Synthesis and structure-activity relationships of (R)-1-alkyl-3-[2-(2- amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils as human GnRH receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.02.004

The synthesis of a series of (R)-1-alkyl-3-[2-(2-amino)phenethyl]-5-(2- fluorophenyl)-6-methyluracils is discussed. SAR around N-1 of the uracil was explored, which led to the discovery that an electron-deficient 2,6-disubstituted benzyl group is required

Full text of DOI:10.1016/j.bmcl.2004.02.004

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2269–2274
Synthesis and structure–activity relationships of (R)-1-alkyl-3-[2-
(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils as human
GnRH receptor antagonists
Martin W. Rowbottom,a,* Fabio C. Tucci,a Yun-Fei Zhu,a Zhiqiang Guo,a
Timothy D. Gross,a Greg J. Reinhart,b Qui Xie,b R. Scott Struthers,b John Saundersa
and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
bDepartment of Exploratory Discovery, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
Received 21 November 2003; accepted 2 February 2004
Abstract—The synthesis of a series of (R)-1-alkyl-3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils is discussed. SAR
around N-1 of the uracil was explored, which led to the discovery that an electron-deficient 2,6-disubstituted benzyl group is
required for optimal receptor binding. The best compound from the series had binding affinity of 0.7 nM (Ki) for the human GnRH
receptor, which was 8-fold better than the 2,6-difluorobenzyl analog.
Ó 2004 Elsevier Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH), also known
as luteinizing hormone-releasing hormone (LHRH), is a
decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-
Gly-NH2), which is produced and secreted by the
hypothalamus in a pulsitile manner.1;2 Through interac-
tion with specific GnRH receptors in the pituitary, both
follicle-stimulating hormone (FSH) and luteinizing hor-
mone (LH) are released from this site. These hormones,
in turn, regulate the production of steroids and gametes.
antagonists act immediately at the receptor, quickly
suppressing the release of FSH and LH. A number of
peptide antagonists are currently available, represented
by Cetrotidee.1 Due to the very low oral bioavailability
of these peptides, administration is normally via injec-
tion or depot formulation. In response to the need for a
more convenient route of administration, intensive
efforts have been initiated toward the development of
orally bioavailable small-molecule GnRH antagonists.4
A number of disease states can be controlled via the
regulation of this pituitary–gonadal hormonal axis, in
particular endometriosis, uterine fibroids, and prostate
cancer. Suppression of both FSH and LH production
can be achieved via the agonism or antagonism of the
GnRH receptor. Continuous activation ultimately leads
to down regulation of the receptor and a number of
peptide agonists are commercially available, represented
by Leuprorelinâ.3 However, treatment with GnRH
agonists initially leads to overproduction of both FSH
and LH with a concomitant Ôflare effectÕ, which tends to
exacerbate symptoms in patients. In contrast GnRH
We had previously reported the discovery of a new class
of uracils as orally bioavailable, human GnRH
[hGnRH] receptor antagonists exemplified by 3-(2-ami-
noethyl)-5-aryl-6-methyluracils 3,5 which were evolved
from
6-aminomethyl-7-aryl-pyrrolo[1,2-a]pyrimid-4-
ones 16 and 2-aryl-3-aminomethyl-imidazolo[1,2-a]pyri-
mid-5-ones 2a,b7 (Fig. 1). The synthetic route developed
for 3 allowed for rapid SAR studies around the 3-posi-
tion (of 3) and potent analogs such as 4 were identified.
We were also particularly interested in varying the
substituent at N-1. Due to the synthetic route employ-
ed,5a in which this substituent was introduced early on
(Fig. 2), only a very small number of variations at N-1
were screened. In this letter we report on an alternative
synthesis of highly substituted 6-methyluracils, in which
the N-1 substituent is introduced in the last step. This
modification subsequently enabled us to efficiently
* Corresponding authors. Tel.: +1-858-658-7799; fax: +1-858-658-7601;
e-mail: mrowbottom@neurocrine.com
URL: http://www.neurocrine.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.004

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M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2269–2274
1
1
R
R
2
2
R
R
N
N
O
O
O
6
4
6
5
5
3
R
R
5
N
OR
2
N
3
7
3
6
3
R
R
N
8
N
N
1
1
8
X
4
4
R
R
2a: R6 =CO2R7, R5=H
2b: R6=Ar, R5=CH3
1: X=CN, F or H
2
R
N
O
O
5
5
3
N
3
N
1
Ar
Ar
R
6
6
H2N
O
N
Me
O
N
Me
1
1
3
R
R
4
3
Figure 1. General structures of GnRH antagonists.
O
N
R2
N
O
N
O
N
1
R
Ar
N
N
O
N
H
Me
O
F
Me
O
F
Me
F
F
Figure 2. Previous synthetic route employed.
explore the SAR around N-1 and the results from this
study are described.
ers9 gave 5-(2-fluorophenyl)-6-methyl-1,3-oxazine-2,4-
(3H)-dione 8, which was isolated in 33% yield.10 Sub-
sequent N-3 alkylation of 8 by (R)-N-Boc-2-phenylgly-
cinol, via a Mitsunobu protocol, gave 1,3-oxazine-2,4-
dione 9 in 81% yield. Reaction of 1,3-oxazine-2,4-dione
9 with alkyl amines at 100 °C for 4 h followed by N-Boc
deprotection, gave the corresponding uracils (10), which
were isolated in 5–60% yield as the trifluoroacetate
salts.11 Primary aryl amines and very sterically hindered
primary alkyl amines failed to give the desired uracil
products under these conditions.
The synthesis of substituted uracils via the condensation
of primary amines with 1,3-oxazine-2,4-diones had been
reported previously by a number of groups.8 Reaction
of ammonia8a;b;k or primary alkyl amines8b–i;k with 1,3-
oxazine-2,4-diones proceed efficiently under relatively
mild conditions to yield the corresponding N-1(H) or N-
1(alkyl) uracils, respectively. As outlined in Scheme 1
nucleophilic addition of the amine nitrogen to C-2 of the
1,3-oxazin-2,4-dione ring (5) with subsequent ring
opening presumably leads to intermediate 6,8g which
upon loss of water results in formation of uracil ring 7.
Due to the much reduced nucleophilicity of primary aryl
amines, acid catalysis and/or high temperatures are
normally required to facilitate their reaction with 1,3-
oxazine-2,4-diones.8j
Over 100 6-methyluracils (10) were prepared using the
methodology described and a wide variety of substitu-
tion at N-1 explored. A selection of compounds (11–36)
is listed in Tables 1 and 2. All 6-methyluracil derivatives
11–36, were assayed for their ability to bind competi-
tively to the cloned hGnRH receptor expressed in
HEK293 cells, using a 96-well filtration apparatus12;13
We proposed that synthesis of the novel and highly
substituted 1,3-oxazine-2,4-dione 9 (Scheme 2) and its
subsequent condensation with a variety of primary alkyl
amines to give the corresponding uracils, would enable
us to rapidly screen a large number of substituents at N-
1. Reaction of (2-fluorophenyl)acetone with chloro-
sulfonyl isocyanate (CSI), according to the general
method previously described by Hassner and co-work-
and des-Gly10
[
125I-Tyr,5 DLeu,6 NMeLeu,7 Pro9-
NEt]GnRH as the radiolabeled ligand. Selected results
are summarized in Tables 1 and 2. The functional
antagonism of the described compounds was confirmed
by their ability to inhibit GnRH stimulated Ca2þ flux in
the transfected cells in a dose-dependent manner (Fig.
3).7c

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2269–2274
2271
O
O
O
O
R1
O
R 2
R3
R1
O
R1
O
R 2
R3
R4NH2
N
2
N
R3
4
N
5
R2
∆
6
N
R4
O
NH
R4
6
7
5
Scheme 1. Mechanism of reaction for the synthesis of uracils from 1,3-oxazin-2,4-diones.
F
F
O
O
F
b
a
N
O
HN
NH
boc
Me
O
O
Me
O
O
Me
8
9
c, d
F
O
N
H2N
O
N
R
Me
10
Scheme 2. Reagents and conditions: (a) chlorosulfonyl isocyanate, Et2O, rt, 12 h, 33%; (b) (R)-N-Boc-2-phenylglycinol, DEAD, PPh3, THF, rt, 12 h,
81%; (c) 1° Alkyl amine, neat, 100 °C, 4 h; (d) TFA, DCM, rt, 2 h, 5–60% over two steps.
From this study, it is clear that the N-1 substituent of
6-methyluracil 10, participates in a key binding inter-
action with the hGnRH receptor. For instance, alkyl
substituents containing polar groups as illustrated by
11–13 (Table 1), resulted in compounds with poor
affinity for the receptor. In particular those compounds
with a basic amine (11 and 12) and/or hydrogen bond
donating group in this region (12 and 13) were not well
tolerated. The introduction of lipophilic functionality,
such as small alkyl and cycloalkyl groups, improved
potency (as indicated by compounds 15–19). Uracil 19
(which contains cyclohexyl methyl at N-1) had a Ki
value of 200 nM and was approximately 5-fold more
potent than the corresponding cyclopropyl methyl
compound (15). This suggested that not only was lipo-
philicty important, but both the size and shape pre-
sented by cyclohexyl methyl to the binding pocket were
also key factors. The introduction of pyridylethyl (20) or
phenethyl (24) gave compounds with low affinity for the
receptor (>10,000 and 1000 nM, respectively). However,
moving the aromatic group toward the uracil core (by
one carbon atom) resulted in markedly enhanced bind-
ing affinities (21–23, 25, and 26). This data suggests that
an important aromatic p–p interaction between a certain
residue in this region of the receptor and ligand must be
taking place. In order to fully determine the nature of
this interaction, a number of compounds containing a
substituted benzyl ring at N-1 (27–36, Table 2) were
prepared. It was clear that 2-substituted benzyls were
preferred,14 2-fluorobenzyl (29) being approximately
3-fold more potent (Ki ¼ 20 nM) than the corresponding
3- and 4-substituted isomers (28 and 27, respectively).
The results also indicated that an electron-deficient ring
was preferred (compare compounds 30–33). Compound
30 (2-methoxybenzyl) was approximately 60-fold less
potent than that containing 2-(trifluoromethyl)benzyl
(33, Ki ¼ 4 nM). This may suggest that an important p–
p charge-transfer complex is being formed between the
benzyl ring and an electron-rich aromatic, such as a
tyrosine residue located in the binding pocket.15 The
addition of a second electron-withdrawing substituent at
position 6 of the benzyl ring, further improved binding
affinity (34 and 35). Compound 35 proved to be the
most potent 6-methyluracil discovered from this study
(Ki ¼ 0:7 nM). Moving the 6-fluoro substituent of
compound 35 to the 4-position of the benzyl ring (36)
led to significant loss in potency, up to 50-fold
(Ki ¼ 33 nM), thus highlighting the importance of the
2,6-disubstitution pattern.
In summary, we have prepared a series of novel N-1-
alkyl-N-3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-

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M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2269–2274
Table 1. Binding affinities of N-1-alkyl-6-methyluracils 11–26 toward
the hGnRH receptor
Table 2. Binding affinities of N-1-benzyl-6-methyluracils 27–36 toward
the hGnRH receptor
F
F
O
O
N
N
H2N
H2N
O
N
R
Me
O
N
R
Me
.TFA
.TFA
Compd
R
Ki (nM)
Compd
R
Ki (nM)
Me
N
11
>10,000
27
75
Me
HN
F
F
28
29
67
20
12
>10,000
N
F
HO
13
14
>10,000
1700
MeO
OMe
Me
30
31
230
35
15
16
1100
500
Me
Me
Cl
17
260
32
33
27
4
Me
18
19
230
200
CF3
F
N
N
34
35
6
20
>10,000
F
F
21
22
700
77
0.7
N
Cl
Cl
36
33
F
23
24
O
430
Me
1000
2000
1000
Me
25
26
140
57
methyluracils, via the condensation of a variety of alkyl
amines with a novel and highly substituted 1,3-oxazin-
2,4-dione. A number of these compounds have been
shown to be potent antagonists of the hGnRH receptor.
The SAR around N-1 of the uracil ring was explored,
which led to the discovery that an electron-deficient 2,6-
disubstituted benzyl group was preferred for optimal
binding with the receptor. In addition the 2-chloro-6-
fluorobenzyl group was found to be 8-fold more potent
than the previously favored 2,6-difluorobenzyl group.
We believe that the benzyl group is sitting in a lipophilic
binding pocket and is involved in an important p–p
charge-transfer interaction with a tyrosine residue of the
receptor.
0
-11
-10
-9
-8
-7
-6
Log concentration (M)
Figure 3. Inhibition of GnRH (5 nM) stimulated Ca2þ release, by
compound 35. Compound 35 has a measured IC50 of 2.8 nM.
Acknowledgements
We are indebted to Mr. John Harman for LC–MS
support and Dr. Warren Wade for technical advice. This

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2269–2274
2273
Y.-F.; Guo, Z.; Gross, T. D.; Connors, P. J., Jr.; Struthers,
R. S.; Reinhart, G. J.; Wang, X.; Saunders, J.; Chen, C.
Bioorg. Med. Chem. Lett. 2002, 12, 3491.
work was partly supported by NIH grants 1-R43-
HD38625-01 and 2-R44-HD38625-02.
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10. The remaining material contained unidentified decompo-
sition products.
11. Typical experimental procedure for the preparation of 6-
methyluracils from 1,3-oxazin-2,4-dione 9: Synthesis
of (R)-N-3-[2-(2-amino)phenethyl]-N-1-(2-chloro-6-fluoro-
benzyl)-5-(2-fluorophenyl)-6-methyluracil trifluoroacetate
35;
a
mixture of 1,3-oxazin-2,4-dione
9
(40 mg,
0.091 mmol) in neat 2-chloro-6-fluorobenzylamine
(160 mg, 1.0 mmol) was heated at 100 °C in a sealed vial
for 4 h. The reaction was allowed to cool to rt and
trifluoroacetic acid (1 mL) was slowly added. After stirring
for a further 2 h, the mixture was concentrated in vacuo.
Direct purification via preparative LC–MS afforded 35
(23 mg, 43%) as a colorless oil; 1H NMR dH (300 MHz;
CDCl3) 6.91–7.37 (12H, m), 5.47 and 5.35 (1H, d,
J ¼ 16:5 Hz), 5.24 and 5.14 (1H, d, J ¼ 16:5 Hz), 4.44–
4.65 (2H, m), 4.07 (1H, m), 2.06 (3H, s); HRMS calcd for
C26H22ClF2N3O2 482.14469 (M+H). Found: 482.14354
(M+H).
12. Perrin, M. H.; Haas, Y.; Rivier, J. E.; Vale, W. W. Mol.
Pharmacol. 1983, 23, 44.
13. On each assay plate, a standard antagonist of comparable
affinity to those being tested was included as a control for
plate-to-plate variability. Overall, Ki values were highly
reproducible with an average standard deviation of <45%
for replicate Ki determinations. Key compounds were
assayed in 3–8 independent experiments.
14. One possible explanation is that the presence of the
6-methyl group on the uracil ring may (for steric reasons)
help orient the 2-substituted benzyl ring into the preferred
conformation, this being out of the plane of the uracil
ring. This steric interaction between the 6-methyl (of the
uracil) and the benzyl group is not as pronounced in
compounds 27 and 28.
6. (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. Bioorg. Med. Chem. Lett. 2002,
12, 399; (b) Zhu, Y.-F.; Wilcoxen, K.; Saunders, J.; Guo,
Z.; Gao, Y.; Connors, P. J., Jr; Gross, T. D.; Tucci, F. C.;
Struthers, R. S.; Reinhart, G. J.; Xie, Q.; Chen, C. Bioorg.
Med. Chem. Lett. 2002, 12, 403; (c) Tucci, F. C.; Zhu,
15. Mutational studies performed on the human GnRH
receptor suggest that tyrosine residues 283 and 284
(located on TM domain 6) are crucial for binding of both

2274
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2269–2274
€
peptide agonists and antagonists. See: Hovelmann, S.;
obtained from the crystal structure of b-rhodopsin, a
possible interaction was observed between this benzyl
group and tyrosine 283 or tyrosine 284. Unpublished
results.
€
€
Hoffmann, S. H.; Kuhne, R.; Laak, T.; Reilander, H.;
Beckers, T. Biochemistry 2002, 41, 1129. Based on
the docking study using a GnRH homology model