A novel synthesis of 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-ones as potent, stable GnRH receptor antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00745-X

A new class of small molecule GnRH antagonists, the 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-ones, was designed and a novel synthesis for these compounds was developed. The synthesis utilizes a base-catalyzed intramolecular cyclization of fluoromethyl pyrimidone 5 to generate the bicyclic core. Amongst the compounds synthesized, we discovered some highly potent GnRH receptor antagonists (e.g., 12, K(i)=9 nM), which showed enhanced stability towards acidic physiological conditions compared to the des-fluoro analogues.

Full text of DOI:10.1016/S0960-894X(02)00745-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3491–3495
A Novel Synthesis of 7-Aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-ones
as Potent, Stable GnRH Receptor Antagonists
Fabio C. Tucci,* Yun-Fei Zhu, Zhiqiang Guo, Timothy D. Gross,
Patrick J. Connors, Jr., R. Scott Struthers, Greg J. Reinhart,
Xiaochuan Wang, John Saunders and Chen Chen*
Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121, USA
Received 18 June 2002; accepted 23 July 2002
Abstract—A new class of small molecule GnRH antagonists, the 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-ones, was designed and a
novel synthesis for these compounds was developed. The synthesis utilizes a base-catalyzed intramolecular cyclization of fluoro-
methyl pyrimidone 5 to generate the bicyclic core. Amongst the compounds synthesized, we discovered some highly potent GnRH
receptor antagonists (e.g., 12, Ki=9 nM), which showed enhanced stability towards acidic physiological conditions compared to the
des-fluoro analogues.
# 2002 Elsevier Science Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH) is a deca-
peptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-
NH2) produced in and secreted by the hypothalamus in
a pulsatile manner.1 Through interactions at specific
GnRH receptors, it stimulates the gonadotrophes in the
pituitary gland to release follicle stimulating hormone
(FSH) and luteinizing hormone (LH). These gonado-
tropins, in turn, regulate the production of sex steroids
and gametogenesis. Antagonism of the GnRH receptor
in the pituitary gland, therefore, constitutes a valid and
effective way to treat diseases that are reproductive in
nature, such as endometriosis, uterine fibroids and
prostate cancer. Currently, these conditions are treated
with peptide agonists, represented by Leuprorelin1.2
Even though these are effective, they have to be admi-
nistered in depot form since such peptides are not orally
bioavailable. In response to that need, intensive efforts
have been initiated for the development of small
molecule GnRH antagonists.3,4
good potency, preliminary studies indicated that this
class of compounds is relatively unstable under acidic
conditions. Incubation of 2 with 0.2 N HCl at 37 ꢀC for
2 h results in the fast disappearance of the parent
compound and the formation of a major degradation
product, which was identified as 3 (Fig. 2). This degra-
dation results from an acid catalyzed retro-Mannich
reaction on the basic side-chain. Compound 1, on the
other hand, was much more stable under the same con-
ditions. We reasoned that the presence of the cyano
group at the 8-position of the bicyclic system reduces its
electron density, which is also felt by the basic side
chain, resulting in a compound with increased stability
towards acid. Introduction of the cyano group however,
led to a decrease in potency in the order of 5–13-fold,5a,b
possibly because of a steric encumberance with the
Very recently, we described our initial efforts towards
potent, orally bioavailable small molecule GnRH
antagonists, as exemplified by the 7-aryl-8-cyano-pyr-
rolo[1,2-a]pyrimid-4-one 15a and 7-arylpyrrolo[1,2-
a]pyrimid-4-one 25b (Fig. 1). In the case of 2, despite its
*Corresponding author. Tel.: +1-858-658-7677; fax: +1-858-658-7601;
e-mail: ftucci@neurocrine.com; website: http://www.neurocrine.com
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00745-X

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F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3491–3495
receptor and this moiety, when compared to the smaller
hydrogen. A stable, more potent antagonist could be
obtained if a smaller electron-withdrawing group, such
as a fluoride is used. In this letter we report on the novel
synthesis of 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-
ones and their stability profile as potent GnRH receptor
antagonists.
separately with a-bromo-30-methoxyacetophenone or
a-bromo-40-isobutoxyacetophenone,7 which were cho-
sen based on previous knowledge from our work.5 The
reactions were performed in the presence of tetra-
butylammonium fluoride (TBAF) as a base, to provide
mixtures of the respective N1-alkylated products 5a–b
and the O-alkylated side products 6a–b, in 60 and 65%
yield, respectively. After chromatographic separation,
the ratios in both reactions were determined to be 2:1 in
favor of the unwanted products 6a–b. The structural
assignment of regioisomers was performed by NOE
experiments. 5a–b were each then subjected to the key
cyclization step in the presence of sodium ethoxide in
ethanol, to give the desired core 7-aryl-8-fluoro-pyr-
rolo[1,2-a]pyrimidone 7a–b. Each were alkylated with 2-
The synthetic approach is depicted in Scheme 1.
a-Fluoroacetamidine hydrochloride was prepared in
32–43% by the action of HCl and EtOH on a-fluoro-
acetonitrile, according to a published procedure.6 The
acetamidine was then cyclized in the presence of diethyl
ethoxymethylenemalonate, under basic conditions, to
afford hydroxy pyrimidine 4. This was then alkylated
Figure 2. Degradation pathway of compound 2 in the presence of 0.2 N HCl at 37 ꢀC.
Scheme 1. Reagents and conditions: (a) (i) HCl (g), 15 ꢀC, 45 min; (ii) EtOH, ꢁ50 ꢀC to rt, 16 h; (b) NaOH, EtOH, diethyl ethoxymethylene mal-
onate, reflux, 7 h; (c) (i) TBAF, DME/THF, rt, 30 min; (ii) a-bromoacetophenones; (d) NaOEt, EtOH, rt, 4 h; (e) (i) TBAF, DME/THF, rt, 30 min;
(ii) 2-fluorobenzyl bromide, 16 h; (f) R0NHMe, CH2O aq, HOAc, rt, 1 h.

F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3491–3495
3493
fluorobenzyl bromide, in the presence of TBAF, to give
8a–b with combined yields over both steps of 25 and
30%. Finally, Mannich reactions with dialkylamines
and formaldehyde in glacial acetic acid led to the target
compounds 9–14.
replacement of the (2-pyridyl)ethyl group on the basic
nitrogen by either a benzyl (13) or a (2-furyl)methyl (14)
led to a decrease of 5- and 10-fold in potency, respectively.
This trend was also confirmed with compounds 9–11,
where the differences were even greater. When com-
pared to the 8-cyano analogue 1,5a the 8-fluoropyrrolo-
pyrimidone 12 shows 10-fold increase in affinity for the
GnRH receptor. This difference confirms our initial
Compounds 9–14 were assayed for their ability to bind
competitively to the cloned human GnRH receptor
expressed in HEK293 cells using a 96-well filtration
setup8,9 and des-Gly10[125I-Tyr5, DLeu6, NMeLeu7,
Pro9-NEt]GnRH as the radiolabel (Table 1). Their
functional antagonism was confirmed by their ability to
inhibit GnRH stimulated Ca2+ flux in a dose-dependent
manner.10 For instance, 1 mM of 12 completely blocked
the influx of Ca2+ ions after stimulation with 10 nM of
GnRH (Fig. 3).
Table 1. Binding affinities of 8-fluoro-pyrrolo[1,2-a]pyrimidones
9–14 towards the human GnRH receptor9
The most potent compound in this series is 12, which
shows a Ki value of 9.0 nM. In general, compounds
containing the 7-(40-isobutoxy)aryl group were 20–260-
fold more potent than their analogues with 7-(30-meth-
oxy)aryl, suggesting a possible lipophilic interaction
with the receptor. In the 7-(40-isobutoxy)aryl series,
Compd
R
R0
Ki (nM)
9
3-MeO–
200
10
11
12
13
14
3-MeO–
3-MeO–
4-iBuO–
4-iBuO–
4-iBuO–
2600
24,000
9
42
92
Figure 3. Inhibition of GnRH stimulated Ca2+ release by compound 12.
Figure 4. Alignment of minimum energy conformations of compounds 1, 2 and 12.

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F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3491–3495
Figure 5. Electron density surface for compounds 1, 2 and 12. The 1-(2-fluorobenzyl) groups are highlighted in the circles. The surfaces are colored
to reflect the electostatic potentials, with color changes indicating their magnitude and polarity. The gray and cyan represent the richest electron
density, whereas the white means poorest electron density domain, according to the legend on the right.
of compounds 1,2 and 12 are presented in Figure 5,12 and
provide another useful qualitative tool to help rationa-
lize the experimental data. The impact of the substituent
at the 8-position on the electronics of the bicyclic core is
not surprising—1 and 12 are more electron-deficient
than 2. In fact, we deliberately used this notion to
improve their stability towards acid, as alluded to
earlier. Less obvious is the effect of the 8-substituent on
the electronics of the 1-(2-fluorobenzyl) group. In the
case of 8-cyano compound 1, the charge density on the
Figure 6. Stability curve of compound 12 after incubation with 0.2 N
HCl for 2 h at 37 ꢀC.
1-(2-fluorobenzyl) group is the highest, followed by
unsubstituted compound 2. The 1-(2-fluorobenzyl) moi-
ety of the 8-fluoro substituted pyrrolo-pyrimidone 12
has the lowest charge density among these compounds,
which may help explain why it is a better binder.
According to a 3-D model of the human GnRH recep-
tor built based on the X-ray crystal structure of rho-
dopsin,13 it is believed that in the binding pocket, the 1-
(2-fluorobenzyl) group may interact with one of the two
tyrosine residues on the transmembrane VI (Y283 and
Y284) of the human GnRH receptor, either by p stack-
ing or by edge-to-face interactions. Since the aromatic
ring on the tyrosine is electron-rich, the binding of a
ligand is more energetically favorable if it possesses an
electron-deficient aromatic.
hypothesis that only a small substituent at the 8-posi-
tion of the bicyclic system is tolerated so as to avoid an
unfavorable steric interaction with the receptor.
Another aspect of the presence of a small substituent at
the 8-position is its impact on the orientation of the
1-(2-fluorobenzyl) group. The conformations of
compounds 1, 2 and 12 were compared using AM1/
semi-empirical quantum mechanics. The most preferred
conformation of 12 was used as a template to align with
conformers of 1 and 2, and the relative differences in
energies between these conformers were then calculated
(Fig. 4). The depicted conformations of 2 and 12 are
energetically very similar (ꢂ0 kcal/mol difference),
whereas the one for 1 is approximately 7 kcal/mol
higher in energy. In the case of 2 and 12, the 1-(2-fluoro-
benzyl) group can reside perpendicularly on either face
of the plane established by the bicycle (Fig. 4), whereas
in the case of 1, the presence of the larger 8-cyano sub-
stituent forces the ring to adopt different orientations,
which may not be the preferred mode of binding. This
observation may help explain the differences in affinity
of 1,2 and 12 for the GnRH receptor.
The stability of compound 12 under acidic conditions
was then determined (Fig. 6). As expected, 12 showed
decreased degradation when incubated with a 0.2 N HCl
aqueous solution at 37 ꢀC for up to 1 h—up to 63% of
the parent compound remained after this time. This is in
contrast to stability of 2, which after 1 h showed more
than 90% degradation.14
In conclusion, we have designed and achieved the novel
synthesis of 7-aryl-8-fluoro-pyrrolo[1,2-a]pyrimid-4-
ones, which behave as potent antagonists of the human
GnRH receptor. In addition, we have demonstrated
that introduction of a small electron-withdrawing
group, such as a fluoride, is able to stabilize the pyr-
rolo[1,2-a]pyrimidone core, while maintaining the
potency towards the human GnRH receptor.
In addition to the conformational aspect, we conducted
semi-empirical calculations using the MOPAC/AM1
method from ‘Quantum CAChe 5.0’,11 in order to
investigate other possible causes for the better affinity
exhibited by 12 and 2 over 1. The charge density surfaces

F. C. Tucci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3491–3495
3495
Acknowledgements
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We are indebted to Dr. Tao Hu, Dr. Xin-Jun Liu and
Mrs. Mila Lagman for assistance in obtaining the sta-
bility data. This work was partly supported by NIH
grants 1-R43-HD38625-01 and 2-R44-HD38625-02.
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3–8 independent experiments.
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12. The charge density surfaces of compounds 1, 2 and 12,
represent the charge distribution on each molecule as the
probability of electron availability. These were obtained
directly from the conformations used in Figure 4, and may not
represent the conformation of these compounds in the bound
state. The charge density surfaces of compounds 1, 2 and 12
are shown in colors according to the scale provided—the gray
color at the bottom represents the highest possible electron
density (ꢁ0.060, in arbitrary units) while the top white corre-
sponds to the other end of the scale (0.090, in arbitrary units),
with all the other colors in between.
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14. Unpublished results.