The discovery of novel small molecule non-peptide gonadotropin releasing hormone (GnRH) receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(02)00755-2

A novel series of non-peptide derivatives 1, 14, and 15 that bind with high affinity to the human GnRH receptors is discussed. The discovery was made from screening our in-house libraries that contained the active structure 2 along with a trace amount of a second active structure 1 that was derived from an acid-induced rearrangement. From this structure type 1, a series of guanidine and non-guanidine containing analogues were prepared and tested as GnRH receptor antagonists. Compounds derived from this series bind to both human and rat GnRH receptors and antagonize GnRH-mediated increases in inositol phosphate production in cells containing recombinant human receptors. These compounds or their analogues may be useful as therapeutic agents for the treatment of hormone-dependent pathologies including prostate, breast and ovarian cancers.

Full text of DOI:10.1016/S0960-894X(02)00755-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3467–3470
The Discovery of Novel Small Molecule Non-peptide
Gonadotropin Releasing Hormone (GnRH)
Receptor Antagonists
David R. Luthin,* Yufeng Hong, Ved P. Pathak, Genevieve Paderes,
Karen D. Nared-Hood, Mary A. Castro, Haresh Vazir, Haitao Li, Eileen Tompkins,
Lance Christie, 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—Anovel series of non-peptide derivatives 1, 14, and 15 that bind with high affinity to the human GnRH receptors is
discussed. The discovery was made from screening our in-house libraries that contained the active structure 2 along with a trace
amount of a second active structure 1 that was derived from an acid-induced rearrangement. From this structure type 1, a series of
guanidine and non-guanidine containing analogues were prepared and tested as GnRH receptor antagonists. Compounds derived
from this series bind to both human and rat GnRH receptors and antagonize GnRH-mediated increases in inositol phosphate
production in cells containing recombinant human receptors. These compounds or their analogues may be useful as therapeutic
agents for the treatment of hormone-dependent pathologies including prostate, breast and ovarian cancers.
# 2002 Elsevier Science Ltd. All rights reserved.
GnRH1 is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-
Leu-Arg-Pro-Gly-NH2) that is synthesized in the hypo-
thalamus and acts upon receptors in the anterior pituitary
where it triggers2 the synthesis and release of LH and fol-
licle-stimulating hormone (FSH). LH and FSH enter the
systemic circulation and bind to receptors in the gonads
where they stimulate steroidogenesis and gametogenesis
in testes and ovaries.3 Peptide GnRH receptor antago-
nists have been studied by various groups,4 due to their
potential therapeutic benefit5À8 in treating hormone-
dependent diseases such as prostate, breast and ovarian
cancers with additional possibilities in fertility disorders,
endometriosis, uterine fibroids, and precocious puberty.
Several companies have recently reported the discovery
of nonpeptide GnRH antagonists.9À14 We report here
the identification of a novel series of non-peptide GnRH
receptor antagonists that might have utility as thera-
peutic agents for treating hormone-dependent disease.
The synthesis involves the slow addition of the solid bis-
BOC protected N-(1H-pyrazol-1-yl)guanidine 4 to a
dilute THF solution of the diamine 3 in order to ensure
monofunctionalization to a bis-BOC protected guani-
dine 5 in an overall good yield. Once 5 was prepared,
2-furoyl chloride 10 was used to acylate the free amine
followed by deprotection to give N-{[4-({[amino(imino)
methyl]amino}methyl)cyclohexyl]methyl} - 5 - [(3,5,5,8,8-
pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl]-
2-furamide 1 (Scheme 3) using TFAin dichloromethane
at room temperature. Deprotected 1 was then purified
by HPLC. The preparation of 9 involved a Friedel–
Crafts alkylation of toluene using 2,5-dichloro-2,5-
dimethylhexane 615 which gave 1,1,4,4,6-pentamethyl-
1,2,3,4-tetrahydronaphthalene 7. This alkylation was
followed by a second Friedel–Crafts alkylation using
methyl 5-(chloromethyl)-2-furoate 8. The resulting
methyl 2-furoate 9 was saponified to its corresponding
carboxylic acid and then transformed to 2-furoyl chlo-
ride 10 using thionyl chloride (Scheme 2). Additional
analogues like methyl 4-[({5-[(3,5,5,8,8-pentamethyl-
5,6,7,8-tetrahydronaphthalen-2-yl)methyl]-2-furoyl}amino)
methyl]cyclo hexanecarboxylate 14 (Scheme 4) were pre-
pared in a similar fashion and are shown in Table 1.16
The synthetic pathway for the preparation of the gua-
nidine containing core structure is shown in Scheme 1.
*Corresponding author. Tel.:+1-858-526-4968; fax: +1-858-526-
4121; e-mail: david.luthin@pfizer.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00755-2

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D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3467–3470
Although the guanidine-containing analogues were
potent GnRH receptor antagonists (Fig. 1), it was per-
ceived that guanidines, due to potential absorption
issues, may be a liability in developing them as potential
therapeutics. Therefore, we wanted to determine if we
could remove and/or replace the guanidine moiety with
a small variety of other functional groups and still
retain activity against the rat and human GnRH recep-
tors. Two clear examples of potent guanidine-contain-
ing compounds stand out from our efforts. The initial 1
was a mixture of cis and trans isomers and bound to
human and rat receptors with Ki’s of 40 and 520 nM,
respectively. However, separation of the cis 11 and trans
12 isomers did not result in appreciable potency changes
for either pure isomer, suggesting that the cis or trans
configuration did not play a significant role in binding
affinities in these specific examples. However, removal of
the guanidine functionality completely as in 13, reduced
affinity at both human and rat receptors with Ki’s of 180
and 1550 nM, respectively. Adding a simple methyl ester
14 afforded comparable affinity at human but slightly
reduced affinity at rat receptor with Ki’s of 24 versus
830 nM, respectively, whereas amide 15 afforded similar
activity to the guanidine moiety with human and rat
Ki’s of 13 and 270 nM, respectively. In general, most of
these compounds showed a marked 5–60Â preference
for the human GnRH receptor with the exceptions of
19, 20, and 21 that showed similar affinities between
species. Some of the higher-affinity compounds at the
human receptor (1, 11–13, and 15–18) were tested for
their ability to antagonize GnRH-stimulated increases in
total inositol phosphate production and were determined
to be antagonists with comparable affinities to their
binding Ki’s (see Fig. 1; 1: KB=30 nM, Ki=40 nM).
Although these are potent against the human GnRH
receptor, most of these compounds did not have suffi-
cient binding affinity to the rat GnRH receptor to war-
rant extensive animal studies. Further exploration of
Scheme 1. Reagents and conditions: (a) 0.01 M solution in THF (ratio A/B 1/1.1 equiv), 2 h, rt.
Scheme 2. Reagents and conditions: (b) 0.5 equiv AlCl3, CH2Cl2, reflux, 97% yield; (c) 1.0 equiv AlCl3,CH2Cl2, reflux, 2 h 68%; (d) 5 equiv LiOH,
50:25:25 THF/MeOH/H2O, 4 h, 2. 20 equiv SO2Cl2 CH2Cl2 reflux, 5 h overall 71%.
Scheme 3. Reagents and conditions: (e) ethyl acetate, triethyl amine, 1–12 h, rt; (f) 50% TFA, 30 min, rt.
Scheme 4. Reagents and conditions: (e) ethyl acetate, triethyl amine, 1–12 h, rt.

D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3467–3470
3469
these series is under investigation to improve multi-spe-
cies metabolic stability and potency. Substitution
around the cyclohexyl ring system exhibited species dif-
ferences as well as clear potency differences. The SAR
suggests that the guanidine portion was not critical to
the overall binding affinity of the structure. These com-
pounds represent the seminal set of chemical leads from
which to develop potential GnRH receptor antagonists.
In this study, 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetra-
hydronaphthalen-2-yl)methyl]-2-furoyl chloride 10 was
a key structural building block. The combination of this
building block with the amines shown in Table 1 was
responsible for the general activity in the GnRH
receptors. Compounds were potent antagonists of
human GnRH receptors in vitro. These agents or their
Figure 1. Inhibition of GnRH-stimulated total inositol phosphate
accumulation by 1 or the peptide antagonist, antide.16
Table 1. Binding affinity constants of various compounds binding to human and rat GnRH receptors
Analogue
R
Human Ki (nM)
40Æ5
Rat Ki (nM)
520Æ70
Analogue
R
Human Ki (nM)
140Æ11
Rat Ki (nM)
280Æ50
1
22
11
12
40Æ6
16Æ3
730Æ44
385Æ12
23
24
79Æ20
140Æ35
240Æ40
102Æ14
13
14
180Æ16
24Æ3
1550Æ340
830Æ120
25
26
42Æ7
66Æ5
730Æ95
2720Æ210
15
13Æ4
270Æ23
27
180Æ30
1200Æ220
16
17
250Æ26
51Æ7
3000Æ630
2730Æ380
28
29
24Æ2
865Æ230
890Æ120
174Æ13
18
19
20
17Æ3
83Æ4
91Æ17
560Æ46
60Æ13
94Æ30
30
31
32
20Æ2
250Æ12
5210Æ710
2040Æ300
700Æ130
220Æ21
21
56Æ16
55Æ8
33
54Æ6
3260Æ410

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D.R.Luthin et al./ Bioorg.Med.Chem.Lett.12 (2002) 3467–3470
analogues may serve as useful therapeutic agents for
treating hormone-dependent pathologies including
hormone-dependent prostate and breast cancer. Further
investigations of analogues in this area are being
pursued.
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Acknowledgements
We thank Sandra Barnum for her excellent cell culture
work and Stuart Sealfon (Mt. Sinai School of Medicine)
for providing the cell line expressing recombinant
human GnRH receptors.
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