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A series of 6α- and 6β-substituted benztropines were synthesized. A marked enantioselectivity was observed for the 6β-methoxylated benztropines, the (1R)-isomers being more potent than the corresponding (1S) compounds. The racemic 6α-methoxy-3-(4‘,4‘ ‘-difluorodiphenylmethoxy)tropane (5g) was the most potent compound. It has been found that modifications at the 6-position of benztropine might reduce the DAT binding affinity, maintaining otherwise a significant dopamine uptake inhibitory activity. A reinvestigation of the absolute configuration of 6β-methoxytropinone proved the 6R configuration for the (+)-enantiomer.

Here we report the synthesis of 8-heterocycle-substituted xanthines as potent and selective A2B adenosine receptor antagonists. The structure−activity relationships (SAR) of the xanthines synthesized in binding to recombinant human A2B adenosine receptors (ARs) in HEK-293 cells (HEK-A2B) and at other AR subtypes were explored. The synthesized compounds showed A2B adenosine receptor affinity in the nanomolar range and good levels of selectivity evaluated in radioligand binding assays at human (h) A1, A2A, A2B, and A3 ARs. We introduced several heterocycles, such as pyrazole, isoxazole, pyridine, and pyridazine, at the 8-position of the xanthine nucleus and we have also investigated different spacers (substituted acetamide, oxyacetamide, and urea moieties) on the heterocycle introduced. Various groups at the 3- and 4-positions of phenylacetamide moiety were studied. This study allowed us to identify the derivatives 2-(3,4-dimethoxyphenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl]acetamide (29b, MRE2028F20) [Ki(hA2B) = 38 nM, Ki(hA1,hA2A,hA3) >1000 nM], N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (62b, MRE2029F20) [Ki(hA2B) = 5.5 nM, Ki(hA1,hA2A,hA3) > 1000 nM], and N-(3,4-dimethoxyphenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (72b, MRE2030F20) [Ki(hA2B = 12 nM, Ki(hA1,hA2A, hA3) > 1000 nM], which showed high affinity at the A2B receptor subtype and very good selectivity vs the other ARs. Substitution of the acetamide with an urea moiety afforded bioisosteric xanthines with good affinity and selectivity comparable to the acetamide derivatives. Substitution at the para-position of a 4-benzyloxy group of the phenylacetamido chain enhanced affinity at the A2B receptor [compound 30b (Ki(hA2B) = 13 nM) vs compound 21b (Ki(hA2B = 56 nM)] but did not favor selectivity. The derivatives with higher affinity at human A2B AR proved to be antagonists, in the cyclic AMP assay, capable of inhibiting the stimulatory effect of NECA (100 nM) with IC50 values in the nanomolar range, a trend similar to that observed in the binding assay (62b, IC50 = 38 nM; 72b, IC50 = 46 nM). In conclusion, the 8-pyrazolo-1,3-dipropyl-1H-purine-2,6-dione derivatives described herein represent a new family of selective antagonists for the adenosine A2B receptor.

A new series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines bearing various substituents at both the N5-pyrimidinyl and N8-pyrazolyl positions have been synthesized, and their binding affinities at the four human adenosine receptor subtypes (hA1, hA2A, hA2B, and hA3) have been evaluated. All the described compounds contain arylacetyl moieties at the N5 position and arylalkyl substituents at the N8 position. Surprisingly, all the compounds present their most potent affinities at the hA2B adenosine receptor with a range of selectivities against the other subtypes. When bulky groups are present simultaneously at the N5 and N8 positions (e.g., compound 9), the best selectivity for the hA2B receptor was observed (Ki(hA1) = 1100 nM; Ki(hA2A) = 800 nM; Ki(hA2B) = 20 nM; Ki(hA3) = 300 nM, Ki(hA1/A2B) = 55, Ki(hA2A/A2B) = 40, Ki(hA3/hA2B) = 15). To understand the molecular significance of these results, we compared the putative TM (transmembrane) binding motif of compound 9 on both hA2B and hA3 receptors. From our docking studies, compound 9 fits neatly inside the TM region of the hA2B receptor but not in the corresponding hA3 region, illustrating significant differences between the two subtypes. The study herein presented permits an understanding of why the bioisosteric replacement of an −NH, present in previously reported hA3 receptor antagonists, with a −CH2 group at the N5 position induces such large differences in hA2B/hA3 affinity. In the molecular structure of the hA3 receptor, two residues, Ser243 (TM6) and Ser271 (TM7), create a hydrophilic region, which seems to permit a better accommodation of the phenylurea series into this putative hA3 binding site than the phenylacetyl series.

In the past few years, our group has been involved in the development of A2A and A3 adenosine receptor antagonists which led to the synthesis of SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, 61), potent and very selective at the A2A receptor subtype, and N⁸-substituted-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N⁵-urea or amide (MRE series, b), very selective at the human A3 adenosine receptor subtype. We now describe a large series of C⁹- and C²-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines to represent an extension of structure−activity relationship work on this class of tricyclic compounds. The introduction of a substituent at 9 position of the tricyclic antagonistic structure led to retention of receptor affinity but a loss of selectivity in respect to the lead compounds b, N⁸-substituted-pirazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N⁵-urea or -amide. The substitution of the furanyl moiety of compound 61, necessary for receptor binding, with a phenyl or a substituted aromatic ring (compounds 5a−d, 6−8), caused a complete loss of the affinity at all the adenosine receptor subtypes, demonstrating that the furanyl ring is a necessary structural element to guarantee interaction with the adenosine receptor surface. The introduction of an ethoxy group at the ortho position of the aromatic ring to mimic the oxygen of the furan (compound 5c, 5-amino-7-(2-phenylethyl)-2-(2-ethoxyphenyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) did not enhance affinity. The introduction of the cycloaminomethyl function by Mannich reaction at the 5‘ position of the furanyl ring of 61 and the C⁹-substituted compound 41 (5-amino-8-methyl-9-methylsulfanyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) resulted in complete water solubility but a loss of receptor affinity. We can conclude that modifications or substitutions at the furanyl ring are not allowed and the introduction of a substituent at the 9-position of the core pyrazolo-triazolo-pyrimidine structure caused a severe loss of selectivity, probably due to an increased steric hindrance of the radical introduced.

A new series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcarbamoyl moieties at the N5 position, being highly potent and selective human A3 adenosine receptor antagonists, is described. The compounds represent an extension and an improvement of our previous work on this class of compounds (J. Med. Chem. 1999, 42, 4473−4478; J. Med. Chem. 2000, 43, 4768−4780). All the synthesized compounds showed A3 adenosine receptor affinity in the subnanomolar range and high levels of selectivity in radioligand binding assays at the human A1, A2A, A2B, and A3 adenosine receptors. In particular, the effect of the substitution and its position on the phenyl ring have been studied. From binding data, it is evident that the unsubstituted derivatives on the phenyl ring (e.g., compound 59, hA3 = 0.16 nM, hA1/hA3 = 3713, hA2A/hA3 = 2381, hA2B/hA3 = 1388) showed the best profile in terms of affinity and selectivity at the human A3 adenosine receptors. The introduction of a sulfonic acid moiety at the para position on the phenyl ring was attempted in order to design water soluble derivatives. However, this substitution led to a dramatic decrease of affinity at all four adenosine receptor subtypes. A computer-generated model of the human A3 receptor was built and analyzed to better interpret these results, demonstrating that steric control, in particular at the para position on the phenyl ring, plays a fundamental role in the receptor interaction. Some of the synthesized compounds proved to be full antagonists in a specific functional model, where the inhibition of cAMP-generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A3 receptor with IC50 values in the nanomolar range, with a statistically significative linear relationship with the binding data.

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A3 adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768−4780). A derivative having a methyl group at the N⁸ pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N⁵ position, displayed a Ki value at the hA3 receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(β-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA3 receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N⁸ pyrazole nitrogen. The bis-(β-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A3 receptor was built and analyzed to help interpret these results. The model of the A3 transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.