Potent antagonists of gonadotropin releasing hormone receptors derived from quinolone-6-carboxamides

Article abstract of DOI:10.1016/S0960-894X(00)00024-X

SAR studies which focused upon the C-6 position of a recently described series of quinolone gonadotropin releasing hormone antagonists are reported. Synthetic access to diverse quinolone-6-carboxamides was achieved via the palladium-catalyzed amino-carbonylation reactions of iodide 4 with various amines. Amides related to 9y were especially potent, functional antagonists of rat and human GnRH receptors. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00024-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 443±447
Potent Antagonists of Gonadotropin Releasing Hormone
Receptors Derived from Quinolone-6-Carboxamides
Thomas F. Walsh, a,* Richard B. Toupence, a Jonathan R. Young, a Song X. Huang, a
Feroze Ujjainwalla, a Robert J. DeVita, a Mark T. Goulet, a Matthew J. Wyvratt, Jr., a
Michael H. Fisher, a Jane-Ling Lo, b Ning Ren, b Joel B. Yudkovitz, b Yi Tien Yang, b
Kang Cheng b and Roy G. Smith b
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
bDepartment of Biochemistry & Physiology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
Received 23 November 1999; accepted 22 December 1999
AbstractÐSAR studies which focused upon the C-6 position of a recently described series of quinolone gonadotropin releasing
hormone antagonists are reported. Synthetic access to diverse quinolone-6-carboxamides was achieved via the palladium-catalyzed
amino-carbonylation reactions of iodide 4 with various amines. Amides related to 9y were especially potent, functional antagonists
of rat and human GnRH receptors. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Chemistry
Peptidic antagonists of gonadotropin releasing hormone
(GnRH) receptors are of current interest for their ability
to block the release of the anterior pituitary hormones
luteinizing hormone (LH) and follicle-stimulating
hormone (FSH), which results in suppressing the bio-
synthesis of gonadal steroid hormones. The resulting
gender-independent, reversible blockade of the pitui-
tary±gonadal axis has been shown to reduce gonadal
steroid hormone concentrations to castrate levels, which
has utility in the management of some disease states
exacerbated by the presence of those hormones.1±3 The
discovery of the ®rst non-peptidic GnRH receptor
antagonist was reported last year by workers from
Takeda.4 In previous communications from this
laboratory, preliminary structure±activity relationships
(SAR) for a new series of non-peptide GnRH antago-
nists based upon a quinolone screening lead were dis-
closed.5,6 In this letter we present further SAR studies
surrounding the quinolone 6-position substituent which
have a€orded highly potent, functional antagonists of
rat and human GnRH receptors.
At the time we initiated this study, SAR studies for the
quinolone series had established the importance of a
substituted phenyl at the C-3 position, the 6-nitro and 7-
chloro substituents, and a 4-O-alkyl ether derived from
a piperidinyl substituted ethanol. Parallel e€orts within
our laboratory indicated that potency could be further
enhanced through derivatization of the 6-position nitro
group.7 In particular, certain 6-ureido derivatives bear-
ing nitrogen-containing heteroaromatic substituents had
been found to have low nanomolar anity in the rat
GnRH receptor binding assay. We sought to further
extend the SAR for this series by examining 6-amido
substituted quinolones wherein the amide formally
derives from a quinolone-6-carboxylic acid. To this
end, it was recognized that palladium-catalyzed amino-
carbonylation reactions8 of 6-iodoquinolones with var-
ious amines would provide convenient access to diverse
quinolone-6-carboxamides.
The synthesis (Scheme 1) begins with the silver(I)-medi-
9
ated iodination of methyl 4-chloroanthranilate (1) to
a€ord its 5-iodo derivative 2. N-Acylation of 2 with 3,5-
dimethylphenylacetyl chloride a€ords amide 3 which
cyclizes under basic conditions to a€ord the 4-hydroxy-
quinolone 4. O-Alkylation of quinolone 4 with either
(‹)-5 or (S)-5 using our previously described Mitsunobu
*Corresponding author. Tel.: +1-732-594-5232; fax: +1-732-594-
2210.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00024-X

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T. F. Walsh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 443±447
Scheme 1. Reagents and conditions: (a) I2, Ag2SO4, EtOH, rt, 1 h, 100% crude yield; (b) 3,5-dimethylphenylacetyl chloride, 1,2-dichloroethane,
80 ꢀC, 3 h, 85%; (c) LiN(TMS)2, THF, 0 ꢀC, 2 h, 88%; (d) PPh3, DEAD, THF, 0 ꢀC to rt, 4 d, 55%; (e) amines 7a±z, (PPh3)2PdCl2, CO (1 atm),
Et3N, DMF, 95 ꢀC, 16 h, 50±70%; (f) CF3CO2H:CH2Cl2 (1:1), rt, 3 h, 100%.
protocol5 a€ords the 4-O-alkyl ethers (‹)-6 and (S)-6.
Palladium-catalyzed amino-carbonylation of iodo com-
pound 6 with a variety of primary and secondary
amines (7a±z) led to the protected quinolone-6-carbox-
amides 8a±z in 55±70% yield.10 Final deprotection of
the BOC-protected piperidines 8a±z using tri¯uoroacetic
acid furnished the targeted amides 9a±z.11 Evaluation of
the N-methylpiperidine derivative (14) of one of the
more potent antagonists 9y discovered in this series
was also undertaken, since it was known from our pre-
vious studies that N-methylation of the heterocyclic
substituent at C-4 also a€orded potent compounds.
Synthesis of 14 was accomplished as shown in Scheme 2
by alkylating the 4-hydroxyquinolone 4 with (S)-2-
(2-chloroethyl)-1-methylpiperidine (12) followed by
amino-carbonylation of intermediate 13 with 4-amino-
pyrimidine. Ester 10, derived from (l)-pipecolic acid as
previously described,6 provided the requisite alkylating
agent 12 in two steps as shown.
Discussion
During the course of this investigation, a radioligand
binding assay in CHO cell-expressed cloned human
GnRH receptors12 supplemented the rat pituitary
membrane binding assay5,6 which had been previously
used in our program. Additionally, new compounds
were evaluated for their ability to functionally antag-
onize GnRH-stimulated phosphatidyl inositol (PI)
hydrolysis in human cloned receptors13 and LH
release14 from rat primary pituitary cells. Inspection of
the in vitro binding and functional antagonism for
examples 9a±p (Table 1) selected from a broad survey of
racemic quinolone-6-carboxamides revealed that the
amides derived from aliphatic amines (9a±b) are weakly
potent antagonists.15 Furthermore, heterocyclic amines
yielding basic amides (i.e. 9f±h) a€orded derivatives that
were generally more potent than those derived from
amines which yielded neutral amide sidechains (9c±e).
Scheme 2. Reagents and conditions: (a) LiAlH4, THF re¯ux, 90%; (b) SOCl2, HCl (1 atm), CHCl3, re¯ux, 5 h, 62%; (c) quinolone 4, K2CO3, DMF,
80 ꢀC, 3 h, 60%; (d) 4-aminopyrimidine, (PPh3)2PdCl2, CO (1 atm), Et3N, DMF, 95 ꢀC, 16 h, 68%.

T. F. Walsh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 443±447
Table 1. In vitro activities for racemic quinolone-6-carboxamides 9a±p
445
Entry
Substituent
GnRH binding
IC50 (nM)
PI turnover
IC50 (nM)
LH release
IC50 (nM)
Entry
Substituent
GnRH binding
IC50 (nM)
PI turnover
IC50 (nM)
LH release
IC50 (nM)
9a
190 (rat)
Ð
>1280
9i
290
>3200
>1280
9b
9c
9d
45 (rat)
Ð
>1280
9j
9k
9l
7
16
2200
23
28
Ð
Ð
>1280
>1280
320
3.2
775
8.3
>1280
240
9e
9f
110
3.6
591
19
>1280
1265
9m
9n
2
6.4
292
Ð
40
486
9g
9h
3.8
32
10.6
130
1089
9o
9p
32
22
Ð
Ð
>1280
860
977
Table 2. In vitro activities for (S)-quinolone-6-carboxamides 9q±z
Entry
Substituent
GnRH binding
IC50 (nM)
PI turnover
IC50 (nM)
LH release
IC50 (nM)
Entry
Substituent
GnRH binding
IC50 (nM)
PI turnover
IC50 (nM)
LH release
IC50 (nM)
9q
2.3
77.5
513
9v
2.9
21.5
521
9r
9s
1.4
14.5
79.2
9w
2.8
44
167
1.8
33.3
87.2
9x
10.9
360
Ð
9t
9
133
250
Ð
Ð
9y
9z
0.9
0.8
5.0
5.9
96.5
23.9
9u
15
Notably, the amides derived from 4-aminopyrimidine
(9l) and structurally related amines (i.e. 9m) a€orded the
greatest potency observed in the series. Concurrent
e€orts in our laboratory demonstrated the dependence
of GnRH binding anity upon the (S)-piperidine
con®guration, thus we sought to focus further SAR
studies of quinolone-6-carboxamides in the non-racemic
series.6 Table 2 illustrates binding and functional
data for representative GnRH antagonists 9q±z pre-
pared from amines (7q±z) selected to further de®ne the

446
T. F. Walsh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 443±447
quinolone-6-carboxamide SAR. In parallel with the
racemic series, the 4-aminopyrimidine (9y) and 3-
amino-1,2,5-thiadiazole (9z) derivatives were again the
most potent analogues, while incorporating a variety of
substituents on the heterocycle failed to e€ect additional
improvement.
6. DeVita, R. J.; Goulet, M. T.; Wyvratt, Jr., M. J.; Fisher, M.
H.; Lo, J.-L.; Yang, Y. T.; Cheng, K.; Smith, R. G. Bioorg.
Med. Chem. Lett. 1999, 9, 2621.
7. Young, J. R.; Chen, I.; Walsh, T. F.; DeVita, R. J.; Wyvratt
Jr., M. J.; Fisher, M. H.; Goulet, M. T.; Ren, N.; Lo, J.-L.;
Yang, Y. T.; Yudkovitz, J. B.; Cheng, K.; Smith, R. G.
Bioorg. Med. Chem. Lett., manuscript in preparation.
8. Schoenberg, A.; Heck, R. F. J. Org. Chem. 1974, 39, 3327.
9. Sy, W.-W. Synthetic Commun. 1992, 22, 3215.
In vitro characterization of analogue 14 con®rmed that
N-methylation of 9y a€ords potent antagonism (IC50
values: hGnRH binding=2.5 nM, hGnRH PI hydro-
lysis=16 nM, rGnRH LH release=85 nM); albeit
somewhat less potent than the parent unsubstituted
piperidine. Subsequent evaluation of 14 in the rat pituitary
membrane binding assay indicated that it is less potent
(IC50=60 nM) at the rat receptor than at the human
clones. This observation may resolve the apparent dis-
crepancy between the functional antagonism of 14
determined in the PI hydrolysis (cloned human recep-
tors) and LH release assays (rat primary pituitary cells).
10. A representative procedure for the amino-carbonylation
reaction: an oven-dried, two-necked 25 mL round-bottom
¯ask was equipped with a magnetic stir bar, a septum and a
three-way stopcock ®tted with a balloon. The ¯ask was
charged with 0.233 g (0.37 mmol) of iodo compound (S)-6,
0.174 g (1.83 mmol) of 4-aminopyrimidine, 102 mL (0.73
mmol) of triethylamine, 0.013 g (0.018 mmol, 5 mol%) of
dichlorobis(triphenylphosphine)palladium(II) and 2 mL of
anhydrous DMF. The atmosphere in the ¯ask was alternately
evacuated in vacuo and ¯ushed with carbon monoxide three
times. The balloon was next ®lled with carbon monoxide, and
the reaction mixture was then magnetically stirred and heated
with an oil bath at 95 ꢀC for 24 h. At the end of this period, the
reaction mixture was cooled to room temperature and parti-
tioned between EtOAc (15 mL) and 10% aqueous NaHSO4
(15 mL). The organic layer was separated, washed with addi-
tional 10% aqueous NaHSO4, saturated NaCl, then dried
(MgSO4), ®ltered and evaporated. The residual oil was pur-
i®ed on a silica gel ¯ash chromatography column using 2.5%
MeOH in CHCl3 as eluent. Evaporation of the puri®ed frac-
tions and drying in vacuo a€orded 0.125 g (54%) of com-
pound 8y as an amorphous pale yellow powder.
Conclusion
SAR studies for a variety of quinolone-6-carboxamides
revealed that the amides derived from 4-aminopyr-
imidine (9l, 9y) and 3-amino-1,2,5-thiadiazole (9m, 9z)
a€ord highly potent functional antagonists of rat and
human GnRH receptors. As anticipated from earlier
studies, N-methylation of the piperidine substituent at
C-4 (14) retains antagonist potency. The advent of
binding and functional assays using both rat and cloned
human receptors revealed that our quinolone-derived
antagonists appear to have somewhat higher anity
towards human receptors. This phenomenon was also
observed with the thieno[2,3-b]pyridin-4-one class of
GnRH antagonists reported by Takeda.4 Additional
SAR and pharmacological characterization for the qui-
nolone series of GnRH antagonists will be reported
from these laboratories in the near future.
11. All ®nal compounds were obtained as tri¯uoroacetic acid
salts following puri®cation by reversed-phase HPLC. Inter-
mediates and ®nal products a€orded satisfactory 400 or 500
1
MHz H NMR and ESI mass spectral characterization. Data
for 9y (TFA salt): ESI-MS m/z 532 (M++1); 1H NMR
(CD3OD, 500 MHz) d 1.15±1.22 (m, 1H), 1.34±1.45 (m, 111),
1.52±1.86 (m, 7H), 1.97±2.06 (m, 1H), 2.37 (s, 6H), 2.82±2.92
(m, 1H), 2.98±3.05 (m, 1H), 3.27±3.34 (m, 1H), 3.73±3.87 (m,
2H), 7.06 (s, 2H), 7.09 (s, 1H), 7.51 (s, 1H), 8.10 (s, 1H), 8.34
(dd, J=l.0, 6.0 Hz, 1H), 8.71 (d, J=6.0 Hz, 1H), 8.89 (d,
J=1.0 Hz, 1H); 13C NMR (CD3OD, 125 MHz) d 21.46 (2C),
23.03, 23.33, 28.95, 34.76, 46.02, 55.38, 70.19, 111.80, 117.14,
117.56, 123.55, 126.00, 129.43 (2C), 130.68, 130.99, 133.38,
134.85, 139.21 (2C), 140.95, 158.79, 159.18, 159.63, 161.14,
165.65, 168.10. Data for 14 (TFA salt): ESI-MS m/z 546
(M++1); 1H NMR (CD3OD, 500 MHz) d 1.22±1.80 (m, 7H),
2.35±2.40 (m, 1H), 2.37 (s, 6H), 2.77 (s, 3H), 2.86±2.90 (m,
2H), 3.42±3.46 (m, 1H), 3.73±3.86 (m, 2H), 7.06 (s, 2H), 7.10
(s, 1H), 7.51 (s, 1H), 8.15 (s, 1H), 8.36 (dd, J=1.0, 6.0 Hz,
1H), 8.72 (d, J=6.0 Hz, 1H), 8.91 (d, J=l.0 Hz, 1H); 13C
NMR (CD3OD, 125 MHz) d 21.47 (2C), 22.95, 24.28, 29.53,
32.23, 41.63, 57.58, 64.13, 70.67, 111.78, 117.07, 117.60,
123.64, 126.18, 129.49 (2C), 130.48, 131.01, 133.45, 134.93,
139.26 (2C), 140.95, 158.26, 158.84, 159.90, 161.32, 165.62,
168.02.
Acknowledgements
We thank Dr. George Doss for helpful discussions
regarding NMR assignments, J. Leone, J. Pisano, S.
Fabian and G. Reynolds for synthetic services and A.
Bernick for FAB-mass spectrometry analysis.
References and Notes
1. Roy, R. A. Curr. Opin. Obstet. Gy. 1994, 6, 262.
2. Behre, H. M.; Nordho€, V.; Nieschlag, E. In Ovul. Induct.
Update `98, Proc. World Conf., 2nd, Meeting, Date 1997; Fili-
cori, M.; Flamigni, C., Eds.; Parthenon Publishing: Carnforth,
UK, 1998, pp 107.
3. Goulet, M. T. In Annual Reports in Medicinal Chemistry;
Bristol, J. A., Ed.; Academic Press: New York, 1995; Vol. 30,
pp 169.
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. DeVita, R. J.; Hollings, D. D.; Goulet, M. T.; Wyvratt, Jr.,
M. J.; Fisher, M. H.; Lo, J.-L.; Yang, Y. T.; Cheng, K.; Smith,
R. G. Bioorg. Med. Chem. Lett. 1999, 9, 2615.
12. Crude membranes prepared from rat pituitary glands or
Chinese hamster ovary K1 cells stably expressing human
GnRH receptors were used as the sources for GnRH receptors.
5-[1251-Tyr]-Buserelin (a peptidyl GnRH agonist obtained
from Woods assays) having speci®c activity of 1000 Ci/mmol
was used as the radiolabelled ligand. Competitive binding was
measured in a 50 mM Tris±HCl based bu€er (pH 7.5) con-
taining 2 mM MgCl2 and 0.1% bovine serum albumin. The
binding activity is reported as an IC50 value which is the
antagonist concentration required to inhibit the speci®c bind-
ing of [2151]buserelin to GnRH receptors by 50%.
13. Chinese hamster ovary cells stably expressing human
GnRH receptors functionally coupled to phospholipase C

T. F. Walsh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 443±447
447
were used to evaluate the functional GnRH antagonism of test
compounds. Clones were seeded at a concentration of 60,000
cells/mL/well in inositol-free F12 medium containing 10%
dialyzed fetal bovine serum, 1% Pen/Strep, 2 mM glutamine,
500 mg/mL G418 and 1 mCi [3H]inositol in 24-well tray. Forty-
eight hours after seeding, cells were washed with 3Â1 mL of
PBS containing 10 mM LiCl and treated with various con-
centrations of test compounds for 2 h at 37 ꢀC before addition
of 0.5 nM GnRH. After incubation at 37 ꢀC for an additional
60 min, the medium was removed and the cells were lysed with
1 mL of 0.1 M formic acid. The trays were freeze±thawed
once and the cell extract was applied to a Dowex AG1-X8
column. The column was washed with 2Â1 mL H2O to remove
free [3H]inositol and [3H]inositol phosphates were eluted
with 3Â1 mL 2 M ammonium formate in 1 M formic
acid. The eluate was counted in a scintillation counter. The
results are reported as an IC50 value which is the antagonist
concentration required to inhibit the GnRH stimulated PI
hydrolysis by 50%.
14. Rat primary pituitary cells were isolated by enzymatic
digestion with a mixture of collagenase and hyaluronidase.
The suspended cells were then cultured for 4 days in 24-well
tissue culture plates. On the day of the assay, the cells were
washed and then treated with medium containing 2 nM
GnRH and the compound of interest at concentrations
between 5 and 5120 nM. After a 3 h incubation, the medium
was removed and centrifuged. The supernatant was then ana-
lyzed for content of LH using a RIA kit speci®c for rat LH.
The results are reported as an IC50 value which is the antago-
nist concentration required to inhibit the GnRH stimulated
LH release by 50%.
15. The IC50 values for GnRH receptor binding shown for 9a
and 9b in Table 1 were determined using rat pituitary mem-
branes. See refs 5 or 6 for a description of the assay.