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

Article abstract of DOI:10.1021/jm049791w

Uracils possessing N-3 side chains derived from various amino alcohols were designed and synthesized as potent human gonadotropin releasing hormone receptor antagonists. The compounds herein presented displayed superior metabolic stability than their pred

Full text of DOI:10.1021/jm049791w

J . Med. Chem. 2004, 47, 3483-3486
3483
Letters
3-(2-Am in oa lk yl)-1-(2,6-d iflu or oben zyl)-5-
(2-flu or o-3-m eth oxyp h en yl)-6-m eth yl-
u r a cils a s Or a lly Bioa va ila ble
An ta gon ists of th e Hu m a n Gon a d otr op in
Relea sin g Hor m on e Recep tor
Fabio C. Tucci,*,† Yun-Fei Zhu,† Zhiqiang Guo,†
Timothy D. Gross,† Patrick J . Connors, J r.,†
Yinghong Gao,† Martin W. Rowbottom,†
R. Scott Struthers,‡ Greg J . Reinhart,‡ Qiu Xie,‡
Ta Kung Chen,§ Haig Bozigian,§
Anne L. Killam Bonneville,§ Andrew Fisher,§
Liping J in,§ J ohn Saunders,† and Chen Chen*,†
Departments of Medicinal Chemistry, Endocrinology, and
Preclinical Development, Neurocrine Biosciences, Inc.,
10555 Science Center Drive, San Diego, California 92121
Received March 15, 2004
Abstr a ct: Uracils possessing N-3 side chains derived from
various amino alcohols were designed and synthesized as
potent human gonadotropin releasing hormone receptor an-
tagonists. The compounds herein presented displayed superior
metabolic stability than their predecessor molecules. Selected
compounds from this series featured good oral bioavailability
in mice and cynomolgus monkeys.
F igu r e 1. Design of new GnRH antagonists.
However, the lack of oral bioavailability and the un-
avoidable “flare effect” are the limitations of these
peptides. In response to the need for convenient admin-
istration, we3 and others4 have initiated intensive efforts
for the development of orally active, small-molecule
GnRH antagonists.
Gonadotropin-releasing hormone (GnRH), also known
as luteinizing hormone-releasing hormone (LHRH), is
a decapeptide that is synthesized in neurons of the
hypothalamus from a 92 amino acid precursor molecule.
GnRH is released to the local vasculature in a pulsatile
manner and activates specific receptors in the anterior
pituitary gland, thus leading to the release of gonado-
tropins, luteinizing hormone (LH), and follicle-stimulat-
ing hormone (FSH). These hormones in turn are re-
leased into the general circulation and bind to receptors
on specialized cells in the ovaries or testes to stimulate
steroidogenesis. The gonads respond to this activation
by synthesizing and secreting the sex steroid hormones
(estrogen and progesterone in the female and testoster-
one in the male), which in turn exert their classical
effects on secondary sexual characteristic development
throughout the body. Elucidation of the hypothalamic-
pituitary-gonadal axis physiology led to the notion that
antagonism of the GnRH receptor in the pituitary gland
is an effective way to treat diseases that are aggravated
by the presence of sex steroid hormones, such as
endometriosis, uterine fibroids, and prostate cancer.1
Currently, these conditions are treated with long-acting
GnRH peptide agonists.2 These work by an initial
stimulation, followed by down-regulation of the receptor.
Recently, we reported on the discovery of a new class
of small-molecule GnRH antagonists, the 1-arylmethyl-
3-(2-aminoethyl)-5-aryl-6-methyluracils, exemplified by
13g (Figure 1). This series of compounds was further
optimized by introduction of a methyl group at the
â-carbon of the N-3 side chain, e.g., 23h,j (Figure 1).
Although potent functional GnRH antagonists such as
(S)-2a and (S)-2b were identified from this series, we
later found that these were relatively metabolically
unstable. For instance, upon incubation with human
liver microsomes (HLM), both (S)-2a and (S)-2b were
rapidly consumed, leading mainly to the side chain
dealkylation metabolite 3. This compound in turn was
much less active as a GnRH antagonist (path A, Figure
1). To obtain orally active, longer-acting small-molecule
GnRH antagonists, we clearly needed to improve the
metabolic stability profile of these uracil compounds. To
accomplish this goal, we envisioned having the lipophilic
side chain (e.g., the benzyl group in (S)-2a or the
cyclopentyl in (S)-2b) originate from the 2-carbon on the
N-3 side chain. This modification would result in
branched primary amines where the side chain is
connected by a carbon-carbon bond (path B, Figure 1),
which we expect should be much more stable toward
HLM metabolism. Here, we report on the synthesis,
SAR studies, and preliminary pharmacokinetics of these
novel GnRH antagonists.
* To whom correspondence should be addressed. For F.C.T.: phone,
+1-858-658-7677; fax, + 1-858-658-7601; e-mail, ftucci@neurocrine.com.
For C.C.: phone, +1-858-658-7634; fax, + 1-858-658-7601; e-mail,
cchen@neurocrine.com. Company Web site: http://www.neurocri-
ne.com.
† Department of Medicinal Chemistry.
‡ Department of Endocrinology.
§ Department of Preclinical Development.
10.1021/jm049791w CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/28/2004

3484 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Letters
Ta ble 1. Binding Affinities of 3 and 4 toward the hGnRH
Sch em e 1a
Receptor
compd
R
stereochemistry
Ki ( SEM (nM)
(R)-3
(S)-3
Me
Me
i-Pr
i-Pr
c-Pent
c-Pent
c-Hex
c-Hex
i-Bu
i-Bu
neo-Pent
Bn
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(S)
(S)
595 ( 87
>10000
51 ( 18
973 ( 226
10 ( 3.3
94 ( 27
6.5 ( 1.2
46 ( 12
6.4 ( 1.0
39 ( 13
71 ( 15
66 ( 11
16 ( 1.1
965 ( 140
(R)-4a
(S)-4a
(R)-4b
(S)-4b
(R)-4c
(S)-4c
(R)-4d
(S)-4d
(R)-4e
(R)-4f
(S)-4f
(S)-4g
Bn
PhCH2CH2
Ta ble 2. Binding Affinities of 2 and 9-11 toward the hGnRH
Receptor
compd
R
R′
Bn
c-Pent
Me
R′′
Ki ( SEM (nM)
(S)-2a
(S)-2b
(R)-9d
(S)-9f
(R)-10b
(R)-10c
(R)-10d
(R)-11b
(S)-11b
(R)-11c
(S)-11f
Me
Me
i-Bu
Bn
c-Pent
c-Hex
i-Bu
c-Pent
c-Pent
c-Hex
Bn
H
H
H
H
H
H
H
Me
Me
Me
Me
51 ( 12
4.5 ( 0.6
1.2 ( 0.3
8.1 ( 0.5
12 ( 0.2
23 ( 3.0
12 ( 2.7
0.6 ( 0.1
2.4 ( 0.7
4.1 ( 1.0
7.9 ( 1.5
Me
i-Pr
i-Pr
i-Pr
Me
Me
Me
a
Reagents: (a) diethyl azodicarboxylate, Ph3P, THF, room
temp, 5 h; (b) 2-fluoro-3-methoxyphenylboronic acid, Pd[PPh3]4,
DME/PhH/EtOH, Ba(OH)2 (aq), 80 °C, 16 h; (c) TFA, CH2Cl2, room
temp, 1 h; (d) aldehyde or ketone, CH2Cl2, HOAc, Na(OAc)3BH,
room temp, 1 h; (e) CH2O (aq), CH2Cl2, HOAc, Na(OAc)3BH, room
temp, 1 h.
Me
a branched primary amine, e.g., 4. Interestingly, the
enantiomeric preference for the receptor varies accord-
ing to the substitution pattern. For instance, the (S)-
enantiomer of the phenylalanine analogue ((S)-4f) is
about 4- fold more potent than its (R)-enantiomer
counterpart ((R)-4f). Homologation of the (S)-isomer
((S)-4g) significantly reduced binding affinity. When
alkyl-substituted side chains were employed, the op-
posite enantiomeric preference was observed. In all
cases, the (R)-isomer was the preferred one. The affinity
for the hGnRH receptor was proportional to the lipo-
philic content of the side chain, with cyclohexyl ((R)-
4c) and isobutyl ((R)-4d ) being the best. The lack (e.g.,
(R)-3) or excess (e.g., (R)-4e) of lipophilicity proved to
be detrimental to binding.
Next, we investigated the effects of having small alkyl
substituents on the basic nitrogen. The results are sum-
marized in Table 2. Generally, these modifications led
to enhancements in potency and in some cases to a re-
versal of enantiomeric preference for the receptor. Mono-
and dimethylation of the phenylalanine derivative (S)-
4f gave (S)-9f and (S)-11f, respectively, which showed
modest improvements in binding affinity. While the de-
rivative from (R)-leucinol ((R)-4d ) was very potent (Ki
) 6.4 ( 1.0 nM), monomethylation of its basic nitrogen
resulted in a 5-fold increase in binding affinity ((R)-9d ,
Ki ) 1.2 ( 0.3 nM). Interestingly, the highly potent di-
methylated cyclopentyl derivatives (R)-11b and (S)-11b
4 and 9-11 were synthesized according to the pro-
cedure depicted in Scheme 1. Thus, the previously
described 5-bromo-1-(2,6-difluorobenzyl)-6-methyluracil
53h was coupled, using Mitsunobu conditions, with the
corresponding Boc-protected amino alcohols 6, which are
available either commercially or from the respective
amino acids by lithium aluminum hydride reduction
followed by Boc protection in an one-pot procedure. The
products 7 were obtained in 80-90% isolated yields.
Subsequent Suzuki couplings of 7 with 2-fluoro-3-
methoxyphenylboronic acid gave 8 in 50-70% yields.
These compounds were deprotected in the presence of
trifluoroacetic acid in dichloromethane to furnish the
primary amines 4 in quantitative yields. This procedure
was also applied to N-methyl derivatives 9 because
N-monomethylation of 4 was very difficult with conven-
tional reductive alkylation methods. Selected com-
pounds 4 were further alkylated under reductive con-
ditions to give N-alkyl derivatives 10 or N,N-dimeth-
ylated 11 in very good yields.
All compounds were then evaluated for their competi-
tive binding to the cloned human GnRH (hGnRH)
receptor expressed in HEK293 cells, using a 96-well
filtration setup5,6 and des-Gly10[125I-Tyr5,D-Leu6,NMeLeu7,-
Pro9-NEt]GnRH as the radiolabel. The results of the
binding assay are presented in Tables 1 and 2.
In Table 1, we examined N-3 side chains containing

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3485
Ta ble 3. Mouse and Cynomolgus Monkey Pharmacokinetics
Profiles of (S)-2a , (R)-4c, (R)-4d , (S)-4f, and (S)-9f
oral
clearance
Vd
t1/2
(h)
compd bioavailability (F, %) (mL min-1 kg-1
)
(L kg-1
)
Mouse
(S)-2a
(R)-4c
(R)-4d
(S)-4f
(S)-9f
3
11
6
11
42
81.3
65.4
65.7
65.6
97.2
7.9
7.3
3.9
4.9
6.9
1.1
1.3
0.7
0.9
0.8
Monkey
(S)-9f
22
13.5
3.6
3.1
clearance of all compounds, the main differences seen
here most probably have to do with their ability to cross
the gut wall. We can only speculate that (S)-9f may have
an advantage in that respect because it only has one
hydrogen bond donor, in contrast with its analogue (S)-
4f, which has two.
To address the species differences with respect to
clearance, we also performed a pharmacokinetic evalu-
ation of (S)-9f in cynomolgus monkeys (Table 3). (S)-9f
showed good oral bioavailability (22%), with a clearance
of 13.5 mL min-1 kg-1, much lower than hepatic blood
flow rate.
We were unable to perform the in vivo evaluation of
efficacy in mice, rats, and cynomolgus monkeys because
of the lack of sufficient affinity of our compounds for
their GnRH receptors. For example, (S)-9f had Ki values
of 200 nM in the cynomolgus monkey receptor and
11 000 nM in the rat receptor. This type of species
selectivity is consistent with our previous findings.3h-j
Further optimization toward good potency on the mon-
key GnRH receptor and in vivo efficacy results of those
compounds will be reported in due course.
In summary, we have designed and synthesized a
novel series of uracils containing modified N-3 side
chains, as potent hGnRH receptor antagonists. These
are metabolically more stable, and selected compounds
from this series displayed good oral bioavailability in
mice and cynomolgus monkeys. Although the half-lives
of these compounds are still short for desirable phar-
macokinetic properties in these species, the in vitro
metabolism data in HLM were encouraging, suggesting
that these compounds should have longer half-lives in
humans because of a lower rate of metabolism.
F igu r e 2. Dose-response curves of (R)-4c and (S)-9f inhibit-
ing GnRH induced Ca2+ flux. The respective IC50 values are
indicated for each case.
(Ki ) 0.6 ( 0.1 and 2.4 ( 0.7 nM, respectively) are vir-
tually equipotent toward the hGnRH receptor, contrast-
ing with the marked preference for the (R)-isomer in
the primary amine parent compounds (R)-4b and (S)-
4b. The observation that N-alkylated or dialkylated de-
rivatives of primary amines were equally or more potent
than their parent compounds offers a good opportunity
to adjust physicochemical properties of this class of
compounds.
The functional antagonism of the compounds herein
synthesized was confirmed by their ability to inhibit
GnRH stimulated Ca2+ flux in transfected cells in a
dose-dependent manner.7 For instance, (R)-4c and (S)-
9f were found to be functional antagonists with IC50
60 and 20 nM, respectively (Figure 2).
)
These new compounds were metabolically more stable
in the HLM assay. For example, upon incubation with
HLM for 20 min, (S)-4f suffered a 4% loss whereas 33%
of (S)-2a was degraded. This same trend was also
observed with other compounds in this series, which
shows that the strategy of connecting the lipophilic side
chain via a carbon-carbon bond was warranted.
Ack n ow led gm en t. This work was partly supported
by NIH Grants 1-R43-HD38625-01 and 2-R44-HD38625-
02. We are grateful to Sue K. Sullivan and Lee Carter
for pharmacological support.
(S)-2a , (R)-4c, (R)-4d , (S)-4f, and (S)-9f were inves-
tigated for pharmacokinetic properties in mice. The
compounds were given orally (po) and intravenously (iv)
at a 10 mg/kg dose, and the data are shown in Table 3.
All compounds showed low to good oral exposure in
mice, despite their relatively high rate of clearance. (S)-
2a , for instance, had the worst exposure among all of
those tested, which can be partly attributed to its
inherent metabolic instability. (S)-9f was far superior
than the other compounds in terms of absolute oral
bioavailability, even though it had the highest rate of
clearance. On the basis of compound concentration in
the hepatic portal vein, we estimated that 76% of (S)-
9f was absorbed, translating in 55% of first-pass me-
tabolism in the mice. Clearly, given the similar rates of
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails of the pharmacokinetic experiments and experimental
procedures for the synthesis and characterization of (R)-4c,
(R)-4d , (S)-4f, and (S)-9f. This material is available free of
charge via the Internet at http://pubs.acs.org.
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refer to the Supporting Information.
J M049791W