New potent and selective human adenosine A3 receptor antagonists

MEETING

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( Jean-Marie Boeynaems, Université Libre, Brussels, Belgium) in platelets are also available. Several other possibilities are also being actively investigated. References 1 Fredholm, B.B. et al. (1994) Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46, 143–156 2 Nicke, A. et al. (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J. 17, 3016–3028 3 Zimmermann, H. and Braun, N. (1999) Ecto-nucleotidases, molecular structures, catalytic properties, and functional roles in the nervous system. Prog. Brain Res. 120, 371–385

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4 Miras-Portugal, M.T. et al. (1999) Diadenosine polyphosphates, extracellular function and catabolism. Prog. Brain Res. 120, 397–409 5 Mulryan, K. et al. (2000) Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors. Nature 403, 86–89 6 Cressman, V.L. et al. (1999) Effect of loss of P2Y2 receptor gene expression on nucleotide regulation of murine epithelial Cl(-) transport. J. Biol. Chem. 274, 26461–26468 7 Fabre, J.E. et al. (1999) Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1deficient mice. Nat. Med. 5, 1199–1202 8 Leon, C. et al. (1999) Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y1 receptor-null mice. J. Clin. Invest 104, 1731–1737

Pier G. Baraldi and Pier Andrea Borea

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Adenosine regulates many physiological functions via specific cell membrane receptors. To date, four adenosine receptor subtypes have been cloned, A1, A2A, A2B and A3, each of which exhibits a unique tissue distribution, ligand affinity and signal transduction mechanism1. A1 and A2A receptors are activated by nanomolar concentrations of adenosine whereas A2B and A3 subtypes become activated only when adenosine levels increase into the micromolar range during periods of inflammation, hypoxia or ischemia2,3. Thus, the pathophysiological role of the A3 receptor might be very different from that of A1 and A2A subtypes, in that A3 receptors could act as endogenous regulators under conditions of more severe challenge4. To fully evaluate the pathophysiological role of these receptors, subtype-selective agonists and antagonists with high affinity are required. In the past ten years, great efforts by medicinal chemists and pharmacologists have been devoted to the design of potent and selective ligands for A1 and A2A receptors, whereas agonists

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Chemical name CGS21680: 2-[p-(2-carbonylethyl) phenylethylamino]-59-Nethylcarboxamidoadenosine

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New potent and selective human adenosine A3 receptor antagonists

P.A. Borea, Professor, Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, E-mail: [email protected] and P.G. Baraldi, Professor, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Via Fossato di Mortara 17–19, I-44100 Ferrara, Italy. E-mail: [email protected]

9 Ledent, C. et al. (1997) Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2A receptor. Nature 388, 674–678 10 Salvatore, C.A. et al. (2000) Disruption of the A3 adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J. Biol. Chem. 275, 4429–4434

and antagonists for the A3 subtype have, only recently, been identified. Although most of the available ligands are only moderately selective for human A3 receptors5, they have been useful probes to elucidate the function of this receptor subtype and have suggested a role for A3 receptors in inflammation6,7, neurodegeneration8, asthma9 and cardiac ischemia10. Indeed, A3 receptor antagonists have been hypothesized to act as potential anti-asthmatic11, anti-inflammatory6,7 or cerebroprotective agents8. Moreover, these antagonists have been reported to induce, at low micromolar concentrations, apoptotic effects in some tumor cell lines12. A3 receptor agonists appear to exert dual and opposite effects, either cytoprotective or cytotoxic, depending on the cell type and the level of receptor activation. At low concentrations (in the nanomolar range) A3 receptor agonists reduce hypoxic heart damage, protect blood eosinophils, HL-60 and U-937 cells from apoptosis, and promote protective mechanisms (e.g. BCLXLdependent reorganization of the cytoskeleton) in astroglial cultures10,12–14.

At micromolar concentrations, the same agonists instead tend to induce death of human eosinophils, cells of the lymphoid lineage, rat cardiac myocytes and cerebellar granule cells10,12–14. The different effects induced by A3 receptor agonists in these experimental models might simply depend on differential coupling to transduction mechanisms or expression of cellspecific factors. Thus, as a result of their ability to regulate cell survival, A3 receptors represent a promising therapeutic target in diseases in which excessive cell death is either undesirable, such as neurodegeneration, or desirable, such as cancer and inflammation. A major challenge of the past two years has been the synthesis of selective A3 receptor ligands (mainly antagonists) that are effective at subnanomolar concentrations and the preparation of a selective, high-affinity radiolabeled compound to help clarify the role of A3 receptors in humans. Here, the most recent results obtained in this field will be summarized. State of the art in the A3 receptor antagonist field

In the past few years, different classes of compounds have been reported to be A3 receptor antagonists. Eight classes of compounds with non-xanthine structures have been synthesized:

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CURRENT dihydropyridine and pyridine analogs15,16 , flavonoid17, isoquinoline18 and triazoloquinazoline derivatives19, and triazolonaphthiridine and thiazolopyrimidine analogs20 (Fig. 1). Recently, a new chemical entity, a 2-arylpyrazolo[3,4-c]quinoline derivative, was reported to display good affinity and selectivity for A3 receptors21. The Ferrara University approach

With respect to the triazoloquinazoline class of compounds, Jacobson and co-workers19 started from the experimental observation that CGS15943 possesses affinity for the human A3 receptor (Ki 5 14 nM). In fact, acylation of CGS15943 with a phenylacetyl group at the amino function at position 5 produced MRS1220, a potent but not highly selective A3 receptor antagonist. In the past few years, we have synthesized (using the general formula in Fig. 2a) more than 100 pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine derivatives that are potent and selective A2A receptor antagonists22. In addition, several N6-substituted phenyl carbamoyl adenosine-59-uronamides have been reported to act as potent agonists of the rat A3 receptor subtype23 (general formula in Fig. 2b). On this basis, we linked the amino group at position 5 of our A2A receptor antagonists (Fig. 2a) with the phenylcarbamoyl moiety that is typical of our A3 receptor agonists in an attempt to modulate the affinity and selectivity of these compounds at human A3 receptor subtypes. A series of new synthesized hybrid molecules, namely 5-N(substituted phenylcarbamoyl)amino-8substituted-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines, was obtained (Fig. 2c). The goal of this project was to evaluate if a moiety characteristic of A3 receptor agonists was able to confer A3 receptor affinity to molecules that are not capable of interacting significantly with this receptor subtype. The best substitution on the phenyl ring of the phenylcarbamoyl moiety was a methoxy in para position or a chlorine atom at meta position, as already demonstrated in the preparation of the A3 receptor agonist series.

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O

O Cl

Cl

H5C2S

CH(CH3)2 O

O

H5C2O2C Cl H3C

MRS1067 rA1 = 36% (10−4 M) rA2A = 19% (10−4 M) hA3 = 561 nM

N

H5C2

HN

N

N

Cl

MRS1334 rA1 = >100 µM rA2A = >100 µM hA3 = 2.69 nM

H3C

O

MRS1523 rA1 = 15 600 nM rA2A = 2050 nM rA3 = 113 nM hA3 = 18.9 nM

MRS1220 rA1 = 52.7 nM rA2A = 10.3 nM hA3 = 0.65 nM

O

N N

N

N

N S

CO2CH3

VUF5574 rA1 = 3870 nM rA2A = 22% (10−5 M) hA3 = 4 nM

NH

NH2

N

HN H3CO

O

NO2

H3CO

N

NN N

O N H

N

HN

O

nC3H7 CO2nC3H7

L249313 hA1 = 4000 nM hA2A = 19 000 nM hA3 = 13 nM

N

N N

O

L268605 hA1 = >10 000 nM hA2A = >10 000 nM hA3 = 18 nM

bA1 = 42% (10−5 nM) bA2A = 3% (10−5 nM) hA3 = 2.1 nM

Fig. 1. Chemical structures of the most representative adenosine A3 receptor antagonists. The affinities to rat (r), bovine (b) or human (h) adenosine receptor subtypes are also shown.

(b) R1

(a)

O H

NH2 N N R N

N

N N N

N

N

O

N H

N

N

R1 = 3-Cl, 4-OCH3

O H5C2HN

A2A receptor antagonist

O OH

OH

A3 receptor agonist

R1

(c) O H N N R

N N

N H N N N

O

A3 receptor antagonist

trends in Pharmacological Sciences

Fig. 2. Rational design for the synthesis of human adenosine A3 receptor antagonists (c) from the general formula for A2A receptor antagonists (a) and the general formula for A3 receptor agonists (b).

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CURRENT OCH3

O HN N

HN N O

N

OCH3

O

N H N N

N N

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N N

MRE3005F20 hA1 = 1026 nM hA2A = 1045 nM hA3 = 0.28 nM

N H N N O

N

[3H]MRE3008F20: a new selective and potent human A3 receptor antagonist radioligand

MRE3008F20 hA1 = 1197 nM hA2A = 141 nM hA3 = 0.29 nM trends in Pharmacological Sciences

Fig. 3. Chemical structures and binding affinities of the most representative adenosine receptor antagonists developed by Ferrara University. Abbreviation: h, human. OCH3

O HN N

3H

N N

N H N N N

[3H]MRE3008F20 hA1 = 1100 nM hA2A = 140 nM hA2B = 2100 nM hA3 = 0.85 nM

O

3H

so-called MRE series, MRE3005F20 and MRE3008F20, and the corresponding affinities towards human A1, A2A, A2B and A3 receptor subtypes.

trends in Pharmacological Sciences

[3H]MRE3008F20

Fig. 4. Chemical structure of and binding affinities towards adenosine A1, A2A, A2B and A3 receptors. Abbreviation: h, human.

Several ligands that belong to this class of compounds were shown to be the most potent and selective A3 receptor antagonists that have been synthesized to date24. Figure 3 shows the chemical structures of the two most representative ligands that belong to the

The lack of a radiolabeled selective A3 receptor antagonist has been the major drawback for an adequate characterization of this receptor subtype. Until now, the agonist [125I]ABMECA has been widely used as a high-affinity radioligand for A3 receptors4, even if it exhibits a moderate A3–A1 selectivity. Antagonists are generally considered to be more acceptable tools than agonists to characterize receptors because results obtained using agonists are complicated by different receptor states and celldependent effector coupling. Starting from the N 8-allyl antagonist, after reduction with tritium gas, we obtained [3H]MRE3008F20 (Fig. 4). This radioligand binds with subnanomolar affinity to the human A3 receptor and displays a selectivity of 1294-fold versus the human A1 receptor, 165-fold versus the human A2A receptor and more than 2400-fold versus the human A2B receptor. Interestingly, [3H]MRE3008F20 does not bind to rat A3 receptors. The saturation of [3H]MRE3008F20 binding to human A3 receptors is shown in Fig. 5.

300

200

200 F/B

[3H]MRE3008F20 (fmol mg protein−1)

300

100

100

0

0

2

4

6

B 0 0

2

4 6 8 [3H]MRE3008F20 concentration (nM)

10

trends in Pharmacological Sciences

Fig. 5. Saturation of [3H]MRE3008F20 binding to human adenosine A3 receptors. The Scatchard plot is shown in the inset. Kd 5 0.80 6 0.06 nM and Bmax 5 300 6 33 fmol mg protein21. Values are the mean of four separate experiments25.

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Concluding remarks

The A3 receptor subtype appears to be the target of drugs that have potential use in several important pathologies. The importance of A3 receptors in the pathophysiology of many disorders is demonstrated by the enormous increase in scientific interest in this field of research. Indeed, in the past few months, several studies on the synthesis of new ligands and the role of the A3 receptor in different diseases have been reported. In particular, the synthesis of 5-N(substituted phenylcarbamoyl)amino-8substituted-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines24 has permitted the tritiation, with consequent pharmacological characterization, of the first selective and high-affinity A3 receptor antagonist [3H]MRE3008F20. This radiolabeled compound could represent a key advance towards the characterization of A3 receptors in native tissues and cells that possess different subtypes of adenosine receptors, opening new and exciting perspectives in this important research area. References 1 Fredholm, B.B. et al. (1994) Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46, 143–156 2 Jacobson, K.A. et al. (1995) A3 adenosine receptors: design of selective ligands and therapeutic prospects. Drugs Future 20, 689–699 3 Olah, M.E. and Stiles, G.L. (1995) Adenosine receptor subtypes: characterization and therapeutic regulation. Annu. Rev. Pharmacol. Toxicol. 35, 581–606 4 Jacobson, K.A. (1998) Adenosine A3 receptors: novel ligands and paradoxical effects. Trends Pharmacol. Sci. 19, 184–191 5 Jacobson, K.A. et al. (1998) A3 adenosine receptors: protective vs damaging effects identified using novel agonists and antagonists. Drug Dev. Res. 45, 113–124 6 Broussas, M. et al. (1999) Inhibition of f MLPtriggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptors. J. Leukoc. Biol. 66, 495–501 7 Salvatore, C.A. et al. (2000) Disruption of the A3 adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J. Biol. Chem. 275, 4429–4434 8 Von Lubitz, D.K.J.E. (1999) Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur. J. Pharmacol. 371, 85–102 9 Forsythe, P. and Ennis, M. (1999) Adenosine mast cells and asthma. Inflam. Res. 48, 301–307 10 Liang, B.T. and Jacobson, K.A. (1998) A physiological role of the adenosine A3 receptor: sustained cardioprotection. Proc. Natl. Acad. Sci. U. S. A. 95, 6995–6999 11 Ezeamuzie, C.I. and Philips, E. (1999) Adenosine A3 receptors on human eosinophils mediate inhibition of degranulation and superoxide anion release. Br. J. Pharmacol. 127, 188–194

CURRENT 12 Yao, Y. et al. (1997) Adenosine A3 receptor agonist protect HL-60 and U-937 cells from apoptosis induced by A3 antagonists. Biochem. Biophys. Res. Comm. 232, 317–322. 13 Kohno, Y. et al. (1996) Activation of A3 adenosine receptors on human eosinophils elevates intracellular calcium. Blood 88, 3569–3574 14 Abbracchio, M.P. et al. (1997) The A3 adenosine receptor mediates cell spreading, reorganization of actin cytoskeleton, and distribution of Bcl-xL. Studies in human astroglioma cells. Biochem. Biophys. Res. Comm. 241, 297–304 15 Jiang, J.L. et al. (1997) Structure activity relationships of 4-phenylethynyl-6-phenyl1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists. J. Med. Chem. 40, 2596–2608 16 Li, A.N. et al. (1999) Synthesis, ComFA analysis and receptor docking of 3,5-diacyl2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists. J. Med. Chem. 42, 706–721 17 Ji, X.D. et al. (1996) Interactions of flavonoids and other phytochemicals with adenosine receptors. J. Med. Chem. 39, 781–788 18 Van Muijlwijk-Koezen, J.E. et al. (2000) Isoquinoline and quinazoline urea analogues as antagonists for the human adenosine A3 receptor. J. Med. Chem. 43, 2227–2238

19 Kim, J.C. et al. (1998) Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes. J. Med. Chem. 41, 2835–2845 20 Jacobson, M.A. et al. (1996) Novel selective non-xanthine selective A3 adenosine receptor antagonists. Drug Dev. Res. 37, 131 21 Colotta, V. et al. (2000) 1,2,4-Triazolo[4,3a]quinoxalin-1-one: a versatile tool for the synthesis of potent and selective adenosine receptor antagonists. J. Med. Chem. 43, 1158–1164 22 Baraldi, P.G. et al. (1998) Synthesis and biological activity of a new series of N6arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-59-N-ethyluronamide as A1 and A3 adenosine receptor agonists. J. Med. Chem. 41, 3174–3185 23 Baraldi, P.G. et al. (1996) Novel N 6(substituted-phenylcarbamoyl)adenosine-59uronamides as potent agonists for A3 adenosine receptors. J. Med. Chem. 39, 802–806 24 Baraldi, P.G. et al. (1999) Pyrazolo[4,3e]1,2,4-triazolo[1,5-c]-pyrimidine derivatives as highly potent and selective human A3 adenosine receptor antagonists. A possible template for adenosine receptor subtypes? J. Med. Chem. 42, 4473–4478

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25 Varani, K. et al. (2000) [3H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A3 adenosine receptors. Mol. Pharmacol. 57, 968–975

Chemical names ABMECA: 4-aminobenzyl-59-Nmethylcarboxamidoadenosine CGS15943: 5-amino-9-chloro-2-(2furyl)-1,2,4-triazolo[1,5-c]quinazoline MRE3005F20: 5-N-(4methoxyphenylcarbamoyl)amino-8ethyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine MRE3008F20: 5-N-(4methoxyphenylcarbamoyl)amino-8propyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine MRS1220: 5-phenylacetylamino-9chloro-2-(2-furyl)-1,2,4-triazolo[1,5-c]quinazoline

HIGHLIGHTS

Fatal attractors: theoretical approaches to tumor differentiation Every new technological advancement in molecular biology, such as the development of massively parallel geneexpression measurements, raises the same fundamental question in tumor biology: how deeply do we need to understand cancer in order to treat it? Until now, the theoretical and experimental approaches to malignancies have developed rather independently, their scientific interaction often being limited to polite curiosity from both sides. This estrangement has been due to an apparent lack of tangible problems that require the united efforts of experimental biology and theory. Experimental studies have not required abundant theorizing because they usually produced a relatively low amount of information that was traditionally analyzed by simple decision trees. Theoretical biology provided some interesting ideas about the nature

of genetic-network rearrangement during malignant transformation. However, these theoretical ideas needed experimental proof to be validated, and for a while remained untested. One such idea is that the altered geneexpression pattern in cancer results from the superimposed mechanisms of genetic instability (e.g. aneuploidy) and self-consistent gene-network regulation. During the initial steps of malignant transformation, the genetic network of a cell undergoes a major perturbation leading to an unstable state. From here, according to theory, the cell will search the ‘gene-expression space’ to find a stable yet dynamic state of the genetic network. This stable state has been termed ‘attractor’. The theory further postulates that the attractor will stabilize the gene-expression pattern of the cell because further perturbations will induce

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regulatory feedback mechanisms that will return the cell to this newly found stable state. However, this elegant theory has not, until now, had any experimental support. Now, an exciting paper by Perou et al.1 provides the first evidence that gene-network attractors might, in fact, exist. The authors have performed cDNA microarray measurements on a series of surgically removed breast tumors. Twenty tumors were sampled twice, before and after the patient underwent chemotherapy. In addition, gene-expression measurements from two primary tumors were also paired with their corresponding lymph-node metastases. Not surprisingly, the geneexpression patterns from different tumors were rather distinct. However, classification by hierarchical cluster analysis showed that tumor samples removed from the same patient, despite presumed profound perturbation by chemotherapy, were always much more similar to each other than to any other sample from another patient. The same findings applied to

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