Synthesis and pharmacological evaluation of indolinone derivatives as novel ghrelin receptor antagonists
Introduction
Ghrelin and its receptor (GHSR) have been shown to affect several important physiological activities such as food intake regulation, energy balance, gastric motility and secretion, as well as glucose metabolism, fat accumulation and cell proliferation.1, 2
Ghrelin is a 28-amino acid hormone predominantly produced by P/D1 cells of the stomach and small intestine, but is also present in minor quantity in the kidneys, pancreas and hypothalamus.3 It was discovered in 1999 and recognized as the endogenous bioactive ligand for the Growth Hormone Secretagogue receptor (GHSR).4 Ghrelin presents an unusual n-octanoyl acetylation at serine 3 essential for its biological activity. It has been postulated that this acetylation is also critical for the transport of the ghrelin molecule across the blood–brain barrier into the brain where the receptor is highly expressed.5
The ghrelin receptor, GHSR, is a member of the G-protein-coupled receptor (GPCR) superfamily.6 Two different splice forms of the human GHSR are known; however, only GHSR1a is activated by ghrelin and its mimetics, whereas the role of GHSR1b, a truncated form of GHSR1a, is unknown.7 GHSR1a is widely expressed in the brain and peripheral tissues, especially in the stomach. It was identified and cloned by Howard et al. in 1996.8 Its discovery was the result of an extensive research on synthetic modulators of GH secretion seen as a new alternative treatment to growth hormone replacement therapy.9, 10, 11 Interestingly, GHSR1a showed a constitutive activity, independent from the presence of its ligand, being able to change into an active conformation in the absence of the agonist.12, 13
GHSR1a, with its ligand, ghrelin, is predominantly involved in the hunger perception prior to mealtimes. Ghrelin circulating levels decrease with feeding and increase by fasting achieving concentrations sufficient to stimulate hunger and food intake. Administration of exogenous ghrelin potently stimulates food intake; this effect was more efficient than that of any other molecule, with the exception of neuropeptide Y.14 Chronic administration of ghrelin in freely feeding mice and rats, increased body weight and adiposity.15
Due to its involvement in feeding behavior, in energy homeostasis and body weight regulation, ghrelin has become an attractive target to treat obesity and feeding disorders. Moreover, ghrelin seems to play a direct role on glucose homeostasis through regulation of insulin secretion.16, 17, 18
Those findings support the potential therapeutic role of ghrelin antagonists in diabetes.
However, while GHSR agonists have been extensively studied, due to their role in GH release, relatively fewer antagonists are described in literature.19, 20, 21, 22, 23 Some of these compounds are shown in Figure 1.
As part of our efforts to develop ghrelin receptor antagonists we identified, through HTS screen of the Sanofi-Aventis compound library, an indolinone scaffold as exemplified by the general formula 1 (Fig. 2).
This class of compounds has been previously identified as Vasopressin (V1) receptor antagonists.24 We designed new molecules devoid of vasopressin activity, with lower molecular weight and improved in vitro ADME profile. We first investigated the influence of N-sulphonylation of the indolinone nitrogen atom on GHSR1a antagonism. This group was known to be essential for V1 antagonism. The urea linker was modified with the aim to ameliorate bioavailability. This synthetic strategy also led to the identification of novel, enantiopure compounds.
The hit compound 2 (Fig. 2), with an IC50 of 47 nM in functional CreLuc assay, represented an excellent starting point for new leads identification.25
Interestingly, during our SAR studies, we could observe that slight modifications in scaffold decoration, could lead to agonist activity. Indeed a series of oxindole derivatives, here represented by SM-130686 (Fig. 3), have already been published by Sumitomo, and reported to be GHSR agonists.26
Our compounds, however, basically differ from Sumitomo’s class of molecules, especially for the presence of a substituted amide at the indolinone C-3 position and for this carbon configuration.
Looking for new molecules we focused our attention on compounds possessing a full GHSR1a antagonist activity only. Modifications both at the basic moiety and at the aromatic portions were largely evaluated and will be discussed hereafter.
Section snippets
Chemistry
The synthetic pathway leading to the Hit compound 2 is reported on Scheme 1.
Starting from commercially available 1,2-dichloro-4-fluoro-5-nitrobenzene and methyl-4-chlorophenylacetate, an aromatic SN reaction provided the intermediate 3, which was transformed in the halogen derivative 4 by the use of phenyltrimethylammonium tribromide. Other reagents more commonly used for benzylic halogenation, such as NBS, failed in giving the desired product. In some cases the halogenations proceeded also to
In vitro SAR studies
Compounds potency and efficacy on GHSR1a were determined by Luciferase reporter assay system employing a hGHSR-1a CHO-Creluc cell line. Binding was performed on membrane preparations from the same cell line in presence of [125I]-ghrelin.
In our preliminary investigation we identified the hit 2; the (+)-enantiomer showed an IC50 of 47 nM, whereas, the corresponding (−)-enantiomer was completely inactive (IC50 >10,000 nM). This big difference in activity was observed for several pairs of enantiomers
Conclusion
Following the HTS identification of new indolinone derivatives with GHSR-1a receptor antagonist properties, we began a comprehensive synthetic program to improve hits profile and establish a robust structure–activity relationship. Compound 14f was identified in vitro as a selective and potent lead (7 nM). Thus it was selected for in vivo further characterization. 14f inhibited the effect of GHSR1a agonist ghrelin on rats gastric emptying and food intake attesting its specificity. It also
General chemistry
Chemicals were purchased from commercial sources and used as supplied, unless otherwise indicated. All reactions were monitored by TLC on VWR glass plates precoated with silica gel 60 F254; spots were visualized through UV light at 220 nm or by treatment with 1% aq KMnO4. Products were purified by flash chromatography on Biotage® SNAP Cartridge KP-Sil or on Supelco VersaPak™ Cartridges, Spherical Silica (20–45 μm). Melting points have been measured with Buchi B-540 instrument. measurements
Acknowledgment
We thank Valentina Ghidelli, Elena Sabbadin, Donatella Picci, Giordano Lesma, Alessandra Silvani, Antonella Larovere, Raffaella Panigada, Massimo Meleri, Elisabetta Ottolina, Giovanni Boccardi, Andrea Bertario, Fausto Gesualdi, Silvia Milanesi, Philippe Ochsenbein, Marc Bianciotto, Laurence Fajas and Sabina Improta for their contribution to this work.
References and notes (36)
- et al.
Trends Endocrinol. Metab.
(2005) - et al.
Bioorg. Med. Chem. Lett.
(2010) - et al.
Regul. Pept.
(2005) - et al.
Pharma. Ther.
(2009) - et al.
Biol. Chem.
(1997) - et al.
Physiol. Rev.
(2005) - et al.
Endocrinol. Rev.
(2004) - et al.
Eur. J. Endocrinol.
(2004) - et al.
Nature
(1999) - et al.
J. Pharmacol. Exp. Ther.
(2002)