Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.08.056

Potent neutral replacements of the basic amine at the 4-position of quinolone non-peptide GnRH antagonists are reported. A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and convergency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identified, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.

Full text of DOI:10.1016/j.bmcl.2004.08.056

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5599–5603
Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH
receptor antagonists
Robert J. DeVita,a,* Mamta Parikh,a Jinlong Jiang,a Jason A. Fair,a Jonathan R. Young,a
Thomas F. Walsh,a Mark T. Goulet,a Jane-L. Lo,b Ning Ren,b Joel B. Yudkovitz,b
Jisong Cui,b Yi T. Yang,b Kang Cheng,b Susan P. Rohrerb and Matthew J. Wyvratta
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Atherosclerosis and Endocrinology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 14 June 2004; revised 20 August 2004; accepted 26 August 2004
Available online 25 September 2004
Abstract—A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and
unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and conver-
gency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identi-
fied, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the
earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
for example, an azetidine at the 4-position of the quino-
lone, was found to contribute significantly to GnRH
receptor binding affinity, functional activity, and in vivo
efficacy in primates.7c Other cyclic amines exhibited
equivalent binding affinity, most notably the 2-piperidin-
yl side chain at the 4-position (i.e., compound 2) for
much of the reported SAR.
Gonadotropin releasing hormone (GnRH) is a decapep-
tide released by the hypothalamus, which binds to
GnRH receptors on pituitary.1,2 The activation of this
G-protein coupled receptor causes the release of lutei-
nizing hormone (LH) and follicle-stimulating hormone
(FSH), which regulate downstream gonadal steroid hor-
mone production.3 A variety of hormone dependent dis-
ease conditions such as prostate cancer may be treated
clinically with peptidyl GnRH agonists, which act by
over stimulation, then desensitization of this pathway,
resulting in suppression of the hypothalamic-pituitary-
gonadal axis.4 Peptidyl antagonists, which act by di-
rectly blocking the effects of GnRH, are currently being
explored in the clinic.5 Over the last decade, there have
been many reports of nonpeptidyl small molecule
GnRH antagonists, which may offer some advantages
such as oral dosing.6
R
NH
NH
H
CH3
CH3
H
CH3
CH3
CH3
CH3
N
O
O
N
O
O
N
N
H
N
N
H
Cl
N
H
O
Cl
N
H
O
2, R = H
1
3, R = CF3
h GnRH
h PI Turn
A series of recent reports from this laboratory described
the syntheses and structure–activity relationships (SAR)
of 3-aryl quinolones, such as compound 1, as nonpep-
tide GnRH antagonists.7 A cyclic basic amine moiety,
IC50 (nM)
IC50 (nM)
Compound
1
2
3
0.44
0.3
2.0
0.9
2.2
18
We were extremely surprised to find that the 6-trifluoro-
methylated piperidine analog 3, did not suffer from a
large decrease in binding affinity in spite of the 3-orders
Keywords: GnRH; Antagonist; Quinolone.
*
Corresponding author. Tel.: +1 732 594 7039; fax: +1 732 594
5966; e-mail: robert_devita@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.056

5600
R. J. DeVita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5599–5603
of magnitude difference in pKa as compared to com-
pound 2.7f We sought to extend the SAR by examining
a variety of analogs containing neutral (nonbasic) side
chains at the 4-position. In this letter,8 we disclose
SAR studies for neutral substituents at the quinolone
4-position leading to the identification of nonbasic
GnRH antagonists possessing high affinity and func-
tional antagonist activity for cloned human receptors.
In addition, new synthetic routes were devised, which af-
forded intermediates that allowed late stage substitution
at the 4-position of the quinolone for rapid analog
synthesis.
substituted ethanols were unsuccessful with this sub-
strate. However, when the pyrimidine moiety was re-
placed with a thiadiazole as in 12b, Mitsunobu
coupling proceeded, albeit in moderate yield to give
the oxetane analog 14b. In the past, it had been observed
that the yields of Mitsunobu couplings with quinolones
such as compound 4 were sensitive to solubility of the
quinolone in tetrahydrofuran. The solubility of the thia-
diazolyl compound 12b in tetrahydrofuran was greater
than that of the pyrimidinyl 12a, thereby enhancing its
reactivity in the Mitsunobu reaction. While these useful
intermediates did allow quick access to two analogs,
they did not provide a general solution for preparation
of 4-substituted quinolones.
2. Chemistry
We then sought an intermediate that would allow late-
stage introduction of 4-position substituents with im-
proved solubility over compounds 12a,b. To that end,
the Mitsunobu coupling of the quinolone 4 with 2-tri-
methylsilylethanol was re-examined (Scheme 3) and it
was discovered that use of excess reagents in the protec-
tion provided exclusive formation of the 2,4-bis-pro-
tected quinolone 9 in nearly quantitative yield. In
addition, the nonpolar nature of this intermediate al-
lowed for easy purification from the reaction by-prod-
ucts, thereby removing a difficulty of the earlier routes.
Subsequent ester hydrolysis to carboxylic acid 15, fol-
lowed by carboxamide formation provided the interme-
diate 16 in a facile manner. We found our original
conditions, tetrabutylammonium fluoride in tetra-
hydrofuran, afforded completely selective deprotection
of the 4-trimethylsilylethoxy group of bis-protected
quinolone 16. The resulting intermediate 17 was versa-
tile in Mitsunobu couplings, exemplified by reaction
with 2-(azetidinon-4-yl)ethanol to give the azetidinone
ether 18. The subsequent deprotection of the 2-(2-tri-
methylsilylethoxy)quinolone was effected with trifluoro-
acetic acid completing a novel and efficient route to
4-substituted quinolones such as lactam analog 19.
The 2-trimethylsilylethyl protected quinolone 17 pro-
vided a general improvement in the Mitsunobu reaction
as compared to quinolones 4 and 12a,b presumably due
to its improved solubility in tetrahydrofuran and facile
purification of subsequent reaction products.
The general syntheses for quinolone compounds had
been well established by the previous work.7 Our initial
efforts in synthesizing 4-substituted analogs utilized the
published route (Scheme 1).9 Thus, Mitsunobu coupling
of known quinolone 47c with tetrahydrofuran-2-yletha-
nol cleanly provided 4-alkoxyquinolone 5. Hydrolysis
of the ester at the 6-position under basic conditions gave
the carboxylic acid 6. Standard amide bond formation
with 4-aminopyrimidine gave the desired compound 7.
This earlier route did not provide a convergent ap-
proach to the analogs of ethers at the quinolone 4-posi-
tion of interest to us. Therefore, we desired a synthesis in
which the 4-substituent could be introduced at a later
stage with incorporation of an optimized 6-substituent
at an earlier stage. Thus, the quinolone 4 was protected
by Mitsunobu coupling with 2-trimethylsilylethanol,
resulting in a mixture of the 4-mono- and 2,4-bis-pro-
tected quinolones 8 and 9, respectively (Scheme 2).
The mono-protected quinolone 8 was then treated with
lithium hydroxide to afford the carboxylic acid 10. Cou-
pling of the carboxylic acid with 4-aminopyrimidine or
3-amino-1,2,5-thiadiazole gave the amides 11a,b, which
were efficiently deprotected using tetrabutylammonium
fluoride to give the 6-functionalized 4-hydroxy quinolo-
nes 12a,b. While the quinolone 12a could be alkylated
successfully under basic conditions to give the quinucli-
dine analog 13a, Mitsunobu couplings with a variety of
O
H
CH3
CH3
CH3
CH3
O
OH
O
O
MeO
CH3
MeO
CH3
b
a
Cl
N
H
O
Cl
N
H
O
4
5
O
O
H
H
CH3
CH3
CH3
CH3
CH3
CH3
O
O
N
O
O
c
HO
N
N
H
Cl
N
H
O
Cl
N
H
O
7
6
Scheme 1. (a) 2-Tetrahydrofuran-2-ylethanol, PPh3, DEAD, THF, rt, 80%; (b) LiOH, aq EtOH, rt, 90%; (c) 4-aminopyrimidine, EDAC, HOBt,
CH2Cl2, rt, 95%.

R. J. DeVita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5599–5603
5601
Me3Si
Me3Si
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
O
OH
O
O
N
O
O
MeO
MeO
MeO
CH3
+
a
Cl
N
H
O
Cl
O
Cl
N
H
O
4
8
9
SiMe3
Me3Si
Me3Si
CH3
CH3
CH3
CH3
CH3
O
O
O
O
b
c
d
8
HO
RHN
Cl
N
H
O
Cl
N
H
O
11a, b
10
CH3
CH3
R1
CH3
CH3
CH3
CH3
O
O
O
OH
N
H
N
O
R2
R2
e or f
N
H
N
H
R1
=
=
13a
Cl
N
H
O
Cl
O
12a, b H
1
14b R
H
S
N
N
=
R2
b
R2
=
N
a
N
Scheme 2. (a) 2-TMS–EtOH (1.1equiv), PPh3, DEAD, THF, 8, 35%, 9, 20%; (b) LiOH, EtOH (aq), 85%; (c) 4-aminopyrimidine (for a) or 3-
amino(1,2,5)thiadiazoleÆHCl (for b), DiPEA, EDAC, HOBt, CH2Cl2, 90%; (d) TBAF, THF, 90%; (e) for 13a, 2-(2-chloroethyl)quinuclidine, KHCO3,
DMF, 55%; (f) for 14b, 2-(2-hydroxyethyl)oxetane, PPh3, DEAD, THF, 75%.
Me3Si
Me3Si
CH3
CH3
CH3
CH3
O
O
N
O
O
a
b
4
MeO
CH3
SiMe3
HO
CH3
SiMe3
Cl
O
Cl
N
O
9
15
Me3Si
CH3
CH3
CH3
CH3
c
d
S
S
N
N
O
O
O
OH
N
N
N
H
CH3
SiMe3
N
H
CH3
SiMe3
Cl
N
O
Cl
N
O
O
16
17
O
H
O
H
NH
CH3
NH
CH3
e
CH3
f
CH3
CH3
S
S
N
N
O
O
O
N
N
N
H
CH3
SiMe3
N
H
Cl
N
O
Cl
N
H
O
19
18
Scheme 3. (a) 2-TMS–EtOH (3equiv), PPh3, DEAD, THF, 99%; (b) LiOHÆH2O, EtOH (aq), 90%; (c) 3-amino(1,2,5)-thiadiazoleÆHCl, DiPEA,
EDAC, HOBt, CH2Cl2, 85%; (d) TBAF, THF, 90%; (e) 4-(2-hydroxyethyl)-2-azetidinone, PPh3, DEAD, THF, 95%; (f) TFA, CH2Cl2, 90%.
3. Biological results and discussion
(CHO) cells stably expressing the human GnRH
receptor.7c,d
Compounds were evaluated for their ability to compete
with GnRH receptor agonist [125I]buserelin for binding
to the human GnRH receptor in the presence of 0.1%
BSA. Functional antagonism in vitro was also deter-
mined via inhibition of GnRH-stimulated phosphatidyl
inositol (PI) hydrolysis by cloned Chinese hamster ovary
A number of 4-substituted quinolones were examined,
as shown in Table 1. The azetidine, pyrrolidine, and
piperidine analogs, entries 1–3, show the close range of
binding affinity and functional activity with respect to
amine ring size and carboxamide heterocycle in this

5602
R. J. DeVita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5599–5603
Table 1. Inhibition of human GnRH receptor binding and PI
turnovera
We continued to explore nonbasic heterocycles at the 4-
position. When another heteroatom is introduced as in
the acetal analog (entry 8), binding affinity is maintained
but a 30-fold loss in functional activity is observed. A
similar effect is observed for the morpholine analog indi-
cating diminished functional activity when more than
one heteroatom is incorporated in the heterocyclic ring.
When all heteroatoms are removed, as in cyclopentyl
analog (entry 9), a dramatic loss in binding and function
is observed, with a 300- and 500-fold loss in activity,
respectively, as compared to the pyrrolidine analog. This
further refined the requirement of a polar group such as
a heteroatom, resulting in the creation of a Bronsted or
Lewis basic site, to achieve high binding affinity in this
series.
CH3
N
N
R
a, R1 =
b, R1
CH3
CH3
O
O
R1
N
H
S N
N
=
Cl
N
H
O
Entry
R1
R
hGnRH
IC50 (nM)
PI turn
IC50 (nM)
1b
b
a
b
a
b
b
a
b
b
b
b
b
b
0.32
0.8
2.2
1.6
18
N
H
2b
0.8
0.55
2
N
H
3b
Decreasing the size of the tetrahydrofuran ring to the
oxetane analog (entry 10) resulted in a 15-fold loss in
functional potency while maintaining nearly equal bind-
ing affinity as compared to the tetrahydrofuran analog.
When the cyclic ether is replaced with a cyclic amide, as
in b-lactam (entry 11) or pyrrolidinone (entry 12), bind-
ing affinity is improved and functional activity is re-
stored matching that of the fully basic azetidine.
Methylation of the lactam results in 10-fold decrease
in functional activity indicating the importance of the
lactam N–H to provide functional antagonist activity
for nonbasic heterocycles.
N
H
4b
N
H
CF3
5c
0.6
0.7
0.6
0.6
243
0.9
0.1
0.3
1.4
5.8
32
N
O
6d
N
H
7b
3.3
67
O
O
8c
While it had been previously established that a cyclic
basic amine was important for activity on the quinolone
4-position side chain, we have found nonbasic hetero-
cycles, such as cyclic ethers and cyclic lactams, are also
effective pharmacophores for binding and functional
antagonist activity on the human GnRH receptor. The
original synthesis of quinolones was modified to provide
a general and facile late stage synthesis of 4-substituted
analogs. Useful intermediates for Mitsunobu alkylation
of the 4-hydroxyl group were prepared by bis-protection
at the 2- and 4-positions, installation of the 6-carbox-
amide and subsequent selective deprotection at the 4-
position. The efficiency of Mitsunobu alkylation of the
4-hydroxy quinolone was generally improved with 2-tri-
methylsilylethoxy intermediates due to their increased
solubility in tetrahydrofuran and the ease of purification
of resulting ether reaction products.
O
O
9b
1150
49
10c
11d
12d
13d
O
1.3
0.7
11
N
H
O
O
N
H
N
Me
a In vitro data represent the average of at least two experiments.
b Synthesis of compounds: employed original route (Scheme 1).
c Synthesis of compounds: utilized route Scheme 2 synthesis.
d Synthesis of compounds: utilized route Scheme 3.
Acknowledgements
series GnRH antagonists. Reduction of the amine basic-
ity by addition of the CF3-group (entry 4), formation of
a tertiary amine such as quinuclidine (entry 5), or intro-
duction of a heteroatom for the morpholine analog
(entry 6) resulted in similar binding affinity (hGnRH
binding) but diminished functional activity in the PI
turnover assay. All these analogs necessarily upheld
the previous SAR requiring a basic site, though modi-
fied, at the 4-position of the quinolone. However, the
tetrahydrofuran analog (entry 7) was essentially equipo-
tent in binding and function as compared to the pyrrol-
idine analog, the first indication that a nonbasic
heterocycle at the quinolone 4-position can also serve
as an effective GnRH antagonist.
We would like to thank J. Leone, J. Pisano, S. Fabian,
and G. Reynolds for the preparation of several
intermediates.
References and notes
1. Matsu, H.; Baba, Y.; Nair, R. M. G.; Arimura, A.; Schally,
A. V. Biophys. Res. Commun. 1971, 43, 1334.
2. Conn, P. M.; Janovick, J. A.; Stanislaus, D.; Kuphal, D.;
Jennes, L. In Vitamins and Hormones; Litwack, G., Ed.;
Academic: New York, 1995; Vol. 50, pp 151–214.

R. J. DeVita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5599–5603
5603
3. Stojikovic, S. S.; Reinhart, J.; Catt, K. J. Endocrine Rev.
1994, 15, 462–499.
4. For a review: Conn, P. M.; Crowley, W. F., Jr. N. Engl. J.
Med. 1991, 324, 93.
5. Huirne, J. A. F.; Lambalk, C. Lancet 2001, 358, 1793–
1803.
7. (a) DeVita, R. J.; Hollings, D. D.; Goulet, M. T.; Wyvratt,
M. J.; Fisher, M. H., Jr.; Lo, J.; Yang, Y. T.; Cheng, K.;
Smith, R. G. Bioorg. Med. Chem. Lett. 1999, 9, 2615; (b)
DeVita, R. J.; Goulet, M. T.; Wyvratt, M. J., Jr.; Fisher, M.
H.; Lo, J.; Yang, Y. T.; Cheng, K.; Smith, R. G. Bioorg.
Med. Chem. Lett. 1999, 9, 2621; (c) DeVita, R. J.; Walsh, T.
F.; Young, J. R.; Jiang, J.; Ujjainwalla, F.; Toupence, R.
B.; Parikh, M.; Huang, S. X.; Fair, J. A.; Goulet, M. T.;
Wyvratt, M. J.; Lo, J.; Ren, N.; Yudkovitz, J. B.; Yang, Y.
T.; Cheng, K.; Cui, J.; Mount, G.; Rohrer, S. P.; Schaeffer,
J. M.; Rhodes, L.; Drisko, J. E.; McGowan, E.; MacIntyre,
D. E.; Vincent, S.; Carlin, J. R.; Cameron, J.; Smith, R. G.
J. Med. Chem. 2001, 44, 917; (d) Walsh, T. F.; Toupence,
R. B.; Young, J. R.; Huabg, S. X.; DeVita, R. J.; Hollings,
D. D.; Goulet, M. T.; Wyvratt, M. J., Jr.; Fisher, M. H.;
Lo, J.; Cui, J.; Ren, N.; Yudkovitz, J. B.; Yang, Y. T.;
Cheng, K.; Smith, R. G. Bioorg. Med. Chem. Lett. 2000, 10,
443; (e) Young, J. R.; Huang, S. X.; Chen, I.; Ren, N.;
Walsh, T. F.; DeVita, R. J.; Hollings, D. D.; Wyvratt, M.
J., Jr.; Goulet, M. T.; Ren, N.; Lo, J.; Yang, Y. T.;
Yudkovitz, J. B.; Cheng, K.; Smith, R. G. Bioorg. Med.
Chem. Lett. 2000, 10, 1723; (f) Jiang, J.; DeVita, R. J.;
Goulet, M. T.; Wyvratt, M. J.; Lo, J.-L.; Ren, N.;
Yudkovitz, J. B.; Cui, J.; Yang, Y. T.; Cheng, K.; Rohrer,
S. P. Bioorg. Med. Chem. Lett. 2004, 14, 1795.
6. Zhu, Y.-F.; Chen, C. Expert Opin. Ther. Patents 2004, 14,
187–199; See also (a) Randolph, J. T.; Waid, P.; Nichols,
C.; Sauer, D.; Haviv, F.; Diaz, G.; Bammert, G.; Besecke,
L. M.; Segreti, J. A.; Mohning, K. M.; Bush, E. N.;
Wegner, C. D.; Greer, J. J. Med. Chem. 2004, 47, 1085; (b)
Guo, Z.; Zhu, Y.-F.; Gross, T. D.; Tucci, F. C.; Gao, Y.;
Moorjani, M.; Connors, P. J., Jr.; Rowbottom, M. W.;
Chen, Y.; Struthers, R. S.; Xie, Q.; Saunders, J.; Reinhart,
G.; Chen, T. K.; Killam Bonneville, A. L.; Chen, C. J. Med.
Chem. 2004, 47, 1259; (c) Rowbottom, M. W.; Tucci, F. C.;
Zhu, Y.-F.; Guo, Z.; Gross, T. D.; Reinhart, G. J.; Xie, Q.;
Struthers, R. S.; Saunders, J.; Chen, C. Bioorg. Med. Chem.
Lett. 2004, 14, 2269; (d) Tucci, F. C.; Zhu, Y.-F.; Guo, Z.;
Gross, T. D.; Connors, P. J., Jr.; Gao, Y.; Rowbottom, M.
W.; Struthers, R. S.; Reinhart, G. J.; Xie, Q.; Chen, T. K.;
Bozigian, H.; Killam Bonneville, A. L.; Fisher, A.; Jin, L.;
Saunders, J.; Chen, C. J. Med. Chem. 2004, 47, 3483; (e)
Rowbottom, M. W.; Tucci, F. C.; Connors, P. J., Jr.;
Gross, T. D.; Zhu, Y.-F.; Guo, Z.; Moorjani, M.; Acevedo,
O.; Carter, L.; Sullivan, S. K.; Xie, Q.; Fisher, A.; Struthers,
R. S.; Saunders, J.; Chen, C. Bioorg. Med. Chem. Lett.
2004, 14, 4967–4973.
8. Originally disclosed at 219th ACS National Meeting, San
Francisco CA, Mar 26–30, 2000.
9. All compounds characterized by LC–MS and/or NMR.