Orally bioavailable, indole-based nonpeptide GnRH receptor antagonists with high potency and functional activity

Article abstract of DOI:10.1016/S0960-894X(01)00512-1

Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24, which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys.

Full text of DOI:10.1016/S0960-894X(01)00512-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2597–2602
Orally Bioavailable, Indole-Based Nonpeptide GnRH Receptor
Antagonists with High Potency and Functional Activity
Wallace T. Ashton,a,* Rosemary M. Sisco,a Gerard R. Kieczykowski,a Yi Tien Yang,b
Joel B. Yudkovitz,b Jisong Cui,b George R. Mount,b Rena Ning Ren,b Tsuei-Ju Wu,b
Xiaolan Shen,c Kathryn A. Lyons,d An-Hua Mao,d Josephine R. Carlin,d
Bindhu V. Karanam,d Stella H. Vincent,d Kang Chengb and Mark T. Gouleta
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
bDepartment of Biochemistry and Physiology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
cDepartment of Comparative Medicine, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
dDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
Received 29 May 2001; accepted 20 July 2001
Abstract—Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived
series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary
amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24,
which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys. # 2001 Elsevier
Science Ltd. All rights reserved.
Orally active, nonpeptide receptor antagonists of gona-
dotropin-releasing hormone (GnRH; also known as
luteinizing hormone-releasing hormone, or LHRH)
represent a new class of experimental pharmaceuticals.
They could possess advantages over existing therapies
for chronic suppression of sex hormone levels, as in the
treatment of certain hormone-dependent cancers, uter-
ine fibroids, and endometriosis, or for short-term appli-
cations, as in assisted reproduction protocols.1 Building
upon previous work from these laboratories,2 we have
reported3 that the tryptamine derivative 1a is an effec-
tive GnRH antagonist in vitro. Introduction of a chiral
methyl substituent, as in 1b, has recently been
achieved.4 We now present a structure–activity study of
amides related to 1b4 and its chain-shortened analogue
1c.4
For the first series of compounds, containing a four-
carbon tether between the side-chain amine and the
pyridine ring, the (S)-b-methyltryptamine derivative 24a
was reacted with 4-(4-pyridyl)butyraldehyde (3)3 under
reductive amination conditions2e,3 to give 4 (Scheme 1).
Saponification of the ester and coupling with amines in
the presence of PyBOP reagent as previously described2e,3
*Corresponding author. Fax: +1-732-594-5350; e-mail: wally_
ashton@merck.com
Scheme 1. Conditions: (i) (a) MgSO4, CDCl3, ꢀ5 to ꢀ10 ꢁC; (b)
NaBH4, MeOH, ꢀ5 ꢁC; (ii) as described in refs 2e and 3.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00512-1

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W. T. Ashton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2597–2602
yielded the desired amides 5–20. Noncommercial amines
were prepared by literature methods.5ꢀ15
Fukuyama variation of the Mitsunobu reaction,16 28
was N-alkylated with heterocyclic ethanols 29. Depro-
tection of the resulting 30 with neat propylamine16
furnished the new derivatives 31–37 and also provided
an alternative route to 24.
Analogues with a two-carbon, side-chain tether were
obtained by acylation of 2 with pyridylacetic acids in
the presence of PyBOP (Scheme 2). Reduction of the
amides 21 with borane furnished the secondary amines
22. Further transformation as described for Scheme 1
yielded the targets 23–25.
The synthesis of intermediate alcohols 29 is illustrated
in Scheme 4. Isomeric pyridones 38 and 39 were pre-
pared and separated by the method of Mohrle and
Weber.17 Hydrogenation of 39 over palladium on car-
bon furnished 40. By a procedure analogous to that
reported for 39,17 2-(3-pyridyl)ethanol 4117 was qua-
ternized by treatment with chloromethyl methyl ether to
give 42, which was then reacted with potassium ferri-
cyanide and sodium hydroxide. The desired pyridone
43, after separation from the isomeric product 44, was
converted to 33 by the methods of Scheme 3. Acid
hydrolysis removed the methoxymethyl group to yield
34. Commercial aminopyridine 45 was diazotized and
hydrolyzed to 46,18 then N-methylated with methyl
iodide in the presence of methanolic potassium hydrox-
ide to give 47.19 Next, the vinyl group was introduced
by a Stille coupling, yielding 48. Reaction with N-bro-
mosuccinimide in aqueous DMSO provided bromo
alcohol 49. Treatment with potassium carbonate resul-
ted in formation of an epoxide (not isolated), which was
reduced regiospecifically to alcohol 50 with ammonium
formate and palladium catalyst. A similar reaction
sequence converted 5-bromo-2-methoxypyridine (51) to
the hydroxyethyl derivative 52.
Scheme 3 outlines the preparation of a series of analo-
gues of 24 and 25 in which the unsubstituted pyridine
ring is replaced by related heterocycles. Boc protection
of the amino group of 2 followed by saponification of
the ester yielded 26. Treatment with isoquinuclidine
hydrochloride13 in the presence of PyBOP gave 27.
After deprotection with trifluoroacetic acid, the result-
ing amine was reacted with 2,4-dinitrobenzenesulfonyl
chloride under modified Schotten–Baumann conditions
to afford the activated sulfonamide 28. Using the
Scheme 2. Conditions: (i) 4- or 3-pyridylacetic acid hydrochloride,
PyBOP, Et3N, 1,2-dichloroethane; (ii) (a) BH3, THF; (b) MeOH; (c)
Me2N(CH2)2OH, Á; (iii) as described in refs 2e and 3.
Scheme 4. Conditions: (i) H2, Pd/C, EtOH; (ii) MeOCH2Cl, CH2Cl2,
0 ꢁC; (iii) K3Fe(CN)6, NaOH, H2O, 0 ꢁC; (iv) as in Scheme 3; (v) (a)
concd HCl–EtOH (1:3), Á; (b) aq Na2CO3; (vi) NaNO2, aq H2SO4, 0–
50 ꢁC; (vii) MeI, KOH, MeOH, Á; (viii) Bu3SnCH¼CH2,
(Ph3P)2PdCl2, DMF; (ix) NBS, DMSO–H2O; (x) K2CO3, DMF–H2O;
(xi) ammonium formate, Pd/C, EtOH.
Scheme 3. Conditions: (i) Boc2O, K2CO3, THF–H2O; (ii) (a) KOH,
MeOH–H2O, Á; (b) H+; (iii) isoquinuclidine hydrochloride, PyBOP,
Et3N, CH2Cl2; (iv) (a) TFA, anisole, CH2Cl2; (b) aq Na2CO3; (v) 2,4-
dinitrobenzenesulfonyl chloride, satd aq NaHCO3, CH2Cl2; (vi) Ph3P,
DEAD, benzene; (vii) PrNH2.

W. T. Ashton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2597–2602
2599
Table 1. Inhibition of GnRH receptor binding and PI turnover by tryptamine derivatives with four-carbon tether to pyridine ring
Compd
R1R2N
Amine source
Ref 5
R3
H
hGnRH IC50 (nM)a
1.4
PI turnover IC50 (nM)b
1ac
18
1bd
5
Ref 5
Ref 6
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.7
0.7
1.3
2.7
0.6
6.1
4.4
5.6
1.4
21
5.7
21
6
Ref 7
39
7
Ref 8
93
8
Comm.e
Comm.e
Ref 9
66
9
220
124
14
10
11
12
13
14
15
16
17
18
19
20
Comm.e
Ref 10
Comm.e
66
31
Ref 11
0.8
0.4
1.0
1.0
2.1
4.8
4.8
8.8
8.7
4.0
2.8
13
f
f
Ref 13
Ref 14
Ref 15
Comm.e
6.8
6.4
aInhibition of [125I]buserelin binding to human pituitary GnRH receptor.
bInhibition of GnRH-stimulated [3H]inositol phosphate hydrolysis.
cRef 3.
dRef 4a.
eCommercial.
fMade from commercial lactam precursor by method in ref 12.

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W. T. Ashton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2597–2602
Table 2. Inhibition of GnRH receptor binding and PI turnover by
tryptamine derivatives with two-carbon tether to pyridine ring
It was anticipated that shortening the side-chain tether
between the pyridine ring and the secondary amine
might improve oral bioavailability. In an earlier series
lacking an indole 5-substituent and the b-methyl group,
such a modification was significantly detrimental to
receptor binding affinity.2d Remarkably, compound 1c,4b
the two-carbon tether analogue of 1b, not only main-
tained excellent potency in the in vitro assays (Table 2)
but also had oral bioavailability of 23% in rats. How-
ever, its bioavailability in dogs (14%) and rhesus mon-
keys (6.4%) was still deficient. Incorporation of some of
the best bicyclic amino groups from Table 1 into this
series gave 23–25, which all displayed high potency in
the binding and functional assays (Table 2). Although
oral bioavailability in dogs was still unsatisfactory for
23 (8.5%) and 25 (12%), compound 24, the iso-
quinuclidine amide bearing a 4-pyridyl terminus, had
promising pharmacokinetic properties. Thus, the oral
bioavailability of 24, while only 8% in rats, was 25% in
dogs and 21% in rhesus monkeys. The terminal half-life
of 24 was determined to be 1 h in rats, 3.9 h in dogs, and
3.3 h in rhesus monkeys.
Compd
R1R2N
Pyridine
linkage
hGnRH
IC50 (nM)a
PI turnover
IC50 (nM)b
1cc
23
24
25
4
4
4
3
0.8
1.2
0.6
0.6
7.0
1.9
7.2
2.6
aInhibition of [125I]buserelin binding to human pituitary GnRH
receptor.
bInhibition of GnRH-stimulated [3H]inositol phosphate hydrolysis.
cRef 4b.
Replacement of the pyridine by a pyridone (or related
heterocycle) was also investigated (Table 3). Of the iso-
meric 1-methylpyridin-2(1H)-ones 31 and 32, linkage at
the 5-position (32) resulted in a dramatic potency
advantage over linkage at the 3-position (31). All of the
analogues with the preferred substitution pattern (32–34
and 36) maintained very high potency. This suggests
that there is no requirement for the side-chain terminus
to be basic, provided that it is a suitable hydrogen-bond
acceptor. The methoxypyridine 35, an isomer of 32,
was somewhat less active. Finally, the reduced analogue
37, tested as a mixture of diastereomers, was only
moderately less potent than its unsaturated parent 32.
Although some of the compounds in this pyridone series
had satisfactory oral bioavailability in dogs (e.g., 35% for
36), none of those tested had acceptable bioavailability in
rhesus monkeys (e.g., 4% for 36).
Compounds were evaluated for their ability to compete
with the GnRH receptor agonist [125I]buserelin for
binding to the human GnRH receptor20 in the presence
of 0.1% BSA. In addition, functional antagonism in
vitro was determined via inhibition of GnRH-stimu-
lated phosphatidylinositol (PI) hydrolysis in cloned
Chinese hamster ovary (CHO) cells stably expressing
the human GnRH receptor.20
The beneficial effect of the (S)-b-methyl substituent on
the tryptamine side chain was apparent (cf. 1b4 vs 1a;3
Table 1). Optimization of the tertiary amide substituent
at the indole-5-position, which had been studied in the
absence of the b-methyl substituent,3 was reinvestigated
here (Table 1). Compared to the 7-azanorbornane lead
1b,4 gem-dimethyl or spiro-fused azetidines or piper-
idines (5–8) generally maintained high affinity to the
human GnRH receptor but clearly suffered a reduction
in potency in the PI turnover functional assay. The
piperazines 9 and 10 proved to be even more detri-
mental in the PI assay. In contrast, the diisobutyl amide
11, although not especially potent in the binding assay,
was relatively effective in the functional assay
(IC50=14 nM). Whereas the fused pyrrolidines 12 and
13 did not produce favorable results, the products from
several bicyclic amines 14–20 were effective GnRH
antagonists, with all but 18 having functional IC50
values below 10 nM. Particularly noteworthy was the
isoquinuclidine amide 17, which had IC50 values of 1.0
and 2.8 nM in the binding and functional assays,
respectively. Nevertheless, the oral bioavailability of 17
was less than desired (14% in rats, 7% in dogs), so
further improvement was needed.
The effectiveness of orally administered 24 in blocking
the release of luteinizing hormone (LH) was investigated
in castrated male rats,21 which have relatively stable and
elevated circulating LH levels. It should be noted that
24 had an IC50 value of 1.7 nM against the rat GnRH
receptor in a whole cell binding assay. Compound 24
dose-dependently inhibited LH release for periods ran-
ging from 5–7 h at 5 mg/kg po to >15 h at 20 mg/kg po.
In a representative experiment, a single dose of 24 at
10 mg/kg po completely suppressed plasma LH levels
for 13 h (Fig. 1).
In conclusion, enhancements to our previously reported
series of indole-based GnRH antagonists3 were under-
taken with the goal of improving pharmacodynamic
and pharmacokinetic properties. With the addition of
the (S)-b-methyl substituent to the tryptamine core, the
amino moiety of the tertiary amide was re-investigated.
Amides derived from compact, hydrophobic bicyclic
amines were preferred, with the isoquinuclidine amide
apparently optimum. Subsequently, it was found that,
with these features in place, the side-chain tether linking

W. T. Ashton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2597–2602
2601
Table 3. Inhibition of GnRH receptor binding and PI turnover by
tryptamine derivatives with two-carbon tether to a heterocycle
Acknowledgements
We are grateful to Amy Bernick and Dr. Lawrence
Colwell for providing mass spectral data, to Robert
Frankshun, Amanda Makarewicz, Joseph Simeone,
Mamta Parikh, and Dr. Peter Lin for preparation of
certain intermediates, to Dr. Jonathan Young for sup-
plying conditions for the Fukuyama–Mitsunobu amine
alkylation methodology, and to Joseph Simeone and
Robert Bugianesi for permission to use their unpub-
lished data. We are indebted to Jinchang Chen and
Mellissa Creighton in Drug Metabolism for pharmaco-
kinetics support. For assistance in the animal experi-
ments, we also thank Klaus Schleim in Animal
Pharmacology, as well as Dr. Judy Fenyk-Melody, Dr.
William Feeney, Dr. Susan Iliff, Donald Hora, Paul
Cunningham, Bonnie Friscino, Alison Kulick, and
Allison Parlapiano in Comparative Medicine.
Compd
Het
hGnRH IC50 (nM)a PI turnover IC50 (nM)b
31
5.3
0.2
0.4
0.3
1.0
0.6
0.4
82
32
33
34
35
36
37
1.4
3.9
1.0
11
References and Notes
4.0
8.2
1. (a) Millar, R. P.; Zhu, Y.-F.; Chen, C.; Struthers, R. S.
Brit. Med. Bull 2000, 56, 761. (b) 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. (c) Kut-
scher, B.; Bernd, M.; Beckers, T.; Polymeropoulos, E. E.;
Engel, J. Angew. Chem., Int. Ed. Engl. 1997, 36, 2148.
2. (a) Chu, L.; Hutchins, J. E.; Weber, A. E.; Lo, J.-L.; Yang,
Y. T.; Cheng, K.; Smith, R. G.; Fisher, M. H.; Wyvratt, M. J.;
Goulet, M. T. Bioorg. Med. Chem. Lett. 2001, 11, 509. (b)
Chu, L.; Lo, J.-L.; Yang, Y. T.; Cheng, K.; Smith, R. G.;
Fisher, M. H.; Wyvratt, M. J.; Goulet, M. T. Bioorg. Med.
Chem. Lett. 2001, 11, 515. (c) Lin, P.; Marino, D.; Lo, J.-L.;
Yang, Y.-T.; Cheng, K.; Smith, R. G.; Fisher, M. H.; Wyv-
ratt, M. J.; Goulet, M. T. Bioorg. Med. Chem. Lett. 2001, 11,
1073. (d) Lin, P.; Parikh, M.; Lo, J.-L.; Yang, Y.-T.; Cheng,
K.; Smith, R. G.; Fisher, M. H.; Wyvratt, M. J.; Goulet, M. T.
Bioorg. Med. Chem. Lett. 2001, 11, 1077. (e) Ashton, W. T.;
Sisco, R. M.; Yang, Y. T.; Lo, J.-L.; Yudkovitz, J. B.; Cheng,
K.; Goulet, M. T. Bioorg. Med. Chem. Lett. 2001, 11, 1723.
3. Ashton, W. T.; Sisco, R. M.; Yang, Y. T.; Lo, J.-L.;
Yudkovitz, J. B.; Gibbons, P. H.; Mount, G. R.; Ren, R. N.;
Butler, B. S.; Cheng, K.; Goulet, M. T. Bioorg. Med. Chem.
Lett. 2001, 11, 1727.
aInhibition of [125I]buserelin binding to human pituitary GnRH
receptor.
bInhibition of GnRH-stimulated [3H]inositol phosphate hydrolysis.
the NH to the pyridine ring could be contracted from
four methylene units to two. This modification main-
tained high potency and improved pharmacokinetic
properties, with the result that 24 had an acceptable
half-life and oral bioavailability of >20% in both dogs
and rhesus monkeys. Furthermore, a single oral dose of
24 at 10 mg/kg provided complete and prolonged sup-
pression of circulating LH levels in a castrated rat
model.
4. (a) Simeone, J. P.; Bugianesi, R. L.; Ponpipom, M. M.;
Goulet, M. T.; Levorse, M. S.; Desai, R. C. Tetrahedron Lett.
2001, 42, 6459. (b) Simeone, J. P.; Bugianesi, R. L. Unpub-
lished work from Merck Research Laboratories.
5. Fraser, R. R.; Swingle, R. B. Can. J. Chem. 1970, 48, 2065.
6. Thompson, H. W.; Swistok, J. J. Org. Chem. 1981, 46, 4907.
7. Mimura, M.; Hayashida, M.; Nomiyama, K.; Ikegami, S.;
Iida, Y.; Tamura, M.; Hiyama, Y.; Ohishi, Y. Chem. Pharm.
Bull. 1993, 41, 1971.
8. Najer, H.; Giudicelli, R.; Sette, J. Bull. Soc. Chim. Fr. 1964,
2572.
9. Foye, W. O.; Fedor, L. R., Jr. J. Am. Pharm. Assoc. 1959,
48, 412.
10. Punniyamurthy, T.; Katsuki, T. Tetrahedron 1999, 55,
9439.
11. Banholzer, R.; Heusner, A.; Schulz, W. Liebigs Ann.
Chem. 1975, 2227.
12. Heesing, A.; Herdering, W. Chem. Ber. 1983, 116, 1081.
Figure 1. Effect of oral 24 (administration at time=6 h indicated by
arrow) on LH release in a castrated male rat, as described in ref 21.

2602
W. T. Ashton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2597–2602
13. (a) Werner, L. H.; Ricca, S., Jr. J. Am. Chem. Soc. 1958,
80, 2733. (b) Morris, I. G.; Pinder, A. R. J. Chem. Soc. 1963,
1841. (c) Yokota, M.; Takizawa, E.; Ohkura, Y.; Fukai, C.;
Tomiyama, T. Eur. J. Med. Chem. 1997, 32, 377.
14. Reinisch, G.; Bara, H.; Klare, H. Chem. Ber. 1966, 99, 856.
15. Hewgill, F. R.; Jefferies, P. R. J. Chem. Soc. 1955, 2767.
16. Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan,
T. Tetrahedron Lett. 1997, 38, 5831.
17. Mohrle, H.; Weber, H. Chem. Ber. 1971, 104, 1478.
18. Cook, D. J.; Bowen, R. E.; Sorter, P.; Daniels, E. J. Org.
Chem. 1961, 26, 4949 (by a different route).
19. Comins, D. L.; Lyle, R. E. J. Org. Chem. 1976, 41, 2065
(by a different route).
Y. T; Cheng, K.; Smith, R. G. Bioorg. Med. Chem. Lett. 2000,
10, 443.
21. Castrated Wistar male rats (350–400 g) were surgically fit-
ted with a chronic indwelling catheter inserted into the femoral
artery for blood sampling. After the surgery, the rats were
housed individually in metabolic cages to recover for at least 3
days. On the day of the experiment, the catheter was con-
nected to an automatic blood micro-sampler via a swivel,
which allowed the animal to move freely inside the cage. In
this experiment, the animals were dosed orally with 24 at
10 mg/kg, and a 140 mL blood sample was withdrawn every
30 min for 24 h (6 h for pre-dosing baseline and 18 h post-dos-
ing). After each blood sampling, the same amount of saline
was returned to the animals to maintain the volume of blood
constant. The LH content in serum was determined by a
double antibody radioimmunoassay procedure.
20. Walsh, T. F.; Toupence, R. B.; Young, J. R.; Huang, S. X.;
Ujjainwalla, F.; DeVita, R. J.; Goulet, M. T.; Wyvratt, M. J.,
Jr.; Fisher, M. H.; Lo, J.-L.; Ren, N.; Yudkovitz, J. B.; Yang,