Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.


Introduction
Adenosine is an endogenous nucleoside that mediates a wide range of physiological responses through interaction with specific adenosine receptors (ARs), which are G-proteincoupled receptors (GPCRs) comprising the characteristic seven transmembrane domains connected by three extracellular and three intracellular loops. There are four basic types of ARs that have been cloned and pharmacologically characterized, namely, A 1 , A 2A , A 2B , and A 3 ARs [1]. Each of these ARs is associated with its own distinct biochemical pathways. Typically, the activation of A 1 and A 3 receptors mediates adenylyl cyclase inhibition through an interaction with G i protein, followed by a subsequent decrease in the level of cyclic adenosine monophosphate (cAMP); conversely, the A 2A and A 2B receptors stimulate the adenylyl cyclase activity via the G s protein thereby increasing the level of cAMP [2]. In addition, other signaling pathways involving phospholipases C and D, and Ca 2+ and mitogen-activated protein kinases (MAPK) have also been described [1]. Pharmacologically, the inhibition of A 1 receptors has led to implications in the renal system disorders through regulation of diuresis and neurological disorders such as Alzheimer's disease [3,4]; on the other hand, A 3 receptor antagonists are primarily related to the treatment of glaucoma, renal protection, inflammatory disorders like asthma, as well as cancer [5][6][7]. Studies have also found that A 2A receptor antagonists can reverse Parkinsonian motor deficits in preclinical models of Parkinson's disease, and they do so without inducing or exacerbating dyskinesias in nonhuman primate models [8,9]. As for the A 2B receptor, its antagonists seem to be suitable for the treatment of certain forms of inflammatory processes such as asthma via modulation of mast cell degranulation [10,11].

International Journal of Medicinal Chemistry
In the last 15 years, intensive efforts in medicinal chemistry to design and synthesize new AR antagonists have led to the discovery of potent and selective ligands (with either agonistic or antagonistic properties) for the A 1 , A 2A , A 2B , and A 3 ARs. These new derivatives have resulted in a better understanding of the pathophysiological role of these receptors; more precisely, among the AR antagonists, several different types of xanthine-derived and nonxanthine-based polyheterocyclic structures have been identified as potent AR antagonists. Some of them are shown to possess good affinity exclusively towards a particular AR subtype with concomitant improvements in their selectivity profiles. On the other hand, some scaffolds demonstrate good binding affinity across more than one AR subtype, with relatively lower selectivity profile. Among these diverse classes of compounds, nonxanthine pyrazolo derivatives have been reported to show good potency towards ARs, together with a broad range of selectivity. The aim of this review is to briefly summarize the structure-activity relationship profiles of various nonxanthine derivatives containing the pyrazole moiety as AR antagonists to the A 1 , A 2A , A 2B , and A 3 receptor subtypes.

Pyrazolo Derivatives as Potent AR Antagonists
In general, nonxanthine AR antagonists are represented by polyheterocyclic derivatives which are categorized as monocyclic, bicyclic, or tricyclic structures [12]. In this review, we emphasized the structure-activity relationships for some of the representative nonxanthine pyrazolo derivatives (i.e., derivatives with a fused pyrazole ring in their respective core nuclei), which have been identified as potent AR  [13], who identified a compound named 8FB-PTP (1 in Figure 1), which demonstrated good binding to the A 2A AR but lacked selectivity towards the A 1 receptor subtype. Structure-affinity relationship studies showed that the free amino group at 5-position and the effect of the substituents on the pyrazole ring seemed important for both high affinity and selectivity for the A 2A AR subtype. From further studies, substitutions at the 7-position were shown to improve the selectivity for the A 2A receptor while the same substitutions at the 8-position increased affinity to the A 1 and A 2A receptors with low levels of selectivity, as indicated by the N 7 -n-butyl (2) and the N 8 -n-butyl (3) derivatives [14,15]. This again indicated that the presence of a chain (preferably a long (ar)alkyl one) at the N 7 position seemed essential for both affinity and selectivity for the A 2A receptors.
In fact, two selected compounds named SCH 63390 (4) and SCH 58261 (5) proved to be the most potent and selective A 2A AR antagonists ever reported, both in rat and human models [15][16][17]. The latter was further developed into an A 2A antagonist radioligand, [ 3 H]SCH 58261 (5a) with a K D value of about 1 nM. Further studies have suggested that it could be a useful tool for characterization of A 2A receptor subtypes in platelets, autoradiography assays, and labeling of striatal A 2A receptors for studying A 2A receptor occupancy of various antagonists [18,50,51]. Nevertheless, this class of compounds presents a significant problem because of poor water solubility. To overcome this drawback, several polar moieties on the side chain of the pyrazole nucleus have been introduced. In particular, the introduction of a hydroxyl function at the para position of the phenyl ring of compounds (4) and (5) 7), which not only showed a better hydrophilic character but also a significant increase of both affinity and selectivity for the A 2A AR subtype, suggesting that most probably, a hydrogen bond is involved in receptor recognition via this part of the ligand [16].
To understand the nature of such a hypothetical hydrogen bond, compound SCH 442416 (8) was synthesized. This derivative showed even higher affinity and selectivity for the A 2A receptor, thus representing a suitable candidate for positron emission tomography (PET) studies in its 11 Clabeled form [19]. Moreover, it was developed into novel fluorescent tracer MRS5346 (9), which was conjugated to the fluorescent dye Alexa Fluor-488. It has a K D value of 16.5 ± 4.7 nM and could be used in fluorescence polarization competition binding experiments as well as high-throughput screening [20]. On top of that, this SCH 442416 derivative also confirmed the role of a hydrogen bond via the pyrazolo side chain. Nonetheless, the introduction of oxygenated groups could not be considered sufficient to confer water solubility. Hence, carboxylic (10) and sulfonic (11) moieties were introduced, and such structural modifications (the sulfonic moiety in particular) improved water solubility. However, in some cases, a loss of affinity with respect to reference compounds (6, 7) for the A 2A AR was observed. On the other hand, the introduction of an amino group at the para position of the phenyl ring (12) improved both affinity and selectivity towards the A 2A receptor, although with low water solubility [17]. Despite these observations, it was found that the N 7 derivative (such as compound 5) was totally inactive to the human A 2B and A 3 receptors. The N 8 regioisomer (13), however, showed a slight affinity profile for these two receptor subtypes [21,22].
A recent series of pyrazolo-triazolo-pyrimidine derivatives was obtained by modifying the phenylethyl substituent of 5 with substituted phenylpiperazine ethyl groups [23]. Introduction of fluorine atoms in the phenyl ring (14) enhanced the affinity to subnanomolar values and   Figure 1, 16 in Figure 2) possessed high affinity to the human A 2B receptors but completely lacked selectivity. Subsequently, introduction of a polar γamino-butyryl amide (17) at the N 5 -position decreased affinity towards the A 2B receptors but was found to be slightly selective against the A 2A subtype [24]. An improvement of this class of compounds was further achieved by an optimized pattern of substitutions at the N 5 -and N 8positions. In fact, in parallel studies on human A 3 receptor antagonists (to be elaborated in the following section), it was observed that replacement of the phenylcarbamoyl moiety    at the N 5 -position with a phenylacetyl group (compound 18) produced a decrease in affinity to the human A 3 AR and a retention or improvement towards the A 2B subtype. A combination of a naphthyl acetyl moiety at the N 5 -position and a phenyl propyl group (characteristic of A 2A antagonists) at the N 8 position led to a compound (19), which was found to be quite potent and selective towards the A 2B ARs [25]. These findings indicated that bulky substituents at both the N 5 -and N 8 -positions could lead to potent and selective A 2B AR antagonists, thus suggesting the presence of a larger pocket in the receptor binding site.

A 3 AR Antagonists.
The optimization approach to obtain potent A 3 AR antagonists in the series of pyrazolotriazolo-pyrimidines was a hybrid molecule between a human A 2A receptor antagonist [15,16] and an agonist of the A 3 subtype [52,53]. The tricyclic scaffold of a known human A 2A antagonist was substituted at the N 5 position with an aryl carbamoyl moiety. Specifically, this para-methoxyphenyl was demonstrated to be optimal for A 3 affinity when introduced at the N 6 -position of the A 3 agonist NECA (as represented by compound 20; Figure 3). Such rational design led to compound 21, which is one of the most potent and selective human A 3 AR antagonists [21]. Subsequent collation of binding data and molecular modeling studies indicated that small substituents, such as a methyl group at the N 8 -position, the phenyl ring on the N 5 -carbamoyl moiety, and a furyl ring at 2-position, were important (although not crucial, as indicated in the following paragraphs) for A 3 affinity (e.g., compound 23) [22,[27][28][29]. Only small substituents at the para position of the phenyl ring, including fluoro (F), chloro (Cl), and methoxy (OCH 3 ) were tolerated. At the meta-position, only hydrogen was tolerated, while the ortho-position could be substituted by a chlorine atom. Introduction of an allyl chain at N 8 -position, followed by reduction with tritium afforded [ 3 H]MRE-3008-F20 (22), which was the first selective and tritiated human A 3 receptor antagonist radioligand [26]. It showed a K D value of 0.8 nM and exhibited ca. 25% of nonspecific binding at that concentration. Since its discovery, it has been used for the identification of A 3 receptors on various cells, including Jurkat T cells, HL60 cells, and human neutrophils [54,55]. Later, the N 5 -phenyl ring of the tricyclic scaffold was substituted with a pyridinium salt, as represented by compound 24, which not only showed good solubility (15 mM) but also significantly improved hA 3 affinity [30]. In previous studies, substitution of the N 5pyridine moiety with various N 5 -heteroaryl rings resulted in a general loss of hA 3 affinity and selectivity [28]. Substitution at position C 2 of the tricyclic system has not been deeply explored, being essentially limited to a furyl group. The furan ring had been considered to be an essential structural requirement for the binding of antagonists to all of the AR subtypes, since its removal from the tricyclic system was associated with an irreversible loss of affinity and selectivity, regardless of the receptor under investigation. In fact, Baraldi and coworkers [31] found that the substitution of the furan ring in PTPs with phenyl (25) or alkoxyphenyl rings led to a loss of affinity to A 2A , A 2B , and A 3 receptors, while the A 1 subtype in some cases displayed a high nanomolar binding profile. Similarly, the functionalization of the furan ring with polar substituents led to completely inactive derivatives, clearly indicating that an unsubstituted furan ring at the C 2 position played a fundamental role in ligand-receptor recognition [31]. Notably, in most cases, substitution at the pyrazole ring occurred at the N 7 -rather than at the N 8 -position. Recently, a new series of 2-aryl pyrazolo-triazolo-pyrimidines was reported by Cheong et al., in which the previously conserved furan at C 2 was substi-tuted with a 2-aryl ring while substitutions on pyrazole ring were maintained at the N 8 -position [32]. Such bioisosteric replacement at C 2 resulted in improved human A 3 affinity and remarkably enhanced selectivity over other AR subtypes. The para substituents at the 2-phenyl ring were generally well tolerated, except for a para-nitro group, which caused detrimental effects on hA 3 affinity. Particularly, the para-OCH 3 and para-F groups conferred better affinities and selectivities towards the hA 3 receptor. The most potent compound in this series (26) had a methyl group at the N 8position, a phenylacetamide at the N 5 -position, and a phenyl ring at the C 2 -position. Interestingly, Okamura et al. also described a series of pyrazolo-triazolo-pyrimidine analogues with a para-(un)substituted-phenyl ring and an alkyl chain at the C 2 -and C 5 -positions, respectively, that was shown to possess good hA 3 affinity. The selectivity against other AR subtypes was significantly improved in this group of derivatives, especially when a para-substituted-2-phenyl ring was present (as illustrated by compounds 27, 28) [6,33]. It was also observed that the introduction of a substituent  (e.g., NHCH 2 CH 3 (29) and SCH 3 ) at the C 9 -position, induced a loss of both affinity and selectivity towards the A 3 receptor. It was postulated that the introduction of these substituents caused a repulsive effect due to steric hindrance, which hampered the interaction with the binding site of the A 3 AR [31].

A 3 AR Antagonists.
The series of pyrazoloquinolin-4-ones and pyrazolo [3,4-c]quinolines, 4-oxo and 4-amino substituted, shared a similar central scaffold as that of the triazoloquinoxalinones (30, 31) [34][35][36][37][38], and they were found to be potent and selective A 3 AR antagonists ( Figure 4) [39,40]. The substituent on the appended 2-phenyl ring was crucial to modulate A 3 affinity while a nuclear (e.g., oxo group) or extranuclear (e.g., amide group) C=O proton acceptor at the 4-position gave rise to potent and selective  [41]. Some of the synthesized compounds showed good A 3 affinities (nanomolar ranges) and excellent selectivities. Particularly, the substitution of methyl, methoxy, or chlorine at the para-position of the 2phenyl ring, together with the presence of a 4-oxo functionality gave good A 3 affinity and selectivity (35). relationship (SAR) analysis, both the substituents at the C 5and N 2 -positions of the bicyclic nucleus were crucial for the human A 3 affinity and selectivity. The concomitant presence of small alkyl chains, such as methyl group at the C 5position and a para-methoxy-substituted phenyl ring at the N 2 position (as demonstrated by compound 36 in Figure 5) gave rise to the most potent and selective A 3 AR antagonist in this series of derivatives. [43]. The lead compound, 4,6-Bis[α-carbamoylethyl)thio]-1-phenylpyrazolo- [3,4-d]pyrimidine (37 in Figure 6), served as a starting template for the optimization of A 1 affinity and selectivity in this series of compounds. 1-phenyl-pyrazolo- [3,4-d]pyrimidine was modified at C 4 with mercapto, methylthio, and amino groups in order to investigate the hydrogen-bonding and steric tolerance at this position [44]. At C 6 , thioesters containing distal amides with varying lengths of linear and branched alkyl groups extending from the α-carbon were evaluated for steric and hydrophobic tolerance [44]. From the binding data at A 1 receptor, it was found that the simultaneous presence of an amino at C 4 and α-butyl side chain at C 6 gave rise to the most potent compound of the series (38); the least potent compound contained a mercapto and an α-isopropyl side chain at C 4 and C 6 , respectively. These observations suggested that the superiority of the C 4 -amino group was most likely due to a hydrogen-bonding interaction with the receptor binding sites. Although a C 4 -methylthio group was less preferable than the amino species, its presence was still tolerable, thus indicating the existence of a hydrophobic pocket in the A 1 binding site able to accommodate the methyl group. As for the C 6 position, the increase in length of the linear carbon side chain (from ethyl to butyl) was favorably tolerated at the A 1 receptor for each C 4 -substituent. Similarly, the hydrophobic tolerance at C 6 position seemed crucial for the A 1 binding affinity as well.

A 1 AR Antagonists. A series of pyrazolo-[3,4-d]pyrimidines was identified that contains novel A 1 AR antagonists
In an attempt to test for the hypothesis mentioned above, a methyl-amino and an α-butyl side chain were concurrently introduced at the C 4 and C 6 positions, respectively [44]. Accordingly, the derivative 39 displayed improved A 1 affinity and increased A 1 selectivity, which further supported the proposed structural requirements at both the C 4 and C 6 positions.

A 2A AR Antagonists. Pyrazolo-[3,4-d]pyrimidines
were also explored by Gillespie and collaborators as A 2A AR antagonists [45]. In particular, the 4-(furan-2-yl)pyrazolo- [3,4-d]pyrimidine (compound 40 in Figure 7) was identified as a starting point for further investigation. It showed a good affinity for the A 2A receptor subtype and was 13-fold more selective over A 1 . The following introduction of 1-phenyl substitution (41) increased potency at A 2A while either incorporation of heteroatoms or ring saturation did not improve affinity significantly. Extension of spatial linker between the phenyl ring and pyrazole by more than one methylene group was found to provide an hA 2A affinity profile similar to the 1-phenyl derivative. Furthermore, subsequent substitution on the meta-position of phenyl ring with electron-rich and deficient groups was tolerated, with the 3-chlorobenzyl derivative (42) demonstrating the best hA 2A affinity and selectivity in the series. Moreover, compounds 40-42 have also shown in vivo activity in a mouse haloperidol-induced hypolocomotion model of Parkinson's disease. Due to the fact that the 4-(furan-2-yl) moiety in this series of compounds could be easily converted into reactive species under oxidative metabolism, further studies were undertaken to replace such group with other nonfuran-containing heterocycles. Unfortunately, the resulting compounds have showed reduced affinity for the A 2A receptor.

A 3 AR Antagonists.
Pyrazolo- [3,4-d]pyrimidines represent a novel series of bicyclic scaffold-derived A 3 antagonists [46] isosterically related to the imidazole-[1,2a] [1,3,5]triazine (43; Figure 8), which have shown a certain degree of binding affinity at both A 1 and A 3 receptors [56]. Such pyrazolo-pyrimidine analogues displayed improved A 3 affinity and selectivity profiles in comparison to the parent imidazole-triazines. From the binding affinity results, it was suggested that the 6-phenyl substituent at the bicyclic scaffold was a key pharmacophoric element for recognition at the ARs, since its removal led to poor affinity to all the ARs. Besides that, small alkyl groups at the N 2 -position, such as a methyl moiety were found to be more favourable than bulky groups for conferring good human A 3 affinity. The introduction of N 4 -acyl substituents generally resulted in improved human A 3 affinity relative to unsubstituted derivatives. In particular, the presence of a methyl group at N 2 , together with para-methoxy benzoyl substituent at N 4 (44) dramatically increased the potency and selectivity to the A 3 AR. Compound 44 was subsequently tested on human glioma U87MG cells, and it was able to counteract the proliferation of glioma cells mediated by A 3 AR agonists Cl-IB-MECA and IB-MECA through the inhibition of A 3 AR agonist-mediated ERK 1/2 activation. This finding implied that this class of derivatives might represent promising lead compounds for the development of adjuvants for glioma chemotherapy [46].   Figure 9) [47] and FK838 (46) [48] were the typical examples of such derivatives, and they also showed diuretic activity both in vivo and in vitro. Nevertheless, there were some limitations in these two compounds. For FK453, photochemical transcis isomerization at the acryloyl amide moiety and low water solubility (11.9 μg/mL) were two main problems in this type of structure. In FK838, photochemical stability was achieved through the substitution of the acryloyl amide with a pyridazinone ring while water solubility (10 mg/mL) was enhanced by the introduction of the butyric acid group. Nevertheless, this derivative had lower binding affinity and poorer selectivity for A 1 receptor than FK453. Subsequently, further structural modifications to FK838 led to the synthesis of FR166124 (47) [49], which is the most potent and selective A 1 AR antagonist of this series, and it shows high water solubility (>200 mg/mL). In fact, it was designed based on the hypothesis that the high affinity and selectivity of FK453 for the A 1 receptor was due to the presence of the (2R)-2- the acryloyl amide as a conformationally limiting factor. The pyridazinone ring of FK838 was maintained in the structure of FR166124, with the introduction of a ring structure joining the C 3 and C 4 positions of the butyric acid group to limit possible conformations. Overall, the close resemblance of X-ray crystal structures of FR166124 and FK453 to each other, together with the experimental binding assay data, suggested that the presence of a double bond in the cyclohexenyl acetic acid group was essential for high selectivity to the A 1 receptor, with good A 1 affinity and water solubility.

Conclusion
Pyrazolo-containing polyheterocyclic scaffolds have given rise to a group of potent and selective antagonists for the A 1 , A 2A , A 2B , and A 3 AR subtypes. An overview of the structureactivity relationships of each class of derivatives not only clarifies the structural requirements deemed essential for the affinity towards the individual AR subtypes, but it also lends insight into the rational design and optimization of existing structural templates to obtain other new, potent AR antagonists.