Antagonists of the adenosine A2A receptor based on a 2-arylbenzoxazole scaffold: Investigation of the C5- and C7-positions to enhance affinity.

We have recently reported a series of 2-furoyl-benzoxazoles as potential A2A adenosine receptor (A2AR) antagonists. Two hits were identified with interesting pharmacokinetic properties but were find to bind the hA2AR receptor in the micromolar-range. Herein, in order to enhance affinity toward the hA2AR, we explored the C5- and C7-position of hits 1 and 2 based on docking studies. These modifications led to compounds with nanomolar-range affinity (e.g. 6a, Ki = 40 nM) and high antagonist activity (e.g. 6a, IC50 = 70.6 nM). Selected compounds also exhibited interesting in vitro DMPK (Drug Metabolism and Pharmacokinetics) properties including high solubility and low cytotoxicity. Therefore, the benzoxazole ring appears as a highly effective scaffold for the design of new A2A antagonists.


Introduction
Targeting the adenosine A2A receptor (A2AR) has emerged as a promising strategy for the treatment of both Alzheimer's (AD) and Parkinson's diseases (PD) [1]. This receptor, one of the four adenosine receptors with A1, A2B and A3, is coupled to the stimulatory G protein [2].
Interest for A2AR emerged with epidemiological studies showing that people consuming regularly caffeine-based beverages over a lifetime are substantially less likely to develop these two diseases [3][4]. Indeed, in experimental models of AD and PD [5][6][7], caffeine exerts neuroprotective effects notably by controlling the glutamate excitotoxicity and the microgliamediated neuroinflammation [8]. Besides, many A2AR antagonists have been discovered over the past few years (Fig. 1). For example, Istradefylline (KW-6002) was approved in Japan for the treatment of PD [9][10][11] and acts by boosting dopaminergic signaling, reducing thereby motor deficits. Preladenant [9], has also been investigated clinically but was discontinued due to a lack of efficacy. Regarding AD, it is now well established that A2AR antagonists lead to the improvement of spatial memory associated with the decrease of A amyloid burden, Tau hyper phosphorylation and neurotoxicity [7,12].
However, although many A2A antagonists display high potency, constant drawbacks remain such as poor solubility and synthetic tractability and high toxicity [8,[13][14][15]. These drawbacks have limited the development of potential drugs targeting this receptor. Therefore, the main challenge regarding A2A antagonist development is to improve their pharmacokinetic properties and especially their solubility.
We have recently reported a series of 2-arylbenzoxazole derivatives [16] as A2AR antagonists.
Two hits were identified from this study as shown in figure 2 (compound 1 and 2). These ligands display promising pharmacokinetic properties but bind the hA2AR in the micromolar-range. The present work describes the structural investigation of benzoxazole scaffold to improve binding affinity while keeping good pharmacokinetic properties.

Molecular modeling-guided design
Putative binding mode of 2 ( Fig. 3A) showed that benzoxazole scaffold interacts through an aromatic interaction with Phe168 and a hydrogen bond with Asn253. The furan forms a hydrogen bond with Asn253 and interacts with Trp246 and His250 through aromatic interactions [16]. Furthermore, the piperidine moiety of compound 2 creates an additional interaction with Glu169. This putative binding mode suggests that introducing a substituent at the C-7 position of the benzoxazole ring would improve affinity by exploring an unexplored hydrophobic pocket ( Fig. 3A & 3C). This pocket delimited by Ala63, Val84, Ile274 and His278 is occupied by the 2-chlorophenol of reference triazine T4E (PBD: 3UZC, Fig 3B) [17] and not by compound 2 (Fig. 3A). Therefore, we evaluated the impact of the introduction of an aromatic ring at the C-7 position of both benzoxazoles 1 (compounds 6a-i, Fig. 2) and 2 (compounds 11- Fig. 2 & 3C). As an alternative, we introduced a primary amine at this position instead of the aromatic group in order to interact with Asn253, inducing a different orientation of the benzoxazole ring (compound 21, Fig. 3D). We also investigated the nature of the tertiary amine, as well as the length of the linker between the amide function and the tertiary amine.
Compounds 5a-h and 5i were obtained respectively via Suzuki [18] and Buchwald [19] coupling. Finally, the nitro group was reduced with hydrazine hydrate in the presence of Raney Nickel to afford amines 6a-i.
To synthesize molecule 21 and its positional isomer 25 (Scheme 2), a Buchwald-Hartwig coupling was performed on benzoxazole 4 using benzophenone imine in order to generate protected exocyclic amine (18). The nitro function of 18 was then reduced with hydrazine hydrate in the presence of Raney Nickel and directly N-acylated using bromoacetyl bromide to get 19. Classical nucleophilic substitution was then performed to give compound 20 followed by amine deprotection under acidic conditions to afford compound 21.
To get compound 25, similar conditions were used starting from amine deprotection of 18 under acidic conditions. Treatment of 22 with bromoacetylbromide followed by nucleophilic substitution with piperidine and nitro group reduction afforded 25.

Structure-Affinity Relationship studies
Affinities of benzoxazole derivatives for the hA2AR were determined by a competitive radioligand displacement assay using [ 3 H]-ZM241385 [20].
Firstly, comparing molecules 1 (Ki = 10 M) and 6a (Ki = 40 nM) on the one hand and 2 (Ki = 1 M) and 11 (Ki = 210 nM) on the other hand, showed that adding an aromatic ring at C-7 position was beneficial for A2A affinity (table 1). To a lesser extent, introducing an amine at the C-7 position of the benzoxazole of compound 2 (21, Ki = 480 nM) also improved affinity.
Interestingly, the positional isomer of the latter (compound 25) displayed no affinity for the hA2AR (Ki > 10 M).  In order to confirm that these new compounds behave as functional antagonists at the hA2AR, representative compounds 6a, 11 and 21 were evaluated using the GTPγ 35 S binding assay (table 2). Experiments were performed as previously described [21]. Briefly, this technique, not often used to assess functional activity at the A2AR, was developed in our lab using the same hA2AR membranes as for the competitive radioligand displacement assay. Under the assay conditions, reference A2AR agonist CGS241680 increased GTPγ 35
At 10 µM concentration, compounds 12, 14 and 15 significantly bind plasma proteins whereas 6a and 2 showed a slightly better unbound fractions. A lower permeability value was observed for molecules with a basic center (12, 14, 15) compared to 6a which exhibits a high permeability value (Papp of 77×10 −6 cm/s). Interestingly, adding a second furan in position 7 improves metabolic stability (t1/2 > 41 min) in human liver microsomes (e.g. compare 2 with 12). Except for 12, a low cytotoxicity is observed for this series. Finally, with the exception of 15, molecules with a protonable amine (2, 12 and 14) exhibit a high aqueous solubility (≥ 165 µM) which is a considerable advantage as compared to many A2A antagonists (reported solubility for Preladenant and KW6002 is 1.5 µM and 20 nM, respectively) [22][23][24].

Conclusion
The present work deals with the optimization of a new series of benzoxazoles as A2A antagonists. Modulation on the C-7 position of the benzoxazole ring by adding a furan significantly improved binding affinity of 1 and 2 toward hA2AR. Compound 6a, displayed the highest binding affinity for hA2AR (Ki = 40 nM) with high antagonist activity (IC50 = 70.6 nM).
Moreover, addition of a tertiary amine-based chain at the C-5 position resulted in ligands with interesting DMPK properties, especially a high solubility, while keeping a good affinity (e.g.

12:
Ki = 81 nM). Overall, the data presented here show that the benzoxazole ring is a highly effective scaffold for the design of new A2A antagonists.

Chemistry
All reagents and solvents were purchased and used without further purification. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2 mm). Some purification of products was carried out by column chromatography using Raney Ni (70 mg) was added followed by hydrazine hydrate (0.073 mL, 1.5 mmol). The mixture was stirred for 2 h at room temperature and then the catalyst was filtered off. The reaction mixture was hydrolyzed with water and then extracted three times with ethyl acetate.
Combined organic layers were dried over MgSO4, and evaporated in vacuo to afford a solid which was recrystallized using diethyl ether and petroleum ether (10/1), to afford 6g as a beige solid (70 mg, 24%). mp 173 °C. 1