ling, N.; Chen,
C. Initial structure-activity relationship studies of a novel series
of pyrrolo[1,2-a]pyrimid-7-ones as GnRH receptor antagonists.
Bioorg. Med. Chem. Lett. 2002, 12, 399-402. (b) Zhu, Y.-F.;
Wilcoxen, K.; Struthers, R. S.; Connors, P., Jr.; Saunders, J.;
Gross, T.; Gao, Y.; Reinhart, G.; Chen, C. A novel synthesis of
2-arylpyrrolo[1,2-a]pyrimidin-7-ones and their structure-activ-
ity relationships as potent GnRH receptor antagonists. Bioorg.
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F.; Guo, Z.; Gross, T. D.; Connors, P. J., Jr.; Struthers, R. S.;
Reinhart, G. J.; Wang, X.; Saunders, J.; Chen, C. Novel synthesis
of 7-aryl-8-fluoropyrrolo[1,2-a]pyramid-4-ones as potent, stable
GnRH receptor antagonists. Bioorg. Med. Chem. Lett. 2002, 12,
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approximately 3.7-6.5 years of age, and animals were allowed
to recover for at least 4 weeks prior to dosing. Animals were
not sedated for dosing but were temporarily restrained outside
their cages. Oral doses were administered via nasogastric
gavage, and blood samples were collected immediately prior
to and 0.25, 0.5, 1, 1.5, 2, 4, 8, and 24 h after dosing.
Intravenous doses were infused over a 15 min period, and
samples were collected immediately prior to and 0.25, 0.33,
0.5, 1, 1.5, 4, 8, and 24 h after the initiation of the infusion.
Bioactive LH concentrations in serum samples were measured
at the Oregon Regional Primate Center using a previously
reported mouse Leydig cell bioassay, which could detect as
little as 3 ng LH/mL using cynomolgus LH RP-1 as the
reference preparation.25
Acknowledgment. The authors thank Dr. David
Hess (Oregon Regional Primate Center) for bioactive
monkey LH determinations, Ms. Julie K. Meyer (Sierra
Biomedical) and Ms. Robin LaChappell for their as-
sistance in coordinating the castrate monkey experi-
ments, and Mr. Patrick Connors for the synthesis of
compounds 19d and 19e.
(13) (a) Wilcoxen, K. M.; Zhu, Y.-F.; Connors, P. J., Jr.; Saunders,
J.; Gross, T. G.; Gao, Y.; Reinhart, G. J.; Struthers, R. S.; Chen,
C. Synthesis and initial structure-activity relationships of a
novel series of imidazolo[1,2-a]pyramid-5-ones as potent GnRH
receptor antagonists. Bioorg. Med. Chem. Lett. 2002, 12, 2179-
2183. (b) Gross, T. D.; Zhu, Y.-F.; Saunders, J.; Wilcoxen, K. M.;
Gao, Y.; Connors, P. J.; Guo, Z.; Struthers, R. S.; Reinhart, G.
J.; Chen, C. Design, synthesis and structure-activity relation-
ships of novel imidazolo[1,2-a]pyramid-5-ones as potent GnRH
receptor antagonists. Bioorg. Med. Chem. Lett. 2002, 12, 2185-
2187. (c) Zhu, Y.-F.; Guo, Z.; Gross, T. D.; Gao, Y.; Connors, P.
J., Jr.; Struthers, R. S.; Xie, Q.; Tucci, F. C.; Reinhart, G. J.;
Wu, D.; Saunders, J.; Chen, C. Design and structure-activity
relationships of 2-alkyl-3-aminomethyl-6-(3-methoxyphenyl)-7-
methyl-8-(2-fluorobenzyl)imidazolo[1,2-a]pyramid-5-ones as po-
tent GnRH receptor antagonists. J. Med. Chem. 2003, 46, 1769-
1772.
(14) (a) Zhu, Y.-F.; Gross, T. D.; Guo, Z.; Connors, P. J., Jr.; Gao, Y.;
Tucci, F. C.; Struthers, R. S.; Reinhart, G. J.; Saunders, J.; Chen,
T. K.; Bonneville, A. L. K.; Chen, C. Identification of 1-arylm-
ethyl-3-(2-aminoethyl)-5-aryluracil as novel gonadotropin-releas-
ing hormone receptor antagonists. J. Med. Chem. 2003, 46,
2023-2026. (b) Guo, Z.; Zhu, Y.-F.; Tucci, F. C.; Gao, Y.;
Struthers, R. S.; Saunders, J.; Gross, T. D.; Xie, Q.; Reinhart,
G. J.; Chen, C. Synthesis and structure-activity relationships
of 1-arylmethyl-3-(2-aminopropyl)-5-aryl-6-methyluracils as po-
tent GnRH receptor antagonists. Bioorg. Med. Chem. Lett. 2003,
13, 3311-3315. (c) Tucci, F. C.; Zhu, Y.-F.; Guo, Z.; Gross, T.
D.; Connors, P. J.; Struthers, R. S.; Reinhart, G. J.; Saunders,
J.; Chen, C. Synthesis and structure-activity relationships of
1-arylmethyl-3-(1-methyl-2-amino)ethyl-5-aryl-6-methylura-
cils as antagonists of the human GnRH receptor. Bioorg. Med.
Chem. Lett. 2003, 13, 3317-3322.
(15) Guo, Z.; Zhu, Y.-F.; Gross, T. D.; Tucci, F. C.; Gao, Y.; Moorjani,
M.; Connors, P. J., Jr.; Rowbottom, M. W.; Chen, Y.; Struthers,
R. S.; Xie, Q.; Saunders, J.; Reinhart, G.; Chen, T. K.; Bonneville,
A. L. K.; Chen, C. Synthesis and structure-activity relationships
of 1-arylmethyl-5-aryl-6-methyluracils as potent gonadotropin-
releasing hormone receptor antagonists. J. Med. Chem. 2004,
47, 1259-1271.
(16) Tucci, F. C.; Zhu, Y.-F.; Guo, Z.; Gross, T. D.; Connors, P. J.,
Jr.; Gao, Y.; Rowbottom, M. W.; Struthers, R. S.; Reinhart, G.
J.; Xie, Q.; Chen, T. K.; Bozigian, H.; Bonneville, A. L. K.; Fisher,
A.; Jin, L.; Saunders, J.; Chen, C. 3-(2-Aminoalkyl)-1-(2,6-
difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methyluracils as
orally bioavailable antagonists of the human gonadotropin-
releasing hormone receptor. J. Med. Chem. 2004, 47, 3487-3486.
(17) Rowbottom, M. W.; Tucci, F. C.; Zhu, Y.-F.; Guo, Z.; Gross, T.
D.; Reinhart, G. J.; Xie, Q.; Struthers, R. S.; Saunders, J.; Chen,
C. Synthesis and structure-activity relationships of (R)-1-alkyl-
3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils as
human GnRH receptor antagonists. Bioorg. Med. Chem. Lett.
2004, 14, 2269-2274.
Supporting Information Available: Synthesis and ana-
lytical data of S-13a,b, R-13c-e, S-14a, R-14b, S-15a-c,
R-15e, 19b-h, 20a,c, and 24a-n,p-w. This material is
available free of charge via the Internet at http://pubs.acs.org.
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