Characterization of mono- and diaminopyrimidine derivatives as novel, nonpeptide gonadotropin releasing hormone (GnRH) receptor antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00756-4

A novel series of derivatives of mono- and diaminopyrimidines 1 potently displaced binding of a radiolabeled GnRH analogue to human and rat GnRH receptors. Analogues from these series competitively antagonized GnRH-stimulated increases in extracellular acidification in vitro and suppressed GnRH-mediated increases in circulating luteinizing hormone (LH) in castrated rats and testosterone in intact rats. These compounds or their analogues may be useful in treating sex hormone-dependent disease.

Full text of DOI:10.1016/S0960-894X(02)00756-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3635–3639
Characterization of Mono- and Diaminopyrimidine Derivatives as
Novel, Nonpeptide Gonadotropin Releasing Hormone (GnRH)
Receptor Antagonists
David R. Luthin*,y Yufeng Hong*,y Eileen Tompkins, Kenna L. Anderes,
Genevieve Paderes, Eugenia A. Kraynov, Mary A. Castro, Karen D. Nared-Hood,
Rosemary Castillo, Margaret Gregory, Haresh Vazir, John M. May
and Mark B. Anderson
Pfizer Global Research and Development-La Jolla/Agouron Pharmaceuticals, Inc., 10724 Science Center Drive,
San Diego, CA 92121, USA
Received 5 June 2002; accepted 12 August 2002
Abstract—A novel series of derivatives of mono- and diaminopyrimidines 1 potently displaced binding of a radiolabeled GnRH
analogue to human and rat GnRH receptors. Analogues from these series competitively antagonized GnRH-stimulated increases in
extracellular acidification in vitro and suppressed GnRH-mediated increases in circulating luteinizing hormone (LH) in castrated
rats and testosterone in intact rats. These compounds or their analogues may be useful in treating sex hormone-dependent disease.
# 2002 Elsevier Science Ltd. All rights reserved.
GnRH plays a central role in the biology of reproduction.
The GnRH decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-
Leu-Arg-Pro-Gly-NH2) is synthesized in the hypothala-
mus and acts upon receptors in the anterior pituitary
where it triggers the synthesis and release of LH and
follicle-stimulating hormone (FSH). LH and FSH enter
the systemic circulation and bind to receptors in the
gonads where they stimulate steroidogenesis and game-
togenesis in testes and ovaries.1 Peptide GnRH receptor
antagonists have been studied by various groups2 due to
their potential therapeutic benefit in treating hormone-
dependent diseases including fertility disorders, endo-
metriosis, uterine fibroids, precocious puberty, and var-
ious hormone-dependent cancers such as prostate,
ovarian, and breast cancer. Several companies have
recently reported the discovery of nonpeptide GnRH
antagonists.3ꢀ6 We report here the identification of a
novel series of nonpeptide GnRH antagonists that
might have utility as therapeutic agents for treating
hormone-dependent disease.
The synthetic pathway for the preparation of com-
pounds 1 and 13–15 is illustrated in Schemes 1 and 2.
Amination of 2,4-dichloropyrimidine with tetrahydro-
furylamine afforded compounds 2 and 3 in a ratio of
3:1. Mono-protection of m-xylene diamine was carried
out in THF in the presence of trifluoroacetate. The
reaction gave 4 in 53% yield and the bis-protected
amine side product was also observed. The remaining
amino group was protected as ethylcarbamate 5 and the
trifluoroacetylamide was cleaved to give mono amino
carbamate 6.7 Amination of compound 3 followed by
cleavage of the carbamate afforded the bis-substituted
pyrimidine 8.8 The synthesis of 1 was accomplished by
coupling 8 with 9. Compounds 16–38 (Table 1) were
prepared following the general chemistry procedure
illustrated in Scheme 3. The acylation and deprotection
steps gave almost quantitative yields. The yields of the
reaction from 11 to 12 were 30–70%, depending upon
the substitution on the pyrimidine.9
*Corresponding authors. Fax: +1-858-526-4121; e-mail: david.
luthin@pfizer.com
yBoth authors contributed equally to this work.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00756-4

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D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3635–3639
In this report, we have optimized the guanidine GnRH
receptor antagonist analogues presented in the accom-
panying report for potency at both human and rat
GnRH receptors. Modifications on the left-hand por-
tion of the amide bond will be presented in future
reports. Although the mono- and diaminopyrimidines
appear in essence as ‘caged’ guanidines, they confer
unique characteristics to the structure–activity relation-
ships of the series. The mono-aminopyrimidine com-
pounds (27–31) had similar affinity and preference (ꢁ5–
40ꢂ) for the human versus the rat receptor as the parent
guanidines reported in the adjoining paper. Others3,6
have reported species differences with very different
chemical series, which can be problematic when utilizing
animal models with surrogate marker endpoints (e.g.,
testosterone suppression). The di-aminopyrimidines were
the most potent compounds generated within the series
and in contrast to the mono-aminopyrimidines (27–31),
several exhibited either similar (16, 17, 20, and 21) bind-
ing between rat and human receptors or a notable pref-
erence (5–10ꢂ; 13–15, 18, 19, and 23) for the rat receptor
versus the human counterpart. 4-amino substitutions
Table 1. Binding affinity constants of various mono- and diaminopyrimidine analogues to human and rat GnRH receptors10
Compd
R
Human
Ki (nM)a
Rat
Ki (nM)
Compd
26
R
Human
Ki (nM)
Rat
Ki (nM)
1
9.3ꢃ0.9
8ꢃ0.8
4.7ꢃ0.4
1.6ꢃ0.7
175ꢃ45
103ꢃ18
235ꢃ49
13
27
2140ꢃ500
14
15
16
17
18
19
20
21
22
23
24
25
56ꢃ8
33ꢃ9
3ꢃ0.6
6ꢃ0.7
28
29
30
31
32
33
34
35
36
37
38
130ꢃ10
67ꢃ7
510ꢃ30
1540ꢃ360
1760ꢃ60
5210ꢃ1250
1490ꢃ340
ND
14ꢃ1.5
11ꢃ1.7
80ꢃ7
7ꢃ1.5
150ꢃ40
170ꢃ16
63ꢃ15
780ꢃ33
220ꢃ23
510ꢃ55
29ꢃ5
11ꢃ0.7
7.5ꢃ1.5
2.5ꢃ1
10ꢃ3
44ꢃ7
22ꢃ2
ND
40ꢃ7
33ꢃ10
400ꢃ40
9ꢃ0.3
ND
110ꢃ10
57ꢃ4
55ꢃ7
35ꢃ10
300ꢃ80
380ꢃ40
ND
74ꢃ19
28ꢃ8
26ꢃ5
370ꢃ90
aValues are means of at least three experimentsꢃSE; Ki values were determined from IC50 values;10 ND=not determined.

D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3635–3639
3637
Scheme 1. Reagents and conditions: (a) Et3N, THF, rt, 2 h, 85%.
Scheme 2. Reagents and conditions: (a) THF, rt, overnight, 53% yield; (b) Et3N, CH2Cl2, rt, 20 min, 95%; (c) 20% K2CO3, MeOH, rt, 3 h, 90%; (d)
Et3N, chlorobenzene, reflux, overnight 60%; (e) 20% KOH, ethyleneglycol, 130 ꢄC, 70%; (f) Et3N, CH2Cl2, rt, 90%.
Scheme 3. Reagents and conditions: (a) Et2N, CH2Cl2, rt, 95%; (b) 20% K2CO3, MeOH, 85%; (c) Et2N, chlorobenzene, reflux, overnight, 30–70%.
Figure 1. Effect of 1 (10, 30 or 100 nM) on GnRH-stimulated increases
Figure 2. Effect of 1 (5, 10, 20 mg/kg iv) on plasma LH in castrated

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D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3635–3639
D2 dopamine12 receptors (160 nM) and sodium channels
(site 2; 200 nM), 1 was 50 to >100ꢂ selective for human
GnRH receptors in all other assays performed.
In this report, we have identified and characterized two
novel series of nonpeptide, mono- and diamino-pyr-
imidine derivatives that potently bind to human and rat
GnRH receptors. Compound 1 was a selective, compe-
titive antagonist of GnRH receptors in vitro and low-
ered testosterone levels in male rats in vivo. These
agents or their analogues may serve as useful ther-
apeutic agents for treating hormone-dependent pathol-
ogies including hormone-dependent prostate and breast
cancer.
Figure 3. Suppression of testosterone in gonad-intact male rats by 1
(20 mg/kg i.m.). *p ꢅ0.05 Compound 1 versus vehicle.
Acknowledgements
We thank Sandra Barnum for her excellent cell culture
work.
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