In vitro and in vivo studies of 6,8-(diaryl)imidazo[1,2-a]pyrazin-3(7H)-ones as new antioxidants

https://doi.org/10.1016/j.bmc.2009.05.025Get rights and content

Abstract

A series of 5-aryl and 3,5-diaryl-2-amino-1,4-pyrazines and the derived imidazopyrazinones has been synthesized to study the chemical oxidative degradation of the bicyclic systems in vitro. Imidazopyrazinones mainly degraded following two independent pathways producing their precursors, namely aminopyrazines, and the corresponding amidopyrazines, respectively. Despite the fact that there is no influence of the substituent of the 3-aryl group on the ratio of the products aminopyrazine/amidopyrazine, diarylimidazopyrazinones and diarylaminopyrazines are good antioxidants in vivo. They protected against microvascular damages in ischemia/reperfusion with similar efficiencies.

Graphical abstract

A series of novel 3,5-diaryl-2-amino-1,4-pyrazine derivatives and related imidazo[1,2-a]pyrazin-3(7H)-ones has been synthesized to study the in vitro chemical oxidative degradation pathways of the hetero-bicycles. The antioxidant potential of those compounds has been evaluated in vivo.

  1. Download : Download full-size image

Introduction

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in aerobic organisms as part of the normal physiological and metabolic processes. They are very important mediators of cell injury or death due to the damages they can inflict if they are produced in excessive concentrations or in wrong locations. The damages that ROS/RNS cause, essentially on biological macromolecules (membrane lipids, proteins, nucleic acids, …), are directly or indirectly implicated in the pathogenesis of various disorders such as cardiovascular diseases, reperfusion injury, Alzheimer’s and other neurodegenerative diseases, cancer development and progression, inflammation as well as in the aging process.1, 2, 3, 4, 5, 6, 7, 8, 9

Therefore the interest for the protective role of antioxidants in medicine has been growing over the last 15 years. Antioxidants are considered as potential drugs due to their ability to reduce or inhibit the free radical reactions initiated by ROS/RNS. Currently available radical scavengers are structurally related to natural antioxidants (vitamin A/β-carotene, vitamin C, vitamin E, green tea extracts, flavonoids, …) and to industrial compounds such as highly hindered ortho-substituted phenols.10, 11, 12, 13, 14

Coelenterazine 1a (Scheme 1) and more generally imidazo[1,2-a]pyrazin-3(7H)-ones (imidazopyrazinones) are bioluminescent substrates of luciferases,15, 16, 17 naturally designed to react with oxygen in light-producing reactions. Numerous studies have been devoted to the oxidation mechanism of coelenterazine and related derivatives 1, essentially in the context of colored light production via an efficient biochemical process and the development of analytical tools in biochemistry.18, 19, 20, 21, 22, 23 A few years ago, our research group considered coelenterazine and other imidazopyrazinones 1 as potential leads in the discovery of novel antioxidants for therapeutic use.24 We showed that imidazopyrazinones have good antioxidant properties as they are highly reactive with radical anion superoxide, lipid radicals, nitrofurantoin-derived radicals and peroxynitrite; LDL protection by compounds 1 has been further demonstrated in vitro. Finally, cellular protection against oxidative stress and UV-irradiation damages has been observed, in the presence of compounds 1, on various cell lines (human keratinocytes, rat hepatocytes, rat neuronal cells, fish erythrocytes).24 During these previous studies, we found that the chemical precursors of imidazopyrazinones synthesis, namely the 2-amino-1,4-pyrazine derivatives 3, are also endowed with remarkable antioxidant properties providing R3 is a para-phenol (or a catechol) moiety.25, 26

The main metabolite of luciferase-catalyzed oxidation of coelenterazine (1a) is coelenteramide (2a) which is initially produced in the excited state; this species emits light during decay (Scheme 1).27, 28 Similarly, numerous synthetic analogs 1 display bioluminescence and chemiluminescence properties under enzyme (luciferase) processing or chemical oxidation conditions.15 The so-produced amides 2, formally derived from aminopyrazines 3, were however inactive in antioxidant tests.24, 25

In the course of chemical and air oxidation studies of coelenterazine (1a), we observed the formation of coelenteramine (3a), in quite significant amounts, together with the expected coelenteramide (2a). This interesting observation was general: the oxidative degradation of imidazopyrazinones 1 led always to both amides 2 and aminopyrazines 3. Thus besides the luminescence pathway of coelenterazine (1) decomposition giving an inactive compound (2), another route can take place which produces in situ a daughter-antioxidant (3) (Scheme 1).

Aiming to optimize this unique antioxidant system acting in cascade (3 prolonging the activity of its mother-compound 1), we studied the outcome of the five-membered ring R2 substituent and the R1 substituents effect on the 2:3 product ratios formed when processing imidazopyrazinones 1 under aerobic conditions in the laboratory. Here we report the HPLC analysis of a model reaction (R1 = H, R2 = Ph), the synthesis of novel derivatives 1 (R2 = Me) with R1 being various para-substituted phenyl groups, their in vitro oxidative decomposition pathways and their in vivo biological activity in the ‘hamster cheek pouch’ assay.

Section snippets

Possible pathways involved in imidazopyrazinone oxidation (Scheme 1)

Results from the literature suggest that bioluminescence and chemiluminescence of imidazopyrazinones (1) might involve a similar mechanism for the production of amides (2) and light.15 However, the formation of aminopyrazines (3) is occasionally mentioned.29, 30 The main questions concern the outcome of the imidazole moiety (which bears the substituent R2) from precursors 1 and the production of 2 and 3 via parallel routes or via consecutive reactions (i.e., formation of 2 and subsequent

Conclusion

Amongst the various strategies of drug discovery, we became interested in the ‘non-natural natural products’ approach which, after about 25 years of ‘combinatorial chemistry technologies’, is nowadays reconsidered as very promising by distinguished scientists in the field.45

Coelenterazine (a marine luciferine, 1a) was our natural ‘lead’ compound for the discovery of potential antioxidants, due to its intrinsic capacity of quenching oxygen and ROS/RNS. Two pharmacophores were identified from our

Chemistry: general methods

1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Varian Gemini-300 spectrometer. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a BRUCKER AM-500 spectrometer. 19F (282 MHz) NMR spectra were recorded on a BRUCKER AVANCE-300 spectrometer. The attributions were established by selective decoupling experiments. Chemical shifts are reported as δ values (in ppm) downfield from TMS for 1H and 13C or from CFCl3 for 19F. The mass spectra (FAB or APCI modes) were obtained using a

Acknowledgements

The authors thank the F.R.S.-FNRS (Belgium), FRIA (Belgium) and IdRS (France) for the financial support. J.M-B. is senior research associate of F.R.S.-FNRS (Fonds de la Recherche Scientifique). F.D.W. is holding a PhD bursary of FRIA (Fonds pour la Formation à la Recherche dans l’Industrie et l’Agriculture).

References and notes (46)

  • P.A. Morrissey et al.

    Int. Dairy J.

    (1998)
  • J.P. Kehrer

    Toxicology

    (2000)
  • C-M. Andersson et al.

    Adv. Drug Res.

    (1996)
  • K. Teranishi

    Bioorg. Chem.

    (2007)
  • C. Wu et al.

    Tetrahedron Lett.

    (2006)
  • M. Kuse et al.

    Tetrahedron

    (2000)
  • K. Kotarsky et al.

    Anal. Biochem.

    (2003)
  • J. Alvarez et al.

    Cell Calcium

    (2002)
  • K. Teranishi et al.

    Anal. Biochem.

    (1997)
  • A. Daiber et al.

    Free Radical Biol. Med.

    (2004)
  • Y. Takahashi et al.

    Tetrahedron Lett.

    (2006)
  • H. Kondo et al.

    Tetrahedron Lett.

    (2005)
  • K. Usami et al.

    Tetrahedron

    (1996)
  • O. Shimomura et al.

    Biochem. Biophys. Res. Commun.

    (1971)
  • I. Devillers et al.

    Bioorg. Med. Chem. Lett.

    (2001)
  • T. Hirano et al.

    Tetrahedron

    (1993)
  • J.-F. Cavalier et al.

    Bioorg. Med. Chem.

    (2001)
  • T. Goto et al.

    Tetrahedron Lett.

    (1969)
  • K. Teranishi et al.

    Carbohydr. Res.

    (2003)
  • M. Adamczyk et al.

    Tetrahedron

    (2003)
  • A. Arrault et al.

    Bioorg. Med. Chem. Lett.

    (2003)
  • Cited by (9)

    • Single-molecule chemiluminescent photosensitizer for a self-activating and tumor-selective photodynamic therapy of cancer

      2019, European Journal of Medicinal Chemistry
      Citation Excerpt :

      Given this, we have performed in vitro toxicity assays in both tumor and “healthy” cell lines, to provide proof-of-concept of the ability of Br-Cla to act as a tumor-selective and self-activating PS for PDT. While Coelenterazine and related imidazopyrazinones can undergo chemiluminescence in aprotic solvents when triggered just by molecular oxygen [29,30,51], in aqueous solutions and biologic media that is only possible in the presence of superoxide anion [23–25,51,52]. Thus, given that tumor cell lines overexpress this radical [26,27], the imidazopyrazinone – superoxide anion reaction should provide to Br-Cla intrinsic tumor-selectivity.

    • Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines

      2013, Protein Expression and Purification
      Citation Excerpt :

      On the other hand, under the same conditions or at alkaline conditions at pH 9, coelenteramide was not hydrolyzed to coelenteramine (data not shown). From these results, the process of the decomposition of coelenterazine to coelenteramide and coelenteramine in aqueous solutions was proposed as shown in Fig. 4, which was similar to what has been proposed using a model compound of coelenterazine [33]. This degradation process could not produce the significant light emission, because the light-emitting species of coelenteramide was not stabilized.

    • Chemistry around imidazopyrazine and ibuprofen: Discovery of novel fatty acid amide hydrolase (FAAH) inhibitors

      2010, European Journal of Medicinal Chemistry
      Citation Excerpt :

      In the course of previous works dedicated to antioxidant compounds belonging to the coelenteramine and coelenterazine families [3,4], we have disclosed an oxidative degradation pathway of imidazopyrazinones A (mother antioxidant) producing a 2-amino-1,4-pyrazine C (daughter antioxidant) and a carboxylic acid D. This route occurs in parallel to the oxidative degradation into 2-amido-1,4-pyrazine B producing light (chemiluminescence pathway) (Fig. 1) [5]. This offers a unique opportunity to possibly combine in the same molecule A an antioxidant activity with an anti-inflammatory activity by choosing adequately the nature of the substituent R. Indeed, upon oxidation, this fragment will be released as the R–CO2H molecule D and a lot of nonsteroidal anti-inflammatory drugs (NSAID) are in fact carboxylic acids [6].

    View all citing articles on Scopus

    Accidentally deceased on January 19, 2004.

    View full text