Original article
Rational design, synthesis and anti-proliferative properties of new CB2 selective cannabinoid receptor ligands: An investigation of the 1,8-naphthyridin-2(1H)-one scaffold

https://doi.org/10.1016/j.ejmech.2012.03.031Get rights and content

Abstract

CB2 receptor ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Using our previously described series of 1,8-naphthyridin-2(1H)-on-3-carboxamides as a lead class, several nitrogen heterocyclic derivatives, characterized by different central cores, were synthesized and tested for their affinity toward the human CB1 and CB2 cannabinoid receptors. The obtained results suggest that the new series of quinolin-2(1H)-on-3-carboxamides, 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides and 1,2-dihydro-2-oxopyridine-3-carboxamides represent novel scaffolds very suitable for the development of promising CB2 ligands. Furthermore, the newly synthesized CB2 ligands inhibit proliferation of several cancer cell lines. In particular, it was demonstrated that in DU-145 cell line these ligands exert a CB2-mediated anti-proliferative action and decrease the CB2 receptor expression levels.

Graphical abstract

Highlights

► In this study we report the synthesis of new CB2R ligands. ► Some ligands possess high affinity and CB2/CB1 selectivity. ► The most active ligands are effective on different tumor cell lines.

Introduction

Over the past 15 years a considerable number of studies have been conducted in order to understand the biological role of the endocannabinoid system (ECS) and its regulatory functions in health and disease. The ECS consists of the G-protein coupled cannabinoid receptors; the endogenous ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG); and the enzymes responsible for endocannabinoid biosynthesis, cellular uptake and metabolism [1], [2], [3].

To date, two distinct cannabinoid receptors, CB1 receptor (CB1R) and CB2 receptor (CB2R), have been identified in mammalian tissues and cloned. CB1R mediates the psychotropic actions of cannabis. It is mainly expressed in neurons of the CNS [4], and in dorsal root ganglion neurons [5], but there is also evidence for the expression of CB1R in non-neural tissues [6]. CB1R inhibits ongoing release of various neurotransmitters such as acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine, γ-aminobutyric acid and glutamate [7]. CB2R was originally described as being restricted to cells of immune origin [3], [8]. However, recent evidence points to a neuronal localization in some regions of the brain [9], and CB2R immunoreactivity has also been shown in activated microglia in affected regions of multiple sclerosis and amyotrophic lateral sclerosis post mortem human spinal cord [10]. Furthermore, various studies have shown that the activation of CB2R could block activation of microglia cells, but has little effect on the normal functioning of neurons within the CNS. Therefore, CB2R agonists may be potential alternatives to immunosuppressants currently used during neuroinflammation [11].

CB1R/CB2R agonists can of course produce adverse effects in patients, and many of these are probably caused by the activation of central CB1R, rather than of CB2R or peripheral CB1R. Several experimental data suggest that an interesting strategy for circumventing the unwanted consequences of cannabinoid CB1R activation, may be to target CB2R [12] The potential therapeutic targets for CB2R-selective agonists include neuropathic and inflammatory pain [13], [14] multiple sclerosis [15], [16], amyotrophic lateral sclerosis [17], [18], Huntington's disease [19], stroke [20], atherosclerosis [21], gastrointestinal inflammatory states [22], [23], chronic liver diseases [23], [24], and cancer [25]. Numerous pharmacological studies have shown that CB2R agonists might directly inhibit tumor growth in vitro and in animal models such as xenograft tumors chemically or genetically induced in mice. The responsible mechanisms may involve cytotoxic or cytostatic effects, apoptosis induction, anti-metastatic effect accompanied by inhibition of neo-angiogenesis, and tumor cell migration [26]. Therefore, the development of CB2R-selective agonists for the treatment of tumors represent an efficacious – and increasingly intriguing – alternative. These agents seem to discern between tumor cells and their non-transformed counterparts, displaying a tumor-selectivity not seen in common cytotoxic agents. The potential adverse effects are acceptable and lie well within the range of those induced by common anti-tumor drugs. Moreover, CB2R-selective agonists could be used in combination with other chemotherapeutic drugs to reduce dosages and exert a more potent clinical impact [27], [28].

Finally, there is evidence that CB2R expression is higher in some human cancer cells than in normal cells [29], and this would justify the development of a diagnostic test based on CB2R levels alone or in association with other recognized prognostics.

In a research program designed with the goal of obtaining CB2R-selective ligands [30], [31], [32], [33], and on the basis of docking studies developed using three-dimensional models of the two CBRs [34], we recently described the synthesis, pharmacological characterization and molecular modeling studies of a series of 1,8-naphthyridin-2(1H)-on-3-carboxamides of general structure A (see Fig. 1) [33]. The docking analysis suggested that the preservation of a good CB2/CB1 selectivity and the improvement of the CB2R affinity required a lipophilic central core connected with a nonaromatic carboxamide group in position 3 capable of interacting in the CB2R with the nonconserved residue F5.46(197). The central core would also need to be connected with a lipophilic substituent in position 1 with an H-bond acceptor atom, capable of interacting in the CB2R with the nonconserved S3.31(112). Some of the new tested compounds exhibited a subnanomolar CB2R affinity and a high selectivity over CB1R. Furthermore, the concentration-dependent inhibitory action on human basophils activation and the concentration-dependent decrease of cell viability in Jurkat cells shown by one of these derivatives strongly suggest that these compounds possess agonist properties at CB2R.

In the present study we evaluated the effects on the CBRs' affinity of the lipophilic central core properties. For this reason, using compounds A as a lead class, the derivatives of general structure BF, characterized by different central cores, were synthesized and tested for their affinity toward the human CB1R and CB2R (see Fig. 1).

On the basis of our previous modeling studies [33], [34], these new scaffolds should correctly orient the nonaromatic carboxamide group in position 3 capable of interacting in the CB2R with the nonconserved residue F5.46(197) and the lipophilic substituent in position 1 with the H-bond acceptor atom capable of interacting with S3.31(112). As shown in Fig. 2, all the derivatives possesses an aromatic feature and are able to orient the two substituents in the hypothesized way.

Finally, the newly synthesized CB2R ligands, showing the best binding profiles in the series, were also evaluated in vitro for their anti-proliferative activity against a large panel of human tumor-derived cell lines selected on the basis of previously published observation [35], [36].

Section snippets

Chemistry

The synthesis of the new compounds BF was accomplished as depicted in Scheme 1, Scheme 2, Scheme 3, Scheme 4. As shown in Scheme 1, the reaction of 2-nitrobenzaldehyde and diethylmalonate in acetic anhydride with NaHCO3 at 110 °C afforded the 2-nitrobenzylidene derivative 1 [37]. Reduction of 1 by iron powder in acetic acid yielded the ethyl quinolin-2(1H)-on-3-carboxylate 2. Heating of 2 with cyclohexylamine or cycloheptylamine at 120 °C in a sealed tube provided the corresponding carboxamide

CB1 and CB2 receptor affinity

The binding affinities (Ki values) of compounds BF was evaluated in competitive binding assays against [3H]CP-55,940 toward both human recombinant CB1R and CB2R expressed in CHO cells as previously described [33]. The results are reported in Table 1 with the Ki values of the previously reported 1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives A1A5 [33]. The Ki values of SR144528 [41] and JWH133 [42] as reference compounds are also included in Table 1.

The low solubility of D2 and D4

Conclusions

Starting from compounds 1,8-naphthyridin-2(1H)-on-3-carboxamides A, in order to evaluate the effects of the modification of the 1,8-naphthyridin-2(1H)-one ring system, new derivatives characterized by a different central scaffold were synthesized and tested on CB1R and CB2R.

The obtained results clearly suggest that the modification of the central scaffold have interesting effects on the CB2R affinity. Compounds B and D emerged as promising CB2R ligands; in particular compounds B5 and D5 are

Chemistry

Melting points were determined on a Kofler hot stage apparatus and are uncorrected. IR spectra in Nujol mulls were recorded on an ATI Mattson Genesis Series FTIR spectrometer. 1H NMR and 13C NMR spectra were recorded with a Bruker AC–200 spectrometer in δ units from TMS as an internal standard. Microwave-assisted reactions were run in a CEM microwave synthesizer. Mass spectra were performed with a Thermo Qest Finningan GCQ plus. Elemental analyses (C, H, N) were within ±0.4% of theoretical

Acknowledgment

This work was partially supported by National Interest Research Projects (PRIN 2008, Grant “Pharmacological characterization of novel direct and indirect agonists of endocannabinoid receptors and their potential use for the treatment of colorectal carcinoma”) and by FISM – Fondazione Italiana Sclerosi Multipla – Cod. 2009/R/3/C1. Authors from the CNR of Pozzuoli thank Marco Allarà for technical assistance. Authors thank Associazione Educazione e Ricerca Medica Salernitana (ERMES).

References (50)

  • T.J. Price et al.

    Neuroscience

    (2003)
  • K. Kim et al.

    Eur. J. Pharmacol.

    (2006)
  • P.L. Ferrarini et al.

    Bioorg. Med. Chem.

    (2004)
  • C. Manera et al.

    Bioorg. Med. Chem. Lett.

    (2007)
  • J.W. Huffman et al.

    Bioorg. Med. Chem.

    (1999)
  • J.W. Huffman et al.

    Bioorg. Med. Chem.

    (2005)
  • D. Piomelli

    Curr. Opin. Invest. Drugs

    (2005)
  • R.G. Pertwee

    Br. J Pharmacol.

    (2006)
  • V. Di Marzo et al.

    Nat. Rev. Drug Discov.

    (2004)
  • L.A. Matsuda et al.

    Nature

    (1990)
  • S. Tokanovic et al.

    Br. J. Pharmacol.

    (2007)
  • B. Szabo et al.

    Effects of cannabinoids on neurotransmission

  • S. Munro et al.

    Nature

    (1993)
  • E.S. Onaivi et al.

    Ann. N.Y. Acad. Sci.

    (2006)
  • Y. Yiangou et al.

    BMC Neurol.

    (2006)
  • P. Thored et al.

    Stroke

    (2007)
  • C. Manera et al.

    Mini Rev. Med. Chem.

    (2008)
  • J. Guindon et al.

    Br. J. Pharmacol.

    (2008)
  • G.T. Whiteside et al.

    Curr. Med. Chem.

    (2007)
  • R.G. Pertwee

    Mol. Neurobiol.

    (2007)
  • B.N. Dittel

    Br. J. Pharmacol.

    (2008)
  • J.L. Shoemaker et al.

    J. Neurochem.

    (2007)
  • O. Sagredo et al.

    Mol. Neurobiol.

    (2007)
  • P. Pacher et al.

    Br. J. Pharmacol.

    (2008)
  • F. Mach et al.

    Br. J. Pharmacol.

    (2008)
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