Structure-activity relationships of the oxindole growth hormone secretagogues

Article abstract of DOI:10.1016/j.bmcl.2005.02.042

A series of substituted oxindole derivatives of SM-130686 was synthesized and evaluated as ghrelin receptor agonists. Modification of the substituents on the C3-aromatic part of the oxindole led to compounds with subnanomolar binding affinities. Compound

Full text of DOI:10.1016/j.bmcl.2005.02.042

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1789–1792
Structure–activity relationships of the oxindole growth hormone
secretagogues
Teruhisa Tokunaga, W. Ewan Hume, Jun Nagamine, Tetsuya Kawamura,
Mutsuo Taiji and Ryu Nagata*
Research Division, Sumitomo Pharmaceuticals Co., Ltd, 1-98 Kasugade Naka 3-chome, Konohana-ku, Osaka 554-0022, Japan
Received 14 January 2005; revised 14 February 2005; accepted 15 February 2005
Abstract—A series of substituted oxindole derivatives of SM-130686 was synthesized and evaluated as ghrelin receptor agonists.
Modification of the substituents on the C3-aromatic part of the oxindole led to compounds with subnanomolar binding affinities.
Compound 4i (IC50 = 0.02 nM) was orally active at low doses and showed in vivo activity when orally administered, 2 mg/kg twice a
day for 4 days, as evidenced by significant body weight gain.
Ó 2005 Elsevier Ltd. All rights reserved.
Ghrelin, a 28-amino acid gastric hormone, was discov-
ered in human and rat stomach tissues in 1999 and iden-
tified as the endogenous ligand for the Growth hormone
secretagogue receptor (GHS-R).1 Ghrelin has an
unprecedentedly unique structure in which the hydroxyl
group of the Ser3 residue is acylated by n-octanoic acid.
This acylation is essential for the biological activity and
is supposed to be critical for the transport of the ghrelin
molecule into the CNS area across the blood–brain bar-
rier, where the targeted GHS receptors are located.2
Ghrelin exhibits a wide range of biological activities.
In humans as well as in rodents, ghrelin not only stimu-
lates pituitary growth hormone (GH) secretion3 but also
increases food intake and body weight gain and regu-
lates energy balance.4,5 Prior to the discovery of ghrelin,
much effort has been made to identify peptidomimetic
and nonpeptidic small molecular GH secretagogues
(GHSs) as an alternative to GH replacement therapy.
Such synthetic GHSs have been reported to stimulate
the secretion of GH through the GHS-R, with a mech-
anism distinct from growth hormone releasing hormone
(GHRH).6 Among the GHSs reported so far,7 MK-
677,8 NN703,9 and CP-42439110 are noted as extensively
studied GHSs and indeed proceeded into clinical trials.
In a previous paper, we demonstrated that certain oxin-
dole derivatives exemplified by the structurally distinct
SM-130686 had potent GH releasing activity (Fig.
1).11–13 We also showed SM-130686 was orally active
with a good in vivo profile. To clarify the structure–
activity relationships (SARs) and understand the
pharmacology of the oxindole class of GHSs, further
Keywords: Ghrelin; Growth hormone secretagogue; SM-130686.
*
Corresponding author. Tel.: +81 6 6466 5193; fax: +81 6 6466
5483; e-mail: rnagata@sumitomopharm.co.jp
Figure 1. Structures of small molecule growth hormone secretagogues.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.042

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T. Tokunaga et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1789–1792
Table 1. Structure–activity relationships of 3-aromatic part of oxin-
dole ring
studies have been conducted. Herein, we describe the
SAR in the C3-aromatic part of the oxindole GHS,
and the discovery of highly potent compounds in this
series.
The compounds were synthesized as shown in Scheme 1.
Addition of aryl magnesium bromides or aryl lithiums
to key intermediary isatin 112,14 afforded oxindoles 2a–
k in 32–71% yields. Compounds 2a–k were treated with
zinc cyanide in the presence of a catalytic amount of tet-
rakis(triphenylphosphine)palladium in DMF at 80 °C to
produce compounds 3a–k in 48–95% yields. Conversion
of the nitrile functionality of 3a–k to a carboxamide was
accomplished by treatment with potassium hydroxide in
anhydrous tert-butyl alcohol at 50 °C to give com-
pounds 4a–k in 61–100% yields.
Compd R1
R2
R3
R4 R5 Binding IC50 (nM)a
4a
4b
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
116
4.0
2.1
15
H
H
(+)-4bb Cl
H
H
4c
4d
H
Cl
H
H
H
Cl
H
83
160
77
All the compounds described in this study were evalu-
ated in a competitive binding assay on a human recom-
binant ghrelin receptor membrane preparation using
125I–ghrelin as a radioligand (Table 1).15 In order to
examine the effect of a substituent at the C3-aromatic
ring of SM-130686 on the activity, we prepared unsub-
stituted phenyl, various monochloro and monomethoxy
substituted phenyl analogues. Removal of the C20
chloro substituent in racemic SM-130686 resulted in a
29-fold loss in binding affinity. Moving the chloro sub-
stituent to the C30 and C40 positions also resulted in a
decrease in activity. These results indicated that exis-
tence of a substituent at the C20 position was important
for the activity. We assumed that the substituent at the
C20 position might help the C3-phenyl ring to adopt a
preferable conformation. Monomethoxy derivatives
4e,f and 4g displayed weaker activity than the corre-
sponding monochloro analogues, suggesting that a
hydrophobic substituent at the C3 aromatic ring was
favourable.
4e
OMe
H
H
4f
4g
OMe
H
H
H
OMe
H
106
4h
4i
Cl
Cl
Cl
Cl
Cl
H
1.6
Cl
H
0.02
0.24
4.2
4j
4k
H
H
H
Ghrelin
0.14
a See Ref. 15.
b SM-130686.
retained activity, compounds 4h,i and 4j showed 2.5,
200 and 17 times enhanced activities, respectively,
compared to 4b. In particular, the IC50 value of 20,40-
dichlorophenyl compound 4i was about ten times supe-
rior to that of ghrelin itself. It was noteworthy that the
introduction of only one substituent on the phenyl ring
influenced the activity to such a great extent.
Compound 4i was evaluated further in vitro and in vivo
assay. In a fluorescent calcium indicator assay (FLIPR)
measuring [Ca2+] accumulation induced by a compound
Next, we assessed more hydrophobic 3-dichlorophenyl
analogues. Gratifyingly, while compound 4k had only
Scheme 1. Reagents and conditions: (a) ArMgBr, THF, 0 °C or ArLi, THF, ꢀ78 °C, 32–71%; (b) Zn(CN)2, Pd(PPh3)4, DMF, 80 °C, 48–95%;
(c) (1) powdered KOH, tert-butyl alcohol, 50 °C, (2) HCl, dioxane, rt, 61–100% (two steps).

T. Tokunaga et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1789–1792
1791
in CHO cells over-expressing GHS-R, compound 4i was
demonstrated to possess a partial agonistic activity,13,16
with 91% of the maximal response obtained by ghrelin
and an EC50 of 17 nM, while 4b displayed 65% of the
maximal response with an EC50 of 11 nM. The reason
for the dissociation of the IC50 value in the binding
assay from the EC50 value in the FLIPR assay was
unclear. Administration of 4i orally increased body
weight gain in a dose dependent manner after 4 days
in rats. When 4i was administered at doses of 2, 6 and
20 mg/kg twice a day for 4 days, the body weight gains
were 11.6, 13.6 and 21.1 g, respectively, whereas the dis-
tilled water administered control group showed body
weight gain of 5.0 g (Fig. 2). These body weight gains
were greater than those of SM-130686 at the corre-
sponding doses.12 In a rat pharmacokinetic study, Cmax
and bioavailability of 4i were 491 ng/mL and 28%,
respectively, after oral dose of 20 mg/kg. When intrave-
nously administered to rats with 5 mg/kg, Cl, Vd and t1/2
were 51 mL/min/kg, 5.4 L/kg and 1.6 h, respectively.
This pharmacokinetic profile was quite similar to that
of SM-130686.12 Although serum protein bindings of
both compounds were not measured, we assume that
the greater in vivo efficacy of 4i compared to SM-
130686 would simply be attributed to the increased
potency.
such as 4i, modification of these compounds may lead
to the identification of a new class of antiobesity drugs
mediated by the modulation of the ghrelin receptor
(GHS receptor).
Acknowledgements
We thank Mr. K. Ushiroda for synthetic contributions.
We also appreciate Mr. T. Watanabe for obtaining the
pharmacokinetic data.
References and notes
1. Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.;
Matsuo, H.; Kangawa, K. Nature 1999, 402, 656.
2. Banks, W. A.; Tschop, M.; Robinson, S. M.; Heiman, M.
L. J. Pharmacol. Exp. Ther. 2002, 302, 822.
3. Takaya, K.; Ariyasu, H.; Kanamoto, N.; Iwakura, H.;
Yoshimoto, A.; Harada, M.; Mori, K.; Komatsu, Y.;
Usui, T.; Shimatsu, A.; Ogawa, Y.; Hosoda, K.; Akamizu,
T.; Kojima, M.; Kangawa, K.; Nakao, K. J. Clin.
Endocrinol. Metab. 2000, 85, 4908.
4. Tschop, M.; Smiley, D. L.; Heiman, M. L. Nature 2000,
407, 908.
5. Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.;
Matsuo, H.; Kangawa, K.; Matsukura, S. Nature 2001,
409, 194.
6. Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.;
Liberator, P. A.; Rosenblum, C. I.; Hamelin, M.; Hreniuk,
D. L.; Palyha, O. C.; Anderson, J.; Paress, P. S.; Diaz, C.;
Chou, M.; Liu, K. K.; McKee, K. K.; Pong, S.-S.;
Chaung, L.-Y. P.; Elbrecht, A.; Dashkevicz, M.; Heavens,
R.; Rigby, M.; Sirinathsinghji, D. J. S.; Dean, D. C.;
Melillo, D. G.; Patchett, A. A.; Nargund, R. P.; Griffin, P.
R.; DeMartino, J. A.; Gupta, S. K.; Schaeffer, J. M.;
Smith, R. G.; Van der Ploeg, L. H. T. Science 1996, 273,
974.
7. Carpino, P. A. Expert Opin. Ther. Pat. 2002, 12, 1599.
8. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
Barkat, K. J.; Johnston, D. B. R.; Cheng, K.; Chan, W.
W.-S.; Butler, B.; Hickey, G.; Jacks, T.; Schleim, K.; Pong,
S.-S.; Chaung, L. Y.-P.; Chen, H. Y.; Frazier, E.; Leung,
K. H.; Chiu, S.-H. L.; Smith, R. G. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7001.
9. Hansen, T. K.; Ankersen, M.; Hansen, B. S.; Raun, K.;
Nielsen, K. K.; Lau, J.; Peschke, B.; Lundt, B. F.;
Thøgersen, H.; Johansen, N. L.; Madsen, K.; Andersen,
P. H. J. Med. Chem. 1998, 41, 3705.
10. Pan, L. C.; Carpino, P. A.; Lefker, B. A.; Ragan, J. A.;
Toler, S. M.; Pettersen, J. C.; Nettleton, D. O.; Ng, O.;
Pirie, C. M.; Chidsey-Frink, K.; Lu, B.; Nickerson, D. F.;
Tess, D. A.; Mullins, M. A.; MacLean, D. B.; DaSilva-
Jardine, P. A.; Thompson, D. D. Endocrine 2001, 14, 121.
11. Nagamine, J.; Nagata, R.; Seki, H.; Nomura-Akimaru,
N.; Ueki, Y.; Kumagai, K.; Taiji, M.; Noguchi, H. J.
Endocrinol. 2001, 171, 481.
In summary, the structure–activity relationships of the
C3-aromatic part of SM-130686 was examined and a
series of 3-dichlorophenyl analogues were identified as
potent ghrelin agonists. Compound 4i, the most potent
compound in the binding assay, showed excellent in vivo
activity and was found to be a ghrelin partial agonist, as
evidenced by the FLIPR assay. Recent accumulating
evidence of the strong orexigenic and adipogenic effects
of ghrelin has raised the possibility that a ghrelin mod-
ulator or antagonist could be used in the treatment of
obesity.17 Owing to the high binding affinity and the
partial agonistic activity of theses oxindole derivatives
25
Vehicle
4i: 2mg/kg
4i: 6mg/kg
4i: 20mg/kg
20
15
10
5
12. Tokunaga, T.; Hume, W. E.; Umezome, T.; Okazaki, K.;
Ueki, Y.; Kumagai, K.; Hourai, S.; Nagamine, J.; Seki,
H.; Taiji, M.; Noguchi, H.; Nagata, R. J. Med. Chem.
2001, 44, 4641.
13. Nagamine, J.; Kawamura, T.; Tokunaga, T.; Hume, W.
E.; Nagata, R.; Nakagawa, T.; Taiji, M. Comb. Chem.
High Throughput Screening, submitted for publication.
14. Hume, W. E.; Tokunaga, T.; Nagata, R. Tetrahedron
2002, 58, 3605.
0
0
1
2
3
4
Time (day)
Figure 2. Effect of compound 4i on the body weight gain. Compound
4i was orally administered twice a day for 4 days in normal female rats.
Data are represented as means SD (n = 6). *: P < 0.05, **: P < 0.01,
Shirley–Williams test (vs vehicle-treatment-group).

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T. Tokunaga et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1789–1792
15. Binding assay: Human recombinant ghrelin receptor
membrane preparation was purchased from Euroscreen.
These were suspended in binding buffer (25 mM Hepes
pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.4% protease free
BSA, 1 mM EGTA, 10 lg/mL leupeptin, 10 lg/mL apro-
tinin, 1 lg/mL pepstain A) to a concentration of 50 lg
protein/mL. These membranes (1 lg protein/tube) were
mixed with 125I–ghrelin (30,000 dpm/tube), with or with-
out different concentrations (0.01–10,000 nM) of test
compounds and the binding buffer up to a total volume
of 100 lL. Nonspecific binding was obtained by adding
500 nM of cold ghrelin. The membranes were incubated at
25 °C for 30 min, and the bound radioligand was sepa-
rated from free radioligand by washing three times with
3 mL of the binding buffer through a 0.5% polyethylen-
imine-presoaked GF/B filter (Whatman). The radioactiv-
ity on the filters was counted in 1470 Wallac Wizard
Gamma Counter (PerkinElmer, MA).
16. All the other compounds in Table 1 displayed partial
agonist activity.
17. Holst, B.; Cygankiewicz, A.; Jensen, T. H.; Ankersen, M.;
Schwartz, T. W. Mol. Endocrinol. 2003, 17, 2201.