Sonja Hinz

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines: Development and Characterization of Adenosine A2B Receptor Antagonists and a New Radioligand with Subnanomolar Affinity and Subtype Specificity

A new series of 1-alkyl-8-(piperazine-1-sulfonyl)phenylxanthines was designed, synthesized, and characterized in radioligand binding and functional assays at A(2B) adenosine receptors. A(2B) antagonists with subnanomolar affinity and high selectivity were discovered. The most potent compounds were 1-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine-1-sulfonyl)phenyl)xanthine (24, PSB-09120, K(i) (human A(2B)) = 0.157 nM) and 8-(4-(4-(4-chlorobenzyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (17, PSB-0788, K(i) (human A(2B)) = 0.393 nM). Moreover, 8-(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (35, PSB-603) was developed as an A(2B)-specific antagonist exhibiting a K(i) value of 0.553 nM at the human A(2B) receptor and virtually no affinity for the human and rat A(1) and A(2A) and the human A(3) receptors up to a concentration of 10 microM. A tritiated form of the compound was prepared as a new radioligand and characterized in kinetic, saturation, and competition studies. It was shown to be a useful pharmacological tool for the selective labeling of human as well as rodent A(2B) receptors (K(D) human A(2B) 0.403 nM, mouse A(2B) 0.351 nM).

Dual Targeting of Adenosine A2A Receptors and Monoamine Oxidase B by 4H-3,1-Benzothiazin-4-ones

Blockade of A2A adenosine receptors (A2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinsons disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)acetamide (13), were identified as a novel class of potent MAO-B inhibitors (IC50 human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17, 27, and 28), showed high affinity and selectivity for A2AARs (Ki human A2AAR: 39.5-69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was N-(4-oxo-4H-3,1-benzothiazin-2-yl)-4-phenylbutanamide (17, Ki human A2A, 39.5 nM; IC50 human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A2AAR/MAO-B dual target approach in PD.

Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and alpha,beta-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB(1) and CB(2) receptors and were identified as new lead structures. In further GTPgammaS binding studies selected compounds were shown to be antagonists or inverse agonists.

Improving Potency, Selectivity, and Water Solubility of Adenosine A1 Receptor Antagonists: Xanthines Modified at Position 3 and Related Pyrimido[1,2,3‐cd]purinediones

The structure–activity relationships of xanthine derivatives related to the adenosine A1 receptor antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) and 1,3‐dipropyl‐8‐(3‐noradamantyl)xanthine (KW3902) were investigated by focusing on variations of the 3‐substituent. Aromatic residues were well tolerated by the A1 receptor in that position. A moderate effect of stereochemistry was found for the 3‐(1‐phenylethyl)‐substituted analogue of DPCPX (S>R) at A1 and A3 receptors, whereas the opposite stereoselectivity was observed at the A2 receptor subtypes. A 3‐hydroxypropyl substituent was found to be optimal for high A1 affinity and selectivity. The most potent compound of the present series was 1‐butyl‐3‐(3‐hydroxypropyl)‐8‐(3‐noradamantyl)xanthine (10 c), which exhibits a Ki value of 0.124 nM at rat, and 0.7 nM at human adenosine A1 receptors, combined with high selectivity (≫200‐fold) versus the other receptor subtypes. The similarly potent 8‐cyclopentyl‐3‐(3‐hydroxypropyl)‐1‐propylxanthine was converted into a water‐soluble phosphate prodrug, which may become a useful pharmacological tool for in vivo studies. 8‐Alkyl‐2‐(3‐noradamantyl)pyrimido[1,2,3‐cd]purine‐8,10‐diones, which can be envisaged as xanthine analogues with a fixed 3‐propyl substituent, were identified as a new class of potent, selective adenosine A1 receptor antagonists. For example, compound 14 (8‐butyl‐substituted) exhibits a Ki value of 13.8 nM at human A1 receptors. A selection of the most potent compounds was investigated in [35S]GTPγS binding assays and showed inverse agonistic activity. Their efficacy was generally lower than that of the full inverse agonist DPCPX, and depended on subtle structural changes. Some of the new compounds belong to the most potent and selective A1 antagonists described to date.

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Magnolia Extract, Magnolol, and Metabolites: Activation of Cannabinoid CB2 Receptors and Blockade of the Related GPR55.

The bark of Magnolia officinalis is used in Asian traditional medicine for the treatment of anxiety, sleeping disorders, and allergic diseases. We found that the extract and its main bioactive constituents, magnolol and honokiol, can activate cannabinoid (CB) receptors. In cAMP accumulation studies, magnolol behaved as a partial agonist (EC50 = 3.28 μM) with selectivity for the CB2 subtype, while honokiol was less potent showing full agonistic activity at CB1 and antagonistic properties at CB2. We subsequently synthesized the major metabolites of magnolol and found that tetrahydromagnolol (7) was 19-fold more potent than magnolol (EC50 CB2 = 0.170 μM) exhibiting high selectivity versus CB1. Additionally, 7 behaved as an antagonist at GPR55, a CB-related orphan receptor (K B = 13.3 μM, β-arrestin translocation assay). Magnolol and its metabolites may contribute to the biological activities of Magnolia extract via the observed mechanisms of action. Furthermore, the biphenylic compound magnolol provides a simple novel lead structure for the development of agonists for CB receptors and antagonists for the related GPR55.

The four cysteine residues in the second extracellular loop of the human adenosine A2B receptor: Role in ligand binding and receptor function

The adenosine A(2B) receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A(2B) receptor with dithiothreitol led to a 74-fold increase in the EC(50) value of the agonist NECA in cyclic AMP accumulation. In the C78(3.25)S and the C171(45.50)S mutant high-affinity binding of the A(2B) antagonist radioligand [(3)H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25-C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A(2B) receptors. In contrast, the C166(45.45)S and the C167(45.46)S mutant as well as the C166(45.45)S-C167(45.46)S double mutant behaved like the wild-type receptor, while in the C154(45.33)S mutant significant, although more subtle effects on cAMP accumulation were observed - decrease (BAY60-6583) or increase (NECA) - depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A(2A) receptor, which showed four disulfide bonds, the present data indicate that in the A(2B) receptor only the C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A(2B) receptor not involved in stabilization of the receptor structure may have other functions.

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors.

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A2A and A2B by exchanging the complete EL2 of the human A2BAR for the EL2 of the A2AAR. Furthermore, single amino acid residues (Asp148(45.27), Ser149(45.28), Thr151(45.30), Glu164(45.43), Ser165(45.44), and Val169(45.48)) in the EL2 of the A2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A2A-selective agonist CGS21680, while being completely inactive at A2BARs, showed high affinity for the mutant A2B(EL2-A2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A2B(EL2-A2A) as compared to the wt AR. Thus, the EL2 of the A2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.

BAY60-6583 Acts as a Partial Agonist at Adenosine A2B Receptors

BAY60-6583 [2-({6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-yl}sulfanyl)acetamide] is the most potent and selective adenosine A2B receptor (A2B AR) agonist known to date. Therefore, it has been widely used for in vitro and in vivo experiments. In the present study, we investigated the binding and functional properties of BAY60-6583 in various native and recombinant cell lines with different A2B AR expression levels. In cAMP accumulation and calcium mobilization assays, BAY60-6583 was found to be significantly less efficacious than adenosine or the adenosine derivative NECA. When it was tested in human embryonic kidney (HEK)293 cells, its efficacy correlated with the A2B expression level of the cells. In Jurkat T cells, BAY60-6583 antagonized the agonistic effect of NECA and adenosine as determined in cAMP accumulation assays. On the basis of these results, we conclude that BAY60-6583 acts as a partial agonist at adenosine A2B receptors. At high levels of the physiologic agonist adenosine, BAY60-6583 may act as an antagonist and block the effects of adenosine at A2B receptors. This has to be considered when applying the A2B-selective “agonist” BAY60-6583 in pharmacological studies, and previous research results may have to be reinterpreted.

Alkynyl-coumarinyl ethers as MAO-B inhibitors.

In this study, alkynyl-coumarinyl ethers were developed as inhibitors of human monoamine oxidase B (MAO-B). A series of 31 new, ether-connected coumarin derivatives was synthesized via hydroxycoumarins, whose phenolic group at position 6, 7 or 8 was converted by means of the Mitsunobu reaction. The majority of the final products were produced from primary alcohols with a terminal alkyne group. The inhibitors were optimized with respect to the structure of the alkynyloxy chain and its position at the fused benzene ring as well as the residue at position 3 of the pyran-2H-one part. A hex-5-ynyloxy chain at position 7 was found to be particular advantageous. Among the 7-hex-5-ynyloxy-coumarins, the 3-methoxycarbonyl derivative 36 was characterized as a dual-acting inhibitor with IC₅₀ values of less than 10 nM towards MAO-A and MAO-B, and the 3-(4-methoxy)phenyl derivative 44 was shown to combine strong anti-MAO-B potency (IC₅₀=3.0 nM) and selectivity for MAO-B over MAO-A (selectivity >3400-fold).