A new class of potent nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists: design and synthesis of 2-phenylimidazo[1,2-a]pyrimidin-5-ones.

Article abstract of DOI:10.1016/S0960-894X(02)00372-4

The design and synthesis of a new class of nonpeptide luteinizing hormone-releasing hormone (LHRH) receptor antagonists, the 2-phenylimidazo[1,2-a]pyrimidin-5-ones, is reported. Among compounds described in this study, we identified the potent antagonist 15b with nanomolar in vitro functional antagonism. The result might suggest that the heterocyclic 5-6-ring system possessing a pendant phenyl group attached to the five-membered ring is the important structural feature for a scaffold of small molecule LHRH antagonists.

Full text of DOI:10.1016/S0960-894X(02)00372-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2073–2077
A New Class of Potent Nonpeptide Luteinizing Hormone-
Releasing Hormone (LHRH) Antagonists: Design and Synthesis
of 2-Phenylimidazo[1,2-a]pyrimidin-5-ones
Satoshi Sasaki,a,* Toshihiro Imaeda,a Yoji Hayase,a Yoshiaki Shimizu,b
Shizuo Kasai,b Nobuo Cho,a Masataka Harada,a Nobuhiro Suzuki,b
Shuichi Furuyac and Masahiko Fujinoa,b,c
aPharmaceutical Research Division, Takeda Chemical Industries, Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan
bPharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17-85 Jusohonmachi 2-chome, Yodogawa-ku,
Osaka 532-8686, Japan
cStrategic Product Planning Department, Takeda Chemical Industries, Ltd., 1-1 Doshomachi 4-chome, Chuo-ku, Osaka 540-8645, Japan
Received 3 April 2002; accepted 18 May 2002
Abstract—The design and synthesis of a new class of nonpeptide luteinizing hormone-releasing hormone (LHRH) receptor
antagonists, the 2-phenylimidazo[1,2-a]pyrimidin-5-ones, is reported. Among compounds described in this study, we identified the
potent antagonist 15b with nanomolar in vitro functional antagonism. The result might suggest that the heterocyclic 5–6-ring sys-
tem possessing a pendant phenyl group attached to the five-membered ring is the important structural feature for a scaffold of small
molecule LHRH antagonists. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
by this achievement, further effort has been devoted
toward searching for new scaffolds of small molecule
LHRH antagonists. Recently, we investigated the
synthesis and biological activity of a new series of
potent nonpeptide LHRH antagonists, the 6-phenyl-
thieno[2,3-d]pyrimidine-2,4-diones 2, which were
designed by substituting the pyrimidine ring for the
pyridine ring of the thienopyridin-4-one nucleus.5
Therefore, we next focused on the thiophene ring of the
thienopyridin-4-one core and decided to replace it with
other five-membered rings. Since the biological activities
and pharmacokinetic profiles of compounds depend on
their physicochemical properties, incorporation of a
five-membered ring with different physicochemical
properties from those of the thiophene ring, into a
bicyclic scaffold was hoped to provide new potent
LHRH antagonists with improved pharmacokinetic
profiles. Accordingly, we designed the 2-phenylimi-
dazo[1,2-a]pyrimidin-5-one 3,6 a heterocyclic 5–6-ring
system in which an imidazole ring is embedded, as a
novel scaffold for nonpeptide LHRH antagonists.
In recent years, luteinizing hormone-releasing hormone
(LHRH, also known as gonadotropin-releasing hor-
mone; GnRH) antagonists are widely acknowledged as
logical candidates for new types of drugs in the treat-
ment of endocrine-based diseases, for example certain
sex hormone-dependent cancers, endometriosis, uterine
leiomyoma and precocious puberty.1,2 Indeed, peptidic
LHRH antagonists, which are expected to directly
reduce the steroid hormone levels without the initial
‘flare effect’ induced by peptidic LHRH agonists, have
achieved clinical success.2,3 Therefore, it is expected that
potent and orally bioavailable nonpeptide LHRH
antagonists may be clinically desirable agents without
the usual liabilities associated with large peptidic ther-
apeutics.
We previously identified the 2-phenylthieno[2,3-b]pyr-
idin-4-one T-98475 (1) as the first, potent, and orally
active nonpeptide LHRH receptor antagonist.4 Inspired
In this letter, we wish to describe the synthesis and bio-
logical evaluation of a series of 2-phenylimidazo[1,2-
a]pyrimidin-5-ones, which led to the identification of a
*Corresponding author. Tel.: +81-298-64-6351; fax: +81-298-64-
6308; e-mail: sasaki_satoshi@takeda.co.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00372-4

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S. Sasaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2073–2077
new class of potent low molecular weight LHRH
antagonists, exemplified by the methoxyurea 15b.
of 2-amino-4-hydroxypyrimidine I with 2-bromopropio-
phenone II provides the 3-alkyl-2-aminopyrimidin-4-one
III, which can then be converted to the desired imidazo-
pyrimidin-5-one IV by intramolecular cyclization.
Chemistry
The methodology of construction of the 2-phenylimi-
dazopyrimidine nucleus is shown in Scheme 1. Initially,
reaction of ethyl 2-amino-4-hydroxypyrimidine-5-car-
boxylate 4 with 2-bromopropiophenone 5 in the pre-
sence of potassium carbonate did not produce the
desired N3-alkylated compound and the cyclized
To our knowledge, there are no reports on the synthesis
of highly functionalized 2-arylimidazo[1,2-a]pyrimidin-
5-ones.7 Hence, we started by developing a synthetic
procedure to obtain the target compound IV, according
to the approach illustrated in Chart 1. Briefly, alkylation
Chart 1.
Scheme 1. Reagents and conditions: (a) K2CO3, KI, DMF (6: 37%, 7: 31%, 8: 1%); (b) Zn, AcOH (78%); (c) 2,6-difluorobenzyl chloride, K2CO3,
KI, DMF (10: 85%, 11: 8%).

S. Sasaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2073–2077
2075
product 9, but instead gave rise to the imidazopyr-
imidine 6 and the O-alkylated pyrimidine 7 (approxi-
mately 1:1 ratio) together with 8 and other minor
products.8 Determination of the chemical structures of 6
and 8 was achieved by measurement of the nuclear
Overhauser effect (NOE) between the two methyl
groups and the proton on the pyrimidine ring. Although
efforts have been made toward preparation of the N3-
alkylated product or 9 directly from 4 and 5 in satisfac-
tory yield, these attempts were unsuccessful. This might
be explained as follows: after initial N3-, O-, or N1-
alkylation of 4 occurred (N3- and O-alkylation exceeded
N1-alkylation), ring-closure reaction of the N3-, N1-
alkylated products and subsequent alkylation with 5
afforded 6 and 8, respectively. Secondary alkylation
may well be attributed to the relatively high reactivity of
the imidazopyrimidines due to their electron-rich nat-
ure. Based on these results, we next examined conver-
sion of 6 to the desired product 9. Gratifyingly,
reductive dealkylation of the phenacyl group using zinc
powder and acetic acid proceeded smoothly to furnish 9
in good yield. Alkylation of compound 9 with 2,6-
difluorobenzyl chloride gave the two regioisomers 10
(85%) and 11 (8%).9 X-ray crystallography showed
the major product to be the desired N8-alkylated com-
pound 10 (see Fig. 1).10
The synthetic procedure for the amides 14a–g and urea
analogues 14h,i and 15a,b is outlined in Scheme 2.
Nitration of 10 with sodium nitrate in sulfuric acid took
place with almost complete regioselectivity toward the
para position of the 2-phenyl ring to afford 12. After
bromination of 12 and incorporation of the N-benzyl-
methylamino group, the nitro compound 13 was
obtained. Reduction of 13 with iron powder-hydro-
chloric acid provided the corresponding aniline, which
in turn was converted to the target amide and urea
derivatives 14a–i via acylation using acyl chlorides,
condensation with carboxylic acids, reaction with ethyl
isocyanate, or reaction of 1,10-carbonyldiimidazole
(CDI) and successive treatment of O-methylhydroxyl-
amine. Finally, transesterification of compounds 14h,i
with titanium(IV) isopropoxide in 2-propanol afforded
the isopropyl esters 15a,b.11
Results and Discussion
The imidazo[1,2-a]pyrimidin-5-one derivatives were
evaluated for inhibition of specific [125I]leuprorelin
binding to the cloned human LHRH receptor12 and the
results are summarized in Table 1. Initially, the para
substituent on the 2-phenyl ring of the imidazopyr-
imidin-5-ones was explored for the 6-ethyl ester deriva-
tives. The isobutyrylamide 14a and propionylamide 14b
were found to show significant 10ꢀ8 M order binding
Figure 1. X-ray structure of compound 10.10
i
Scheme 2. Reagents and conditions: (a) NaNO3, concd H2SO4 (89%); (b) NBS, AIBN, CCl4; (c) N-benzylmethylamine, Pr2NEt, DMF (80%, two
steps from 12); (d) Fe, concd HCl, EtOH (95%); (e) RCOCl, Et3N, CH2Cl2 (58–61%) or RCO2H, PyBOP, iPr2NEt, CH2Cl2 (48–86%); (f) EtNCO,
Py (65%) or (1) CDl, Et3N, CH2Cl2; (2) O-methylhydroxylammonium chloride, Et3N (50%); (g) Ti(OiPr)4, iPrOH (15a: 47%, 15b: 13%).

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S. Sasaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2073–2077
Table 1. In vitro activities of imidazo[1,2-a]pyrimidin-5-one deriva-
tives
Consequently, this study has demonstrated that the
imidazopyrimidin-5-ones, thienopyridin-4-ones, and
thienopyrimidine-2,4-diones constitute a new class of
potent nonpeptide LHRH antagonists. It is expected
that further optimization of the 6-substituent of 15a,b
will lead to identification of potent and orally effective
LHRH antagonists.
Conclusion
Compd
R1
R2
Binding affinity
a
Starting with the thienopyridine-based LHRH antago-
nist T-98475 (1), a series of the 2-phenylimidazopyr-
imidin-5-ones was designed, synthesized, and evaluated
as nonpeptide LHRH antagonists. This study resulted
in the identification of a new class of potent LHRH
antagonists, represented by the methoxyurea 15b pos-
sessing high binding affinity and potent in vitro antagon-
ism, with IC50 values of 0.4 and 7 nM, respectively.
These results led us to conclude that the imidazopyr-
imidin-5-one provides a new scaffold for small molecule
LHRH antagonists devoid of a thiophene ring. Taking
this finding into consideration, it is suggested that the
heterocyclic 5–6-ring system bearing a pendant phenyl
group attached to the five-membered ring is the key
structural motif for a LHRH antagonist scaffold.
IC50 (nM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
iPr
Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
30
10
5
3
20
7
Cyclopropyl
Vinyl
Ph
3-Thienyl
3-Furyl
EtNH
MeONH
EtNH
MeONH
3
0.5
0.7
0.3
0.4
i
15a
15b
CO2 Pr
i
CO2 Pr
aThe binding affinity is reported as the IC50 value, which is the
antagonist concentration required to inhibit the specific binding of
[
125I]leuprorelin to the LHRH receptor by 50%. Chinese hamster
ovary (CHO) cells expressing human LHRH receptors were used as
the source for LHRH receptors. All data are expressed as means of
two or three determinations.
Acknowledgements
affinities. Replacement of the alkyl group of 14a with
cyclopropyl or vinyl moieties (14c,d) gave 6- to 10-fold
enhancement in activity. This indicated that the smaller
alkylamides are favorable. In the arylamide series, the
benzoyl analogue 14e was less potent than 14c,d, how-
ever, substitution of the phenyl group of 14e for the
thiophene (14f) or furan (14g) rings increased the affi-
nity. The high binding affinities of 14f,g might be a
consequence of the smaller ring size and/or introduction
of the hetero atom.
We gratefully acknowledge Dr. H. Onda, Dr. H. Shir-
afuji, Dr. A. Miyake, Dr. H. Sawada, and Dr. C. Kitada
for encouragement and helpful discussion throughout
this work. We also thank Ms. T. Urushibara and Mr. S.
Takekawa for supporting the biochemical experiments,
and wish to express our gratitude for the X-ray crystal-
lographic analysis of compound 10 performed by Mr.
A. Fujishima and Ms. K. Higashikawa.
References and Notes
Incorporation of urea and urea-related moieties onto
the 2-phenyl ring produced an increase in activity. The
ethylurea 14h and methoxyurea 14i displayed sub-
nanomolar affinities and were 14- to 20-fold more
potent than 14b. Moreover, transesterification of 14 h,i
caused further enhancement in affinity. The isopropyl
esters 15a,b were about twice as potent as the ethyl
esters 14h,i. Binding affinities of 15a,b were almost
comparable to that of T-98475 (1) (IC50 value of
0.2 nM). The result revealed that the imidazopyrimidin-
5-one is a new scaffold for nonpeptide LHRH antago-
nists in addition to the thienopyridin-4-one and thieno-
pyrimidine-2,4-dione scaffolds.
1. Filicori, M.; Flamigni, C. Drugs 1988, 35, 63.
2. (a) Huirne, J. A. F.; Lambalk, C. B. Lancet 2001, 358,
1793. (b) Goulet, M. T. In Annual Reports in Medicinal
Chemistry; Bristol, J. A., Ed.; Academic: New York, 1995;
Vol. 30, p 169.
3. Blithe, L. D. Trends Endocrinol. Metab. 2001, 12, 238.
4. Cho, N.; Harada, M.; Imaeda, T.; Imada, T.; Matsumoto, 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.
5. Sasaki, S.; Cho, N.; Nara, Y.; Harada, M.; Endo, S.;
Suzuki, N.; Furuya, S.; Fujino, M. Manuscript in preparation.
6. Furuya, S.; Imaeda, T.; Sasaki, S. WO 99/33831, 1999;
Chem. Abstr. 1999, 131, 87922.
7. (a) There are some reports concerning syntheses of simple
2-arylimidazo[1,2-a]pyrimidines and imidazo[1,2-a]pyrimidin-
5-ones; see: Spitzer, W. A.; Victor, F.; Pollock, G. D.; Hayes,
J. S. J. Med. Chem. 1988, 31, 1590. (b) Groebke, K.; Weber,
L.; Mehlin, F. Synlett 1998, 661. (c) Blackburn, C. A. Tetra-
hedron Lett. 1998, 39, 5469. (d) Sako, M.; Totani, R.; Hirota,
K.; Maki, Y. Chem. Pharm. Bull. 1992, 40, 235.
8. The physicochemical data of compounds 6, 7 and 8 were as
follows. Compound 6: mp 177–178 ꢁC; 1H NMR (300 MHz;
TMS/CDCl3) d 1.42 (3H, t, J=7.1 Hz), 1.83 (3H, d,
J=7.6 Hz), 2.90 (3H, s), 4.41 (2H, q, J=7.0 Hz), 6.82 (1H, q,
The ethylurea 15a and methoxyurea 15b, which exhib-
ited low sub-nanomolar affinities, were next evaluated
for in vitro functional antagonism.13 Compounds 15a,b
potently inhibited LHRH-stimulated arachidonic acid
release from CHO cells expressing the human LHRH
receptor, with IC50 values of 10 and 7 nM, respectively.
From these data, the imidazopyrimidin-5-ones 15a,b
proved to be potent nonpeptide LHRH antagonists in
this context.

S. Sasaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2073–2077
2077
J=7.3 Hz), 7.26–7.43 (3H, m), 7.52–7.70 (5H, m), 8.13 (2H, d,
1
was cloned from a pituitary cDNA library and CHO cells
stably expressing high levels of the recombinant human
LHRH receptor were isolated. [125I][Tyr5]leuprorelin (0.12–
0.15 nM) and the membrane fractions of the CHO cells
(0.2 mg/mL) were incubated at 25 ꢁC for 60 min in 0.2 mL
assay buffer A [25 mM Tris, 1 mM EDTA, 0.1% bovine serum
albumin (BSA), 0.03% NaN3, 0.25 mM phenylmethanesulfo-
nyl fluoride, 1 mg/mL pepstatin A, 20 mg/mL leupeptin and
100 mg/mL phosphoramidon, pH 7.5] containing various con-
centrations of the test compounds. The reaction was termi-
nated by adding 2 mL ice-cold assay buffer A, and the bound
and free ligands were immediately separated by filtration
through a poly(ethylenimine)-coated glass microfiber filter
(Whatman, GF/F). The filter was washed twice with 2 mL assay
buffer A, and radioactivity was measured using a X-ray counter.
Specific binding was determined by subtracting the nonspecific
binding, which was measured in the presence of 1 mM unlabeled
leuprorelin, from the total binding. The concentration of each
test compound that produced 50% inhibition of the specific
binding (IC50 value) was derived by fitting the data into a
pseudo-Hill equation: log[%SPB/(100ꢀ%SPB)]=n[log(C)ꢀ
log(IC50)], where %SPB is the specific binding expressed as a
percentage of the maximum specific binding; n is the pseudo-
Hill constant; and C is the concentration of the test compound.
13. Inhibition of LHRH-stimulated arachidonic acid release
from CHO cells expressing human LHRH receptors was
measured to evaluate the functional LHRH antagonism of the
test compounds according to the previously reported protocol;
see: Masuda, Y.; Sugo, T.; Kikuchi, T.; Kawata, A.; Satoh,
M.; Fujisawa, Y.; Itoh, Y.; Wakimasu, M.; Ohtaki, T. J.
Pharmacol. Exp. Ther. 1996, 279, 675. The human LHRH
receptor-expressing CHO cells were seeded into 24-well plates
at a density of 4ꢂ104 cells/well and cultured for 1 day. The
cells were then incubated with [5,6,8,9,11,12,14,15-3H]arachi-
donic acid (11 kBq/well, NEN Lifescience Products) for 1 day
and washed with Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 20 mM HEPES and 0.2% BSA.
The cells were then pre-incubated with the compounds at
37 ꢁC for 60 min and the reaction was started by addition of
LHRH (1 nM). After incubation at 37 ꢁC for 40 min, radio-
activity in the medium was measured with a liquid scintillation
counter. The assays were repeated twice. IC50 value is the
antagonist concentration required to inhibit the LHRH-sti-
mulated arachidonic acid release from CHO cells by 50%.
J=8.8 Hz), 8.54 (1H, s). Compound 7: H NMR (300 MHz;
TMS/CDCl3)
d 1.35 (3H, t, J=7.2 Hz), 1.70 (3H, d,
J=7.1 Hz), 4.31 (2H, q, J=7.0 Hz), 5.20–5.80 (2H, br), 7.47
(2H, t, J=7.4 Hz), 7.55–7.64 (1H, m), 8.02 (2H, d, J=8.5 Hz),
8.67 (1H, s). Compound 8: mp 190–191 ꢁC; 1H NMR
(300 MHz; TMS/CDCl3) d 1.39 (3H, t, J=7.1 Hz), 1.75 (3H,
d, J=7.1 Hz), 2.62 (3H, s), 4.40 (2H, q, J=7.0 Hz), 6.57 (1H,
q, J=7.0 Hz), 7.26–7.65 (6H, m), 7.70 (2H, d, J=8.7 Hz), 8.04
(2H, d, J=8.8 Hz), 8.77 (1H, s).
9. The 1H NMR spectrum data of compounds 10 and 11 were
1
as follows. Compound 10: H NMR (300 MHz; TMS/CDCl3)
d 1.39 (3H, t, J=7.2 Hz), 2.91 (3H, s), 4.37 (2H, q, J=7.2 Hz),
5.51 (2H, s), 7.00 (2H, t, J=7.9 Hz), 7.31–7.47 (4H, m), 7.68
(2H, d, J=7.6 Hz), 8.37 (1H, s). Compound 11: 1H NMR
(300 MHz; TMS/CDCl3) d 1.40 (3H, t, J=7.2 Hz), 2.64 (3H,
s), 4.38 (2H, q, J=7.1 Hz), 5.34 (2H, s), 6.64 (2H, t,
J=8.1 Hz), 7.10–7.17 (3H, m), 7.36–7.47 (3H, m), 8.77 (1H, s).
10. Crystal data for compound 10: C23H19F2N3O3,
Mr=423.42, rcalcd=1.43 gcmꢀ3, monoclinic, space group P21/n,
˚
a=15.812(3), b=14.321(4), c=17.544(2) A, b=98.70(1),
3
2
˚
V=3926(1) A , Z=8; R1=0.056, wR2 (F ; all data)=0.175,
S=1.05 for 5838 unique data and 562 parametꢀe3rs, final dif-
˚
ference synthesis max=0.55, min=ꢀ0.33 eA
. Intensity
measurement: Rigaku AFC5R diffractometer, Cu-Ka radia-
tion, graphite monochromator, o-2y scan, 2ymax=120ꢁ,
T=293 K. Moderate decay of intensities was observed and
corrected. Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre (CCDC 181750).
Copies of the data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44–
1223–336033; e-mail: deposit@ccdc.cam.ac.uk).
11. The physicochemical data of compound 15b was as fol-
lows: mp 94–96 ꢁC (free amine); 1H NMR (300 MHz; TMS/
CDCl3) d 1.36 (6H, d, J=6.3 Hz), 2.17 (3H, s), 3.66 (2H, s),
3.83 (3H, s), 4.33 (2H, s), 5.19–5.27 (1H, m), 5.50 (2H, s), 6.99
(2H, t, J=8.1 Hz), 7.02–7.26 (5H, m), 7.34–7.43 (1H, m), 7.58
(2H, d, J=8.7 Hz), 8.03 (2H, d, J=8.7 Hz), 8.37 (1H, s). IR
(KBr) 1740, 1597, 1417, 1218, 1036 cmꢀ1. FAB-MS 645
(M+H). Anal. calcd for C34H34N6O5F2: C, 63.34; H, 5.32; N,
13.04. Found: C, 63.64; H, 5.26; N, 12.86.
12. Receptor binding assays were carried out as described
previously (see ref 4). Briefly, human LHRH receptor cDNA