Initial structure-activity relationship studies of a novel series of pyrrolo[1,2-a]pyrimid-7-ones as GnRH receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(01)00779-X

Initial SAR studies on 1-aminomethyl-2-aryl-3-cyano-pyrrolo[1,2-a]pyrimid-7-one-6-carboxylates as human GnRH receptor antagonists were discussed. 2-(2-Methylaminoethyl)pyridine was discovered to be a key feature for generating active compounds. The best compound from the series had 25 nM (K_i) binding affinity to human GnRH receptor.

Full text of DOI:10.1016/S0960-894X(01)00779-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 399–402
Initial Structure–Activity Relationship Studies of a Novel Series of
Pyrrolo[1,2-a]pyrimid-7-ones as GnRH Receptor Antagonists
Yun-Fei Zhu,* R. Scott Struthers, Patrick J. Connors, Jr., Yinghong Gao,
Timothy D. Gross, John Saunders, Keith Wilcoxen, Greg J. Reinhart,
Nicholas Ling and Chen Chen*
Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121, USA
Received 4 October 2001; accepted 12 November 2001
Abstract—Initial SAR studies on 1-aminomethyl-2-aryl-3-cyano-pyrrolo[1,2-a]pyrimid-7-one-6-carboxylates as human GnRH
receptor antagonists were discussed. 2-(2-Methylaminoethyl)pyridine was discovered to be a key feature for generating active
compounds. The best compound from the series had 25 nM (Ki) binding affinity to human GnRH receptor. # 2002 Elsevier Science
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 was originally isolated and character-
ized from porcine1 and ovine2 hypothalami. It acts on
the pituitary gland to stimulate the secretion of both
luteinizing hormone and follicle-stimulating hormone.
These gonadotropines in turn act on the reproductive
organs, where they participate in the regulation of
gonadal steroid production, spermatogenesis in male
and follicular development in female. Several reproduc-
tive disease conditions such as endometriosis, uterine
fibroids and prostate cancer can be treated by suppres-
sion of the pituitary-gonad hormonal axis. As matter of
fact, depot form of peptidic GnRH agonists represented
by leuprorelin# is currently used to treat such condi-
tions through a receptor down-regulation mechanism to
suppress gonadal steroid production.3 However, recent
clinical evidence has showed that peptidic GnRH
antagonists can act immediately at the receptor to lower
steroid level and therefore alleviate disease symptoms
without the concomitant ‘flare effect’, which is exhibited
by the peptide agonists due to their initial over-stimula-
tion of the receptor. Nevertheless peptide antagonists
still have their limitations such as poor oral bioavail-
ability. Recently, small molecule GnRH antagonists
have been described in the literature. Compound 1 (T-
98475) and its analogues were reported4 to have high
potency on the human GnRH receptor (IC50=0.2 nM)
albeit with the reduced binding affinity for the rat
receptor (IC50=60 nM). In addition a series of papers
on quinolones and tryptamines as potent GnRH
antagonists were recently published.5 For example, qui-
nolone 2 possesses high affinity (IC50=0.4 nM) for the
human GnRH receptor, but also shows reduced affinity
(IC50=4 nM) for the rat receptor. While the tryptamine
analogue 3 is a high-affinity inhibitor (IC50=7 nM) of
the human GnRH receptor, but again is less potent for
the rat receptor (IC50=170 nM). In this letter we report
our initial efforts towards the design of orally active
small molecule GnRH antagonists around a novel series
of pyrrolo[1,2-a]pyrimidones.
2-Aryl-3-cyano-6-ethoxycarbonylpyrrolo[1,2-a]pyrimid-
7-one6 (9a, Scheme 1) was the core structure employed
for our initial SAR study. To synthesize the core, the
propanone 4a was a-brominated by CuBr2 in refluxing
ethyl acetate/chloroform (1:1) and the resulting bromide
5a was crystallized in ether at 0 ꢀC. This was reacted
with sodium azide to form azide 6a in THF/H2O at
room temperature. The crude organic layer was trans-
ferred into a Parr bottle diluted with 1% HCl in ethanol
and then hydrogenated over palladium (10% on car-
bon) at 36 psi to give the amino ketone 7a. Subse-
quently, 7a was refluxed with malonitrile under aqueous
basic conditions to give the desired amino pyrrole
*Corresponding author. Tel.: +1-858-658-7745; fax: +1-858-658-
7601; e-mail: fzhu@neurocrine.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00779-X

400
Y.-F. Zhu et al. / Bioorg. Med. Chem. Lett. 12 (2002) 399–402
Scheme 1. Reagents and conditions: (a) CuBr2, EtOAc/CHCl3, reflux; (b) NaN3, THF/H2O; (c) H2, Pd/C, EtOH/HCl; (d) malonitrile, NaOH/H2O–
EtOH, reflux; (e) diethyl ethoxylethylene malonate, EtOH, reflux; (f) Dowtherm, 230 ꢀC; (g) TBAF/THF, R2CH2Br, 60 ꢀC; (h) NBS/CCl4; (i)
R3R4NH, CH3CN, rt.
product 8a. Heating 8a first in ethanol with diethyl
ethoxylethylene malonate and then in Dowtherm at
230 ꢀC yielded the desired pyrrolo[1,2-a]pyrimidone 9a.
products 32–37 as shown in Scheme 2. A simple
approach to simultaneously introduce lipophilic groups
at positions 2 and 6 was developed (Scheme 3). Com-
pound 31a was treated with boron tribromide at À78 ꢀC
to give 38, which was di-alkylated with alkyl bromides
(R5Br) in dry DMF at 90 ꢀC in the presence of K2CO3.
Bromination with NBS and subsequently replacement
by 2-(2-methylaminoethyl)pyridine yielded the desired
products 39 and40.
Having synthesized 9a, parallel synthesis was performed
to explore two points of diversity at postions 1 and 4. In
the presence of tetrabutylammonium fluoride (TBAF)
in THF, 9a underwent N-alkylation with a series of
alkyl bromides (R2CH2Br), followed by radical bro-
mination on the 1-methyl group (NBS, CCl4, benzoyl
peroxide). The bromide intermediates were treated with
a variety of amines (R3R4NH) in CCl4/CH3CN (1:1),
leading to the final products 10–22. After obtaining the
SAR information for the 1-aminomethyl and 4-alkyl
groups, the investigation was expanded by optimizing
the 2-(4-alkoxyphenyl) group. The approach for synth-
eses of compounds 23–30 was identical as that shown in
Scheme 1, where 4-iso-butoxypheny replaced 4-methoxy-
pheny at position 2. Compounds 10–30 were selected
from a library of 400 compounds to facilitate the SAR
discussion here.
All of the synthesized compounds were evaluated for
their ability to compete for des-Gly10[125I-Tyr5,D-
Leu,6NMeLeu,7Pro9-NEt]GnRH radioligand binding
to the cloned human receptor stably expressed in
HEK293 cells using a 96-well filtration assay format
and several compounds were also tested using a cloned
rat GnRH receptor binding assay.7 The binding assay
results for compounds 10–30 are listed in Table 1.8
Notably, all non-basic compounds were not active (data
not shown). Compound 10 with a simple benzyl sub-
stitution at position 4 and a dimethyl amine group at
the 1-methyl position was not active. Weak activity
(Ki=42 mM) was observed when one of the methyl
groups on 10 was replaced by a benzyl group as indi-
cated in 11. Binding activity was significantly enhanced,
however, when a 2-(2-pyridyl)ethyl group was intro-
duced to replace the benzyl group (12, Ki=2.6 mM).
Presumably an extra hydrogen bonding site and the
hydrophobic aromatic ring are preferred in this area.
The ethyl esters 31a and 31b, obtained from 9a and 9b,
respectively, by 4-benzylation with 2-fluorobenzyl bro-
mide in the presence of TBAF in THF, were trans-
esterified by different alcohols and n-butyl lithium under
anhydrous conditions. The same methods outlined in
Scheme 1 were then applied to introduce the 2-(2-
methylaminoethyl)pyridyl group to give the desired

Y.-F. Zhu et al. / Bioorg. Med. Chem. Lett. 12 (2002) 399–402
401
Simultaneous exploration of the benzyl group at posi-
tion 4 led to the 2-fluorobenzyl substitutent as the best
among these analogues with 16 showing 0.5 mM binding
affinity. When a second fluoro group was introduced on
the benzyl ring for all possible positions (17–20), no
improvement on binding affinity was observed. The
importance of benzyl group at position 4 was further
indicated by 21, a cyclopropylmethyl analogue, which
totally lost the activity. Consequently the less active
compound 22 (Ki=1.8 mM) provided further evidence
that the 2-fluorobenzyl group at position 4 and N-[2-(2-
pyridyl)-ethyl]-N-methylaminomethyl group at the
position 1 were the best substituents in this two points
variation parallel synthesis study. Next, optimization of
2-aromatic ring showed that replacing the methoxyl
with a more lipophilic group such as an isobutoxy gen-
erally increased the potency. For example, 23 was 3-fold
more potent than its methoxyl analogue 15. This same
trend was observed for compounds 24–27. Shifting the
nitrogen of the pyridyl from the 2- to 3- or 4-position
(27 vs 28 or 29) resulted in a considerable loss of bind-
ing affinity. The same was true for 30 where a phenyl
was used to replace the 2-pyridyl group. These data
suggested the 2-pyridyl nitrogen might act in concert
with the basic amine to interact with the receptor.
Apparently, none of the other pyridyl analogues can
geometrically ‘coordinate’ such action.
Scheme 2. Reagents and conditions: (a) TBAF/THF, 2-fluorobenzyl
bromide, 60 ꢀC; (b) R5OH, n-BuLi, THF; (c) NBS/CCl4; (d) 2-(2-
methylaminoethyl)pyridine, CH3CN, rt.
Table 2 presents the data for a variety of esters. Chan-
ging the ethyl ester (16, 500 nM) to the isopropyl ester
(32, 400 nM) did not alter the potency. However the
cyclopentyl ester (33, 260 nM) and the 3-pentyl ester
(36, 180 nM), yielded a 2-fold increase in potency. Both
bulky dicyclopropymethyl ester 34 and less lipophoblic
3-tetrahydrofuryl ester 35 were less potent molecules.
These results indicated that a medium sized lipophilic
group was preferred at this position. Not surprisingly,
the molecule with the combination of all four best sub-
stitutents (37) had very good binding affinity (Ki=25
nM). Potent compounds were also obtained when the
cyclopentyl or 3-pentyl moieties were introduced at both
Scheme 3. Reagents and conditions: (a) BBr3, DCM; (b) R5Br,
K2CO3, MeOH, 50 ꢀC; (c) NBS, CCl4, reflux; (d) 2-(2-methyl-am-
inoethyl)pyridine, CH3CN, rt.
2-(4-hydroxyphenyl) and 6-carboxyl groups (39 and 40).
Similar to the literature data,4,5 all the compounds
exhibited much less potency on the rat GnRH receptor
than the human receptor as exemplified by compounds
36–40.
Table 1. Binding affinities of pyrrolo[1, 2-a]pyrimidones (10–30) on the human GnRH receptor8
Compd
R1
R2
R3R4N
Ki (mM) (human)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Ph–
Ph–
Ph–
2-CN–Ph–
2-Cl–Ph–
(CH3)2N
Bn–N–CH3
>50
42
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
Bn–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(2-Pyr)-CH2CH2–N–CH3
(3-Pyr)-CH2CH2–N–CH3
(4-Pyr)-CH2CH2–N–CH3
Ph–CH2CH2–N–CH3
2.6
4.9
8.5
6.6
0.5
2.4
3.7
6.0
2.0
>50
1.8
2.2
0.1
1.2
0.8
0.4
28
2-MeO–Ph–
2-F–Ph–
2.3-di-F–Ph–
2.4-di-F–Ph–
2.5-di-F–Ph–
2.6-di-F–Ph–
Cyclopropyl–
2-F–Ph–
2-MeO–Ph–
2-F–Ph–
2.5-di-F–Ph–
2.6-di-F–Ph–
2.4-di-F–Ph–
2.4-di-F–Ph–
2.4-di-F–Ph–
2.4-di-F–Ph–
>50
>50

402
Y.-F. Zhu et al. / Bioorg. Med. Chem. Lett. 12 (2002) 399–402
Table 2. Binding affinities of 1-N-[2-(2-pyridyl)ethyl-N-methyl]-ami-
nomethyl-pyrrolo[1,2-a]pyrimidones (32–40) on the human and rat
GnRH receptors8
M.; Rivier, J.; Fellows, R.; Blackwell, R.; Vale, W.; Guillemin,
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Compd
R1
R5
Ki (nM) human Ki (nM) rat
32
CH3–
400
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33
34
35
36
37
39
40
CH3–
CH3–
260
2500
2100
CH3–
CH3–
180
25
7700
7300
8900
7600
Me2CH2CH2–
33
31
In conclusion, we have discovered a novel series of 1-
aminomethyl-2-aryl-3-cyano-4-benzyl-pyrrolo[1,2-a]-
pyrimid - 7 - ones as potent human GnRH receptor
antagonists. The continued SAR studies led us to
develop several more potent and novel small molecules,
which will be presented in the following paper.
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References and Notes
8. 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.
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,