Synthesis and initial structure-activity relationships of a novel series of imidazolo[1,2-a]pyrimid-5-ones as potent GnRH receptor antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00370-0

SAR studies of 2-arylimidazolo[1,2-a]pyrimid-5-ones 10a-m, which were derived from initial lead 3a, resulted in the discovery of a series of potent nonpeptide human GnRH receptor antagonists. Compounds with good potency (e.g., 10e, K(i)=7.5 nM) were prepa

Full text of DOI:10.1016/S0960-894X(02)00370-0

Bioorganic & Medicinal ChemistryLetters 12 (2002) 2179–2183
Synthesis and Initial Structure–Activity Relationships of a Novel
Series of Imidazolo[1,2-a]pyrimid-5-ones as Potent GnRH
Receptor Antagonists
Keith M. Wilcoxen,a Yun-Fei Zhu,a,* Patrick J. Connors, Jr.,a John Saunders,a
a
TimothyD. Gross, Yinghong Gao,a Greg J. Reinhart,b
R. Scott Struthersb 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 22 February2002; accepted 26 April 2002
Abstract—SAR studies of 2-arylimidazolo[1,2-a]pyrimid-5-ones 10a–m, which were derived from initial lead 3a, resulted in the
discoveryof a series of potent nonpeptide human GnRH receptor antagonists. Compounds with good potency(e.g.,
10e,
Ki=7.5 nM) were prepared by introduction of a 2-(2-pyridyl)ethyl at the basic nitrogen and a 3-pentyl ester at the 6-position of the
bicyclic core. # 2002 Elsevier Science Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH), or luteiniz-
ing hormone-releasing hormone, is a linear decapeptide
disease symptoms without the concomitant flare effect,
which can be caused bysuper agonists. Several small
molecule GnRH receptor antagonists have appeared in
the literature. T-98475 and its analogues are the first
small molecules reported to have high affinityfor the
human GnRH receptor (IC50=0.2 nM) but are less
potent at the rat receptor (IC50=60 nM).8 An ery-
thromycin A derivative, A-198401, was reported as a
highlypotent GnRH antagonist (p Ki=9.2 and 8.7 on rat
and human receptors, respectively), and having 15.2%
oral bioavailabilityin rats (Fig. 1). 9 Compound 1 has very
potent binding affinityto the human (IC 50=0.44 nM) as
well as rat GnRH receptor (IC50=4 nM),10 and very
recently, indole derivatives such as 2 were reported to be
amide,
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-
NH2, originallyisolated and characterized from por-
cine1 and ovine2 hypothalami. Its action at the level of
the pituitaryis to stimulate the secretion of the gona-
dotropins, luteinizing hormone (LH), and follicle-stim-
ulating hormone (FSH) via interaction with its cell
surface receptor, which belongs to the G-protein couple
receptor superfamily.3 These gonadotropins, in turn, act
on the reproductive organs where theyparticipate in the
regulation of steroid production, gametogenesis and
ovulation.4 GnRH has been the subject of wide interest
in the search for nonsteroidal contraceptive agents
because of its important role in the regulation of both
male and female reproduction.5 Several disease condi-
tions such as endometriosis and prostate cancer can be
treated bysuppression of the pituitary–gonadal axis, and
gonadotropin-releasing hormone superagonists repre-
sented byleuprorelin, 6 which down regulates the receptor,
are currentlyused in the treatment of these conditions. 7
11
potent and orallybioavailable GnRH antagonists.
We have recently reported the SAR of the pyrrolopyr-
imidone core as potent GnRH antagonists (i.e., 3b)
from modification of an initial lead compound 3a.12 For
example, compound 3b had a Ki of 2.7 nM against the
human GnRH receptor and selected compounds from
this series inhibited GnRH stimulated Ca++ flux.12b In
this paper, we describe the design and synthesis of imi-
dazolopyrimidones (10a–m) as potent GnRH antago-
nists. The main focus of this studyis an investigation of
the SAR for the replacement of the pyrrolo ring with an
imizadolo moietyin the belief that such a change would
increase the stabilityof the targeted compounds while
maintaining their GnRH antagonist acivities.13
Clinical evidence shows that peptidic GnRH antagonists
directlylower gonadal sex hormone levels alleviating
*Corresponding author. Fax: +1-858-658-7619; e-mail: cchen@
neurocrine.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00370-0

2180
K. M. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2179–2183
A general synthesis of 7-alkylimidazolo[1,2-a]pyrimid-5-
ones 6 based on a cyclization of a 2-aminopyrimidine
with a-bromoketone is outlined in Scheme 1. Thus,
ethyl 2-amino-4-hydroxypyrimidne-5-carboxylate 4 was
treated with a-bromoacetophenones in the presence of
sodium hydride in tetrahydrofuran at room tempera-
ture, followed byammonium hydroxide. After a simple
aqueous workup, the desired 8H-imidazolo[1,2-a]pyr-
imid-5-ones 5a and 5b were crystallized from methanol.
N-Alkylation of the imidazolo[1,2-a]pyrimid-5-ones 5a
or 5b with 2-fluorobenzyl bromide in the presence of
tetrabutylammonium fluoride in DME at room tem-
perature gave the desired 2-aryl-7-(2-fluorobenzyl)-
imidazolo[1,2-a]pyrimd-5-ones 6 and 7, which were
purified bychromatographyon silica gel (ethyl acetate/
hexanes). The structure of compounds 6 and 7 were
confirmed byNOE experiments in which the NOE
between the benzylic proton and the proton at the 6-
position of the bicyclic system was observed. Trans-
esterification of the ethyl ester 7 in the presence of 3-
hydroxypentane and sodium hydride in THF at room
temperature gave the corresponding 3-pentyl ester 8.
The nitro group of 7 or 8 was easilyconverted to the
corresponding amide 9a or 9b, respectively, by hydro-
genation using Raney-Ni catalyst under hydrogen, fol-
lowed byacylation of the crude anilino compounds with
various acid anhydrides or acid chlorides (Scheme 2).
The imidazolo[1,2-a]pyrimid-5-ones were then subjected
to a Mannich reaction to afford the desired products
10a–m which were purified bypreparative TLC chro-
14
matographyon silica gel (Scheme 3).
The synthesized compounds were evaluated for their
125I-Tyr5,DLeu6,NMeLeu7,Pro9-
abilityto inhibit
[
NEt]GnRH agonist binding to the cloned human
GnRH receptor, and rat GnRH receptor as described
previously.15 All compounds with inhibition better than
50% at 100 mM were titrated on a six-point curve in
duplicate, and their data are reported as Ki values using
the Cheng–Prusoff equation.16 The initial radioligand
binding data for 9a and 9b suggested these two com-
pounds were onlyweaklyactive ( Ki 30 and 28 mM,
respectively); the human GnRH receptor binding data
for the analogues 10 are reported in Table 1.
Figure 1. Small molecule GnRH antagonists.
Scheme 1. Reagents and conditions: (a) a-bromo-40-methoxyacetophenone, or a-bromo-40-nitroacetophenone, NaH, DME, rt; then NH4OH, rt; (b)
TBAF/THF, then 2-fluorobenzyl bromide, rt; (c) NaH/3-hydroxypentane, THF, 0 ꢀC to rt.

K. M. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2179–2183
2181
Scheme 2. Reagents and conditions: (a) RaneyNi, H 2, MeOH; then RCOCl, Et3N, CH2Cl2, rt.
Scheme 3. Reagents and conditions: (a) R1R2NH, CH2O, AcOH, rt to reflux.
The prototypical compound in this series was 2-(4-buty-
roylamidophenyl)-3-(N-benzyl-N-methylaminomethyl)-6-
ethoxycarbonyl-8-(2-fluorobenzyl)imidazolo[1,2-a]pyr-
imid-5-one 10c, which was found to have good affinity
for GnRH receptor (Ki=80 nM). This result indicated
the importance of the basic nitrogen atom and the ben-
zyl group attached (9a had a Ki of 30 mM). Replacement
of the butyroylamido on the 4-position of the phenyl
group (R3) with a methoxygroup resulted in approxi-
mately12-fold loss of activity( 10a, Ki=950 nM). In
addition to a possible hydrogen-bonding site from the
oxygen or nitrogen atom, a small lipophilic group in this
area seems to be veryimportant for high binding affi-
nity. Replacement of the benzyl group on the 3-amino-
methyl functionality of the core structure with a 2-(2-
pyridyl)ethyl group gave an analogue with over 4-fold
increase in activity( 10b, Ki=230 nM). A similar result
was obtained when the benzyl group of 10c was
replaced by the 2-(2-pyridyl)ethyl group (10d,
Ki=12 nM). The ethyl ester in 10d could be replaced by
the more bulky3-pentyl ester with a suggestive, albeit
modest, increase in activity( 10e, Ki=7.5 nM), leaving
open the opportunityfor further exploration at this site.
Replacement of the 2-pyridyl group of compound 10e
with a nonsubstituted phenyl group only slightly
decreased the binding activity( 10f, Ki=14 nM), but
replacement of the 2-(2-pyridyl)ethyl with 3-pyridyl-
methyl group caused a 7-fold loss of activity (10g,
Ki=52 nM). Similar results were obtained when furan-
methyl or 2-methoxyethyl group was used to replace the
2-(2-pyridyl)ethyl group (10h and 10i, Ki=29 and 36,
respectively). However, introduction of cyanomethyl
group caused over 40-fold decrease in binding affinity
for the human GnRH receptor (10j, Ki=300 nM), pos-
siblybecause of the reduction in pKa of the amine by
electron withdrawing cyano group.
Table 1. Structure–activityrelationships for the 2-arylimidazolo[1,2-
a]pyrimid-5-ones
Compd
R1NR2
R3
R4
h-GnRH
Ki (nM)
9a
9b
(ꢁ)
NHCOPr
NHCOPr
OMe
Et
30,000
28,000
950
230
80
(ꢁ)
CH(Et)2
Et
Et
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
PhCH2NMe
2-PyCH2CH2NMe
PhCH2NMe
2-PyCH2CH2NMe
2-PyCH2CH2NMe
PhCH2CH2NMe
3-PyCH2NMe
2-FuranCH2NMe
MeOCH2CH2NMe
NCCH2NMe
OMe
NHCOPr
NHCOPr
NHCOPr
NHCOPr
NHCOPr
NHCOPr
NHCOPr
NHCOPr
Et
Et
12
7.5
14
52
29
36
CH(Et)2
CH(Et)2
CH(Et)2
CH(Et)2
CH(Et)2
CH(Et)2
300
On the other hand, introduction of a cyclic tetrahydro-
isoquinoline group decreased activityalmost 40-fold
(10k, Ki=290 nM), while the non-aromatic pyrrolidine
analogue showed much less activity( 10l, Ki=2.2 mM).
Finally, the 4-isobutyroylamidophenyl analogue gave
similar activity( 10m, Ki=41 nM), compared with the
corresponding n-butyroylamidophenyl analogue such
as 10g and 10h. From these studies, a number of the
10k
10l
NHCOPr
NHCOPr
CH(Et)2
CH(Et)2
290
2200
10m
PhCH2NMe
NHCOPr-i
CH(Et)2
41

2182
K. M. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2179–2183
imidazolo[1,2-a]pyrimid-5-ones having high binding
affinityfor the human GnRH receptor were identified.
5. Zatuchni, G. I.; Shelton, J. D.; Sciarra, J. J. In LHRH
Peptides as Male and Female Contraceptives; Philadelphia:
Harper & Row, 1981.
6. Fujino, M.; Fukuda, T.; Shinagawa, S.; Kobayashi, S.;
Yamazaki, I.; Nakayama, R.; Seely, J. H.; White, W. F.; Rip-
pel, R. H. Biochem. Biophys. Res. Commun. 1974, 60, 406.
7. (a) Garnick, M. B.; Glode, L. M. N. Engl. J. Med. 1984,
311, 1281. (b) Dutta, A. S. Drugs Future 1988, 13, 43. (c)
Karten, M. J.; Rivier, J. E. Endocr. Rev. 1986, 7, 44.
As we expected, this series of compounds are much
more stable than their counterpart from the pyrrolo-
pyrimidone series 3b. For example, When 10f was incu-
bated in 0.2 N HCl solution at 37 ꢀC, there was no
significant degradation (<5% of parent).
8. Cho, N.; Harada, M.; Toshihiro, I.; Imada, T.; Matsu-
moto, H.; Hayase, Y.; Sasaki, S.; Furuya, S.; Suzuki, N.;
Okubo, S.; Ogi, K.; Endo, S.; Onda, H.; Fujino, M. J. Med.
Chem. 1998, 41, 4190.
All the compounds have verylow binding affinity
towards the rat GnRH receptor. For example, 10g had
a Ki of 3.5 mM. This is consistent with the similar
observation byCho and co-workers for the thienopyr-
idinone analogue T98475.8
9. Besecke, L.; Diaz, G.; Segreti, J.; Mohning, K.; Cybulski,
V.; Rao, M.; Bush, E.; Randolph, J. T.; Waid, P.; Haviv, F.;
Wegner, G.; Greer, J. Drug Develop. Res. 2001, 52, 485.
10. DeVita, R. J.; Walsh, T. F.; Young, J. R.; Jiang, J.;
Ujjainwalla, F.; Toupence, R. B.; Parikh, M.; Huang, S. X.;
Fair, J. A. D.; Goulet, M. T.; Wyvratt, M. J.; Lo, J.-L.; Ren,
N.; Yudkovitz, J. B.; Yang, Y. T.; Cheng, K.; Chi, J.; Mount,
G.; Rohrer, S. P.; Schaeffer, J. M.; Rhodes, L.; Drisko, J. E.;
McGowan, E.; MacIntyre, D. E.; Vincent, S.; Carlin, J. R.;
Cameron, J.; Smith, R. G. J. Med. Chem. 2001, 44, 917.
11. 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.; Kar-
anam, B. V.; Vincent, S. H.; Chang, K.; Goulet, M. T. Bioorg.
Med. Chem. Lett. 2001, 11, 2597.
The binding activityof the initial compounds ( 9a and
9b) was greatlyimproved byincorporation of a basic
tertiary amine at the 3-position of the bicyclic system.
This modification, we suspect, mayinitiate a crucial
interaction between the ligand and its receptor, invol-
ving an acidic residue within the putativelyhelical
domains. Based on a predictive model of the membrane
spanning regions within the protein, the candidate for
this site is aspartic acid 302 on helix 7.3 Interestingly,
the corresponding residue in the rat GnRH receptor is
glutamic acid and this maycontribute to the species
selectivitybetween the human and rat GnRH receptors
we observed for 10a–m. The results of the SAR study
suggest that a varietyof tertiaryamines are tolerated
while the 2-(2-pyridyl)ethyl group on the 3-aminomethyl
functionalityof the imidazolo[1,2- a]pyrimid-4-one core
structure gives the best result.
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. Bioorg. Med. Chem. Lett. 2002, 12, 399. (b)
Zhu, Y.-F.; Wilcoxen, K.; Struthers, R. S.; Connors, P., Jr.;
Saunders, J.; Gross, T.; Gao, Y.; Reinhart, G.; Chen, C.
Bioorg. Med. Chem. Lett. 2002, 12, 403.
13. Unfortunately, it was subsequently shown that this com-
pound (3b) and close related analogues suffered from the
instabilitycaused bythe amino group attached to the benzylic-
like position of pyrrole ring. For example, compound 3b,
after removing the electron-withdrawing cyano group at the
3-position of 3a, onlyhad a t 1/2 of 22 min in rat plasma at 37 ꢀC.
14. All final compounds were characterized byHPLC-MS
with two-wavelength (220 and 256) detection. All the com-
pounds reported here passed purityof >85% of both detec-
tion. The following is a typical procedure for synthesis of 10m:
6-Ethoxycarbonyl-8-(2-fluorobenzyl)-2-(4-nitrophenyl)imida-
zolo[1,2-ꢀ]pyrimid-5-one: To a suspended solution of 2-amino-
5-ethoxycarbonylpyrimid-4-one (10 g, 55 mmol) in DMF
(100 mL) was added NaH (60% dispersion in mineral oil,
2.6 g, 65 mmol). The reaction was allowed to stir under nitro-
gen for 45 min and a-bromo-4-nitroacetophenone (14.09 g,
58 mmol) in DMF (20 mL) was added dropwise. This resulted
in a purple then dark solution which was allowed to stir for
45 min at rt. To this solution was then added 50 mL of 30%
aqueous ammonium hydroxide and the mixture was stirred at
room temperature for 2 h. The resulting dark solution was
concentrated in vacuo, and the residue was slowlypoured into
300 mL of 2 M HCl solution, stirred for 0.5 h and the resulting
orange solid was collected byvacuum filtration to yield the
crude 6-ethoxycarbonyl-2-(4-nitrophenyl)-8H-imidazolo[1,2-
a]pyrimid-5-one, which was used for next step, MS m/e 329
(MH+).
In conclusion, a series of 2-arylimidazolo[1,2-a]pyrimid-
5-ones exemplified by 10a–m was discovered, with some
having good binding affinityfor the human GnRH
receptor. A hydrophobic ester at the 6-position is help-
ful for high binding affinity, and a small lipophilic group
with possible hydrogen bonding site at the 4-position of
the phenyl group is required for optimum human
GnRH receptor binding affinity. Further structure
activityrelationships of this series of compounds will be
reported in the following paper.17
Acknowledgements
This work was supported, in part, byNational Institutes
of Health grants 1-R43-HD38625–01 and 2-R44-
HD38625–02.
References and Notes
1. Matsuo, H.; Baba, Y.; Nair, R. M. G.; Arimura, A.; Schally,
A. V. Biochem. Biophys. Res. Commun. 1971, 43, 1334.
2. Burgus, R.; Butcher, M.; Amoss, M.; Ling, N.; Monahan,
M.; Rivier, J.; Fellows, R.; Blackwell, R.; Vale, W.; Guillemin,
R. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 278.
3. Sealfon, S. C.; Weinstein, H.; Millar, R. P. Endocrine Rev.
1997, 18, 180.
6-Ethoxycarbonyl-2-(4-nitrophenyl)imidazolo[1,2-a]pyrimid-
5-one (10 g, 30 mmol) was suspended in DME (100 mL) and
treated with TBAF (1 M in THF, 1.3 equiv) dropwise at rt.
This mixture was allowed to stir for 0.5 h before 2-fluoro-
benzyl bromide (1.1 mmol in 5 mL of DME) was introduced.
The reaction was stirred at room temperature overnight, con-
centrated and extracted with chloroform (2ꢂ200 mL). The
organic layers were combined, washed with brine, filtered,
4. Cheng, K. W.; Leung, P. C. K. Can. J. Phys. Pharmacol.
2000, 78, 1029.

K. M. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2179–2183
2183
dried over sodium sulfate, and concentrated in vacuo to yield
the title compound as a solid. Analytic sample was obtained
by recrystallization of the product from ethyl acetate. 1H
NMR CDCl3 d 1.40 (t, 3H), 4.42 (q, 2H), 5.54 (s, 2H), 7.6–
7.15 (m, 4H), 8.06 (d, 2H), 8.15 (s, 1H), 8.3 (d, 2H), 8.60 (s,
1H); MS m/e 437 (MH+). Anal. (C22H17FN4O5) C, H, N.
3-(N-Benzyl-N-methyl)aminomethyl-6-(3-pentyloxycarbonyl)-
8-(2-fluorobenzyl)-2-(4-isobutyroylaminophenyl)imidazolo[1,2-
a]pyrimid-5-one: To a solution of 3-pentanol (500 mg, 5 mmol)
in THF (10 mL) was added n-BuLi (2.5M, in hexanes, 2 mL)
while stirring at 0 ꢀC. This solution was stirred for 0.5 h and
then warmed to rt. A suspension of ethoxycarbonyl-8-(2-
fluorobenzyl)-2-(4-nitrophenyl)imidazolo[1,2-a]pyrimid-5-one
(1 g, 2.3 mmol) in THF (10 mL) was added to the stirred solu-
tion. The reaction was stirred at rt for 3 h, quenched with
ammonium chloride solution, extracted with chloroform
and washed with brine. The extract was dried and con-
centrated in vacuo to yield 6-(3-pentoxycarbonyl)-8-(2-
fluorobenzyl)-2-(4-nitrophenyl)imidazolo[1,2-a]pyrimid-5-one
as a solid.
centrated. The crude amide was purified bycolumn chroma-
tography to yield 6-(3-pentoxycarbonyl)-8-(2-fluorobenzyl)-2-
(4-isobutyroylaminophenyl)-imidazolo[1,2-a]pyrimid-5-one as
a yellow oil.
6-(3-Pentoxycarbonyl)-8-(2-fluorobenzyl)-2-(4-isobutyoylami-
nophenyl) imidazolo[1,2-a]pyrimid-5-one (1.0 mmol) was added
to a stirred solution of formaldehye (37% in water, 1.5 equiv),
benzylmethylamine (1.5 mmol) and acetic acid (2 mL). The
reaction mixture was heated to 50 ꢀC for 2 h, cooled to rt and
concentrated in vacuo. This crude oil was suspended between
ethyl acetate and saturated sodium bicarbonate solution. The
organic layer was separated and washed with brine, dried over
sodium sulfate and concentrated in vacuo. The title compound
was purified bychromatographyon silica gel (95% ethyl ace-
tate, 5% MeOH). 1H NMR CDCl3 d 1.2 (m, 9H), 1.69 (m,
4H), 1.72 (m, 2H), 2.21 (s, 3H), 2.45 (m, 2H), 3.72 (s, 2H), 4.37
(s, 2H), 4.88 (m, 1H), 5.54 (s, 2H), 7.2–7.5 (m, 11H), 7.89 (m,
2H), 8.54 (s, 1H); MS m/e 652 (MH+).
15. Perrin, M. H.; Haas, Y.; Rivier, J. E.; Vale, W. W. Mol.
Pharmacol. 1983, 23, 44.
The crude solid obtained from above was hydrogenated at
50 psi in the presence of Raneynickel catalyst in ethanol at
room temperature overnight. The reaction mixture was filtered
and concentrated in vacuo to yield a crude semisolid which
was dissolved in dichloromethane and stirred overnight in the
presence of excess isobutyric anhydride. The reaction mixture
was concentrated, partitioned between ethyl acetate and water,
washed with brine, dried over sodium sulfate and con-
16. On each assayplate, a standard antagonist of comparable
affinityto 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.
17. Gross, T.; Zhu, Y.-F.; Struthers, S. R.; Saunders, J.; Gao,
Y.-H.; Connors, P., Jr.; Guo, Z.-Q.; Chen, C. Bioorg. Med.
Chem. Lett. 2002, 12, 2185.