Dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter

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Abstract

Preliminary screening of a minor, non-xanthine constituent of roasted coffee, 3,4-diferuloyl-1,5-quinolactone (DIFEQ), showed inhibition of the adenosine transporter at low micromolar concentration. DIFEQ is a neutral derivative of the chlorogenic acids, i.e. isomeric mono- and di-substituted coumaroyl-, caffeoyl-, and feruloyl-esters of quinic acid, formed in the roasting process of coffee. Displacement of the adenosine transporter antagonist [3H](S)-(nitrobenzyl)-6-thioinosine binding by DIFEQ in cultured U-937 cell preparations, expressing the human adenosine transporter protein (hENT1), showed a Ki of 0.96±0.13 μM. Extracts of regular and decaffeinated coffee showed binding activities equivalent to 30–40 mg DIFEQ per three cups of coffee. Acute administration of a high dose of DIFEQ (100 mg/kg i.p.) reduced open field locomotion in mice for 20 min in correlation with brain levels of DIFEQ. Both 3,4-dicaffeoyl-1,5-quinide and 3,4-dicoumaroyl-1,5-quinide, two close structural analogs of DIFEQ also present in roasted coffee, showed similar affinities for the adenosine transporter, while the corresponding 3- and 4-mono caffeoyl- and feruloyl-quinides were one to two orders of magnitudes less active. This suggests that 3,4-dicinnamoyl-1,5-quinides in coffee could have the potential to raise extra-cellular adenosine levels, thereby counteracting the stimulant effect of caffeine.

Introduction

The main constituents of coffee have been known for almost half a century Clifford, 1975, Herrmann, 1989, and particularly caffeine has been the subject of extensive studies. The stimulant effect of coffee is attributed to the pharmacological activity of caffeine, acting as an antagonist at adenosine receptors in brain (Fredholm et al., 1999). Although a central stimulant drug, caffeine is not generally considered to have abuse potential (Daly and Fredholm, 1998), because pure caffeine elicits a dose-dependent, subjective feeling of anxiety, even at low doses (Kaplan et al., 1997). Beverages made from roasted coffee, on the other hand, are able to elicit a feeling of well being that seems to increase with the strength of the brew, but not with its caffeine content (Quinlan et al., 2000). We and others have postulated that brewed and instant coffees contain pharmacologically active compounds, other than caffeine, in concentrations sufficient to cause significant effects with normal consumption of coffee. Kalsner (1977) reported that decaffeinated coffee contains a vasoconstrictive substance, later proposed but never identified as a muscarinic acetylcholine receptor agonist Tse, 1991, Tse, 1992. Voto and Schaal (1984) reported that regular and decaffeinated coffees produce bradycardia in human subjects, an effect that was not due to caffeine.

We suggest that pharmacologically active compounds may be found among derivatives of the chlorogenic acids. The chlorogenic acids, named after their green color reaction with ferrous chloride and exposure to air, are common to most plants (Clifford, 1975). Due to their relatively high abundance in coffee, these agents must be seriously considered when elucidating potential pharmacological effects of coffee intake. Green coffee beans can contain as much as 10% of dry weight of chlorogenic acids (Herrmann, 1989), i.e. five to eight times the average concentration of caffeine (Fredholm et al., 1999). Chlorogenic acids are mixtures of mono- and di-esters of various 4-hydroxycinnamic acids with the aliphatic alcohols of (−)-quinic acid, i.e. (R,R,S,R)-1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid, a sugar-like molecule (Clifford, 1975). Because both roasting and brewing of coffee cause extensive isomerisation of the quinic acids (Trugo and Macrae, 1984), no single such compound dominates in coffee, except for the most abundant 5-caffeoylquinic acid (formerly called 3-caffeoylquinic acid or chlorogenic acid), which constitutes 4–5% of green coffee beans (Herrmann, 1989).

The pharmacological effects of chlorogenic acids are mostly unknown. Recently, 5-caffeoylquinic acid was found to be a potent antioxidant agent in human erythrocytes (Lekse et al., 2001). Antioxidants in coffee protect against nitroso-induced genotoxicity in the mouse bone marrow model. In fact, the anti-genotoxic effect of 5-caffeoylquinic acid was synergistically enhanced when co-administered with brewed coffee (Abraham, 1996). 3,5-Dicaffeoylquinic acid is a potent inhibitor of human immunodeficiency virus (HIV-1) integrase, an enzyme required for infection with AIDS (Robinson et al., 1996). Brewed and instant coffee is unique in that the high temperature of the roasting process causes some chlorogenic acids in green coffee beans to lose a molecule of water to form an internal ester bond, thereby transforming up to half of the total amount into a mixture of quinolactones (quinides) that lack the carboxylic acid moiety (Hucke and Maier, 1985). Since the quinides are neutral compounds, they would be taken up in blood and brain even more readily than the original quinic acids (Olthof et al., 2001).

Boublik et al. (1983) reported that approximately one-fifth of the concentration contained in a cup of coffee displaced 50% of the binding of the opiate antagonist [3H]naloxone. The molecular entity that caused this activity has yet to be determined, but mass spectrometry on an active HPLC fraction suggested an isomer of feruloylquinide (Wynne et al., 1987). No other pharmacological data on the quinides are available, except that these compounds being α-hydroxy acid derivatives were found to have anti-urease activity useful for treating dermatological conditions Huynh-Ba et al., 1994, Yu and Van Scott, 1996. Because of stereochemical requirements, only chlorogenic acids that lack a cinnamoyl group in the 5-position can form γ-quinides, i.e. internal 1,5-lactones (Hanson, 1965). This excludes the most abundant 5-caffeoylquinic acid. 1-Cinnamoylquinic acids have never been found in coffee, leaving γ-quinides with cinnamoyl esters in the 3- or 4-positions as the only candidates. Di-cinnamoyl esters would be more likely than mono esters to have favorable properties for crossing the blood–brain barrier. For this reason, and for synthetic expediency, 3,4-diferuloyl-1,5-quinide (DIFEQ) was chosen as a suitable model compound to evaluate the potential psychopharmacological activities of such constituents of roasted coffee. We have synthesized DIFEQ from commercially available starting materials. Preliminary screening (NovaScreen) revealed that DIFEQ exhibits low micromolar affinity for the human equilibrium-sensitive (es) adenosine transporter. We have therefore investigated the binding characteristics of DIFEQ, its mono- and di-caffeoyl and di-coumaroyl analogs, and coffee extracts at the human adenosine transporter protein, and the acute behavioral effects of DIFEQ in mice. A preliminary report of this study was presented at the 19th International Conference on Coffee Science, Trieste, Italy (Martin et al., in press).

Section snippets

Drugs

A synthetic sample of DIFEQ was prepared in five steps from quinic acid and ferulic acid according to the methods of Wynne et al. (1986) as described by Huynh-Ba (1995). By using similar methods, 3-caffeoyl-, 3,4-dicaffeoyl- and 3,4-dicoumaroyl-1,5-quinides were also prepared (Huynh-Ba, 1995). 4-Caffeoyl-1,5-quinide was a gift from Dr. Huynh-Ba, Nestle. (S)-(4-Nitrobenzyl)-6-thioinosine (NBTI), 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 2-[4-(2-carboxyethyl)phenyl]ethylamino-5′-N

Synthesis

Synthesis of 10 g DIFEQ in 31% overall yield was accomplished from 15 g quinic acid and 25 g ferulic acid. Since the protected quinide was esterified in both the available 3- and 4-positions, the condensation reaction did not require the low temperature conditions of Huynh-Ba (1995), but was performed at ambient temperature. Melting point of DIFEQ was 132–134 °C after crystallization from a mixture of ethyl acetate and diisopropyl ether (1:2). The 1H and 13C NMR spectra of DIFEQ in DMSO-d6 were

Discussion

The present study shows that DIFEQ not only binds to the human equilibrative sensitive (es) adenosine transporter (hENT1), but inhibits the re-uptake of adenosine with a potency that is approximately three times higher than the antagonist binding affinity of caffeine at the human adenosine A2A receptor (Fredholm et al., 1999). In brain (Jennings et al., 2001) and periphery the adenosine transporter salvages extra-cellular adenosine for use in the biosynthesis of purine derivatives inside the

Acknowledgements

This work was funded by the Vanderbilt Institute for Coffee Studies. The generous support from the National Coffee Association of USA and Association of Coffee Producing Countries is gratefully acknowledged.

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