Valeria Moas Heloire

Nicolas Renault
email: nicolas.renault-3@univ-lille2.fr

Vânia Batalha

Angela Rincon Arias
email: ararias@quim.ucm.es

Mathieu Marchivie
email: mathieu.marchivie@icmcb.cnrs.fr

Saïd Yous

Noémie Deguine
email: noemie.deguine-2@univ-lille2.fr

Luc Buee
email: luc.buee@inserm.fr

Philippe Chavatte
email: philippe.chavatte@univ-lille2.fr

David Blum
email: david.blum@inserm.fr

Valeria Moas-Héloire

Vania Batalha
email: vanialnbatalha@gmail.com

Said Yous
email: said.yous@univ-lille2.fr

Luc Buée

Luisa Lopes

Patricia Melnyk
email: patricia.melnyk@univ-lille2.fr

Laurence Agouridas
email: laurence.agouridas@univ-lille2.fr

" Onco

Design and synthesis of fused tetrahydroisoquinoline-iminoimidazolines

Keywords: guanidines, A2A receptor, privileged structure, iminoimidazoline Tic-H, tetrahydroisoquinoline hydantoin; A2AR, adenosine A2A receptor

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Introduction

In the field of medicinal chemistry, privileged structures are considered as a promising source of ligands able to interact with various targets. [1][2][START_REF] Evans | [END_REF] In fact, these minimal structures constitute a powerful starting point to identify original compounds by playing with the nature of their substituents. They constitute an anchor point enabling to orientate substituents in various positions, giving access to potentially promising structures and to modulate their affinity and activity. Therefore, identification of novel privileged structures appears as an interesting challenge.

Our group described a series of tetrahydroisoquinoline-hydantoin (Tic-H 1, Figure 1) derived compounds with potent affinity for the sigma-1 receptor in the nanomolar range ensuing various therapeutical in vivo applications. [4][5][6][7][8][9] This heterocycle presents an interesting hydrogen bond acceptor group on the hydantoin cycle and is amenable to various decorations. In this study we proposed to identify a new potential privileged structure and decided to evaluate the consequence of replacing the hydantoin's (1) hydrogen bond acceptor by a hydrogen bond donor group resulting in the guanidine moiety of compound 2 (Figure 1). This new heterocycle could be the central core for the design of new active compounds. Guanidines are present in a large variety of natural products with potent biological activities in many fields 10- 13 and more specifically in the central nervous system area. 14,15 This approach was exemplified by preparing the guanidine derivative "Tic-guanidine" and its derivatives (2 to 13) in order to evaluate the affinity of this new series on the adenosine receptor A2AR. 16 In fact, in addition to its affinity towards sigma-1 receptor, Tic-H 1 showed a promising affinity constant towards adenosine receptor A2A (Ki = 44 µM). This receptor is widely expressed in the central nervous system. Expressed at different levels (neurons, astrocytes, microglial cells), [17][18][19][20][21] it acts at various levels of regulation. [22][23][24][25] Therefore, A2ARs are viewed as promising targets in various neurodegenerative diseases, mainly Parkinson's and Alzheimer's diseases. [26][27][28] Docking studies, based on the crystal structure of A2A bound to the high affinity antagonist (ZM241385), 29 showed that Tic-guanidine restored hydrogen bonds that were missing for Tic-H leading us to expect a better affinity of these compounds. 30 We therefore aim to develop a new series of Tic-guanidine compound. We set up an original and efficient chemical synthesis allowing pharmacomodulations. These latter concern the nature of the substituents on the original tricyclic structure Tic-guanidine (series A, figure 1) but also modifications by opening of the central cyclic core (series B, figure 1).

Results and discussion

Modeling studies

Jaakola et al. published in 2008 a crystal structure of A2AR with the high affinity antagonist ZM241385. 29 Tic-H and Tic-guanidine were both docked in A2AR's binding site. Results showed both molecules place in a same manner as ZM241385 does (Figure 2). Mainly, important hydrogen bonds with Glu 169 and Asn 253 missing in the case of Tic-H are being restored with the guanidine function. Finally, upper carbonyl moiety of Tic-H was not maintained, as it doesn't seem to play an essential role in the interactions with amino acids of the binding site. Supported by our docking studies, pharmacomodulations were envisaged: various Nsubstituents on the guanidine core (series A, figure 1) and opening of the central tricyclic core (series B, figure 1). Series A enables to evaluate the effect of the modification of the benzyl group in the hydrophobic upper pocket of the binding site. On the other hand, series B was prepared in order to evaluate the importance of the central core's nature and especially how its geometry impacts compounds' affinity. We wanted to establish whether a planar conformation was essential as described in many A2A's antagonists ref or if less restricted structures could improve affinity as compared to already published non planar A2A antagonists ref. Finally, B series would bring additional informations on structure activity relationships of our Tic-guanidine compounds.

Chemical synthesis

Synthesis of Tic-guanidine derivatives of series A and B was achieved thanks to a unique chemical pathway as depicted in Scheme 1. This enabled us to access desired compounds starting from commercially available amino acids and therefore to easily access various pharmacomodulations. We optimized the synthetic pathway of compound 2 starting from commercially available Boc-protected L-tetrahydroisoquinoline carboxylic acid I whose chemistry is well mastered in our group. 7,9,31 Key step of this synthesis was the final formation of the cyclic guanidine. Various methods are described in the literature to access guanidines 12 and cyclic guanidines [32][33][34] but could not be applied to our strategy. Indeed, we previously described the synthesis of Tic-thiohydantoins, which could be an interesting intermediate for the synthesis of these Tic-guanidines. But their chemical and enantiomeric instability precluded their use in this study. 35 For final formation of guanidine cycle, direct cyclisation of the free diamine in the presence of cyanogen bromide [36][37][38][39][40][41][42] was unsuccessful. Some adjustments were then required and we finally chose to maintain Boc-protection in order to selectively functionalize the free amine of compounds 2b-13b with cyanogene bromide. 43 Expected guanidines were obtained after Boc-deprotection and subsequent cyclization of intermediate.

Synthesis of series

A started from Boc-protected L-tetrahydroisoquinoline carboxylic acid I.

Corresponding aldehyde was obtained via reduction of Weinreb amide II 44,45 using LiAlH4 in THF at 0°C. 44 Reductive amination with appropriate amine in the presence of sodium triacetoxyborohydride in CH2Cl2 gave expected Boc-monoprotected derivatives 2b-6b. 46 Free amine of this last compound was then functionalized using cyanogen bromide in ethanol to give corresponding nitrile derivatives. 43 Finally, guanidine cyclization was achieved thanks to acidic deprotection of the carbamate and in situ cyclization of the intermediate diamine compound with yields from 46 to 88%. Scheme 1.

Figure 3.

Depending on the nature of the amine in the reductive amination step, compounds 2 to 6 were first obtained (scheme 1), as depicted in figure 3. On the other hand, opening of the upper part of the central cycle while maintaining N-benzyl substitution of the guanidine (scheme 2) core resulted from modification of starting amino acid (compounds 7-13, Figure 4). Compound 12, derived from tyrosine, was prepared from Boc-Tyr(tBu)-OH. The tert-butyl protection was removed in the final cyclisation step. Structure of final compounds was confirmed by various analysis methods. LC-MS gave expected mass, discarding the formation of the dimer. FT-IR indicated the disappearance of the nitrile band (2260-2240 cm -1 ) and appearance of the guanidine C=NH bond (1690-1640 cm -1 ) confirming cyclisation of the compound (see supporting informations). Finally, 1D and 2D 1 H and 13 C NMR showed appropriate signals and correlations, especially the guanidine C=NH bond. Some of our compounds were able to crystallize. X-ray spectroscopy thus enabled us to confirm the structure and enantiopurity of compounds 2 and 3 (figure 5) but also compounds 7, 9 and 11 whose absolute configuration was maintained. 47 On the other hand, crystallographic data showed compound 8 was present as a racemic mixture (for details, see supplementary informations). This compound differs from the other ones, as it is N-methylated. N-methylation was achieved following a protocol described in the literature to be a non-epimerizing route. 48 However, in our case, the basic conditions of methylation led to a complete racemization.

Affinity of Tic-guanidine compounds

Affinity of our compounds was evaluated on human A2A receptor membranes stably expressed in HEK293 cells. Results (table 1) showed no improvement of binding compared to initial Tic-H compound 1 (Ki = 44 µM) except for the tricyclic guanidine substituted with phenylbutylamine 5 (Ki = 35 µM). More specifically, affinity was drastically decreased for open guanidines.

Table 1.

Except compound 5 (35 µM) that gives an affinity comparable to the one of Tic-H 1 (44 µM), binding was not improved with our Tic-guanidine compounds. For sure, rigid tricyclic structures are necessary to bind the A2AR receptor as open structures (7 to 13) showed no affinity at all. X-ray analysis helped understanding this observation. In fact, as shown in figure 5, open Tic-guanidine structures adopt a curved conformation that does not allow the binding of the molecule in the pocket of the receptor whereas tricyclic structures keep a flat conformation. 49 Concerning the tricyclic structures (2 to 6), we therefore assume the replacement of carbonyle moiety of 1 by a hydrogen bond donor does not give us expected improved affinity for the A2AR.

Cytotoxicity of Tic-guanidine compounds

Cytotoxicity assays have been conducted on SY5Y cells and showed no toxicity of our compounds at 100 µM (Table 1).

Conclusion

As Tic-H core was of interest for the design of various biologically active compounds, we proposed the Tic-guanidine core as a new privileged structure. This work therefore presented a new and efficient synthesis of guanidine cycles derived from amino acids. We applied this concept to the design of A2AR ligands. Unfortunately, binding results established that our initial hypothesis was not confirmed: replacement of the hydrogen bond acceptor moiety carbonyl of compound 1 by the hydrogen bond donor guanidine did not improve the affinity for A2AR. Other decorations are needed to improve the affinity for A2AR. Of particular importance is the lack of cytotoxicity of this new scaffold. Thus valorization of this new scaffold for other receptors is currently under evaluation.

Experimental section

General information

Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise noted. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2mm). Purification of products was carried out by either column chromatography or thick layer chromatography. Column chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). Thick layer chromatography was performed on glass plates coated with Macherey-Nagel Sil/UV254 (thickness 2 mm), from which the pure compounds were extracted with the following solvent system: DCM/MeOH(NH3), 90:10. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1 Hand 75 MHz for 13 C). Chemical shifts are expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position (δ in ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = double doublet, br = broad and m = multiplet), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of 2D experiments (COSY, HSQC and HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionization and a UV detector (diode array). HRMS-ESI spectra were recorded on a Thermo Scientific Exactive spectrometer. The purity of final compounds was verified by two types of high pressure liquid chromatography (HPLC) columns: C18 Interchrom UPTISPHERE and C4 Interchrom UPTISPHERE. Analytical HPLC was performed on a Shimadzu LC-2010AHT system equipped with a UV detector set at 254 nm and 215 nm.

Compounds were dissolved in 50 µL methanol and 950 µL buffer A, and injected into the system. The following eluent systems were used: buffer A (H2O/TFA, 100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention times (HPLC tR) were obtained at a flow rate of 0.2 mL/min for 35 min using the following conditions: a gradient run from 100% of buffer A over1 min, then to 100% of buffer B over the next 30 min. The melting point analyses were performed on Barnstead Electrothermal Melting Point Series IA9200 and are uncorrected.

Infrared spectra were performed on Bruker FT-IR spectrometer model α. Preparative HPLC were performed using a Varian PRoStar system using an OmniSphere 10 column C18 250 mm × 41.4 mm Dynamax from Varian, Inc. A gradient starting from 20% CH3CN/80% H2O/0.1% formic acid and reaching 100% CH3CN/0.1% formic acid at a flow rate of 80 mL/min was used. Optical rotations were measured at 20°C on a Perkin-Elmer 343 polarimeter.

Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication 1401476, 1401477, 1401478, 1401480, 1401481 and 1401479. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).

Preparation of Weinreb amide compounds.

General method 1.

To a solution of appropriate amino protected carboxylic acid derivative (1 eq.) dissolved in a mixture of CH2Cl2 and CH3CN (1:1, 3,7 mL/mmol of carboxylic acid), EDC (1.3 eq.), HOBt (1.3 eq.), NMM (6.5 eq.) and HN(Me)OMe.HCl (2.1 eq.) were added. The mixture was stirred at room temperature over 24-72 h and then evaporated. The resulting crude product was dissolved in CH2Cl2, washed three times with saturated NaHCO3 solution, three times with 1M HCl and once with brine. Organic layer was dried over MgSO4, filtered and concentrated in vacuo. Purification via flash chromatography was performed.

tert-butyl N-[(1S)-1-benzyl-2-[methoxy(methyl)amino]-2-oxo-ethyl]carbamate (7a).

According to general method 1, 7a was obtained as a brown oil (0.94 g, 66%). 1 (dd, J = 12.0 and 6.0 Hz, 1H) and (dd, J = 15.0 and 9.0 Hz, 1H), Δυ = 60 Hz]; 1.39 (s, 9H). 50

tert-butyl N-[(1S)-1-benzyl-2-[methoxy(methyl)amino]-2-oxo-ethyl]-N-methylcarbamate (8a).

According to general method 1, 8a was obtained as a yellow oil (0.942 g, 86%).

tert-butyl N-[(1S)-1-[(4-fluorophenyl)methyl]-2-[methoxy(methyl)amino]-2-oxoethyl]carbamate (9a).

According to general method 1, 9a was obtained as a colorless oil (0.810 g, 70%).

tert-butyl N-[(1S)-2-(methoxy(methyl)amino)-1-methyl-2-oxo-ethyl]carbamate (11a).

According to general method 1, 11a was obtained as a yellow oil (0.566 g, 64%). Analyses similar to literature description. 51

tert-butyl N-[(1S)-1-[(4-tert-butoxyphenyl)methyl]-2-[methoxy(methyl)amino]-2-oxoethyl]carbamate (12a).

According to general method 1, 12a was obtained as a yellow solid (0.722 g, 76%). Analyses similar to literature description. 52

tert-butyl N-[(1S)-2-[methoxy(methyl)amino]-1-methyl-2-oxo-ethyl]carbamate (13a).

According to general method 1, 13a was obtained as a yellow solid (0.225 g, 48%). Analyses similar to literature description. 53 4.3. Preparation of amine compounds 2b-13b.

General method 2.

To a solution of Weinreb amide (1 eq.) in THF was added LiAlH4 (0.9 eq. of commercial solution 1M in THF) dropwise under N2 at 0 o C and stirred for 1 h at 0 o C. Then saturated KHSO4 solution was added dropwise. THF was evaporated off. The resulting crude product was dissolved in CH2Cl2, washed twice with saturated NaHCO3 solution, twice with 1M HCl, once with brine. Organic layer was dried over MgSO4, filtered and concentrated in vacuo. No further purification was performed. To a solution of resulting aldehyde (1 eq) in CH2Cl2, benzylamine (1.2 eq) was added under N2. The mixture was stirred at room temperature for 30 min, NaBH(OAc)3 (3 eq) was added portion wise (at least for 10 min). The mixture was stirred overnight, diluted with CH2Cl2 (3 mL) and saturated NaHCO3 solution (3 mL) was added. The mixture was stirred for 10 min. The organic layer was separated and the aqueous phase washed twice with CH2Cl2 (15 mL). Combined organic layers were dried over MgSO4, filtered and evaporated in vacuo. Purification via flash chromatography was performed.

tert-butyl (3S)-3-[(benzylamino)methyl]-3,4-dihydro-1H-isoquinoline-2-carboxylate (2b).

According to general method 2, 2b was obtained as a colorless oil (0.55g, 95%).

tert-butyl (3S)-3-[[2-(2-pyridyl)ethylamino]methyl]-3,4-dihydro-1H-isoquinoline-2carboxylate (3b).

According to general method 2, 3b was obtained as a light brown oil (0.386g, quantitative). 1

tert-butyl (3S)-3-[(2-morpholinoethylamino)methyl]-3,4-dihydro-1H-isoquinoline-2carboxylate(4b).

According to general method 2, 4b was obtained as a colorless oil (0.428 g, 99%). 1

tert-butyl (3S)-3-[(4-phenylbutylamino)methyl]-3,4-dihydro-1H-isoquinoline-2carboxylate (5b).

According to general method 2, 5b was obtained as a colorless oil (0.416 g, 92%).

tert-butyl (3S)-3-[(hexylamino)methyl]-3,4-dihydro-1H-isoquinoline-2-carboxylate (6b).

According to general method 2, 6b was obtained as a colorless oil (0.386 g, 97%).

tert-butyl N-[(1S)-1-benzyl-2-(benzylamino)ethyl]carbamate (7b).

According to general method 2, 7b was obtained as a brown solid (0.498 g, 90%). Analyses similar to literature description. 54

tert-butyl N-[(1S)-1-benzyl-2-(benzylamino)ethyl]-N-methyl-carbamate (8b).

According to general method 2, 8b was obtained as a light yellow oil (0.120 g, 52%).

tert-butyl N-[(1S)-1-[(benzylamino)methyl]-2-(4-fluorophenyl)ethyl]carbamate (9b).

According to general method 2, 9b was obtained as a light yellow oil (0.510 g, 70%).

tert-butyl (2S)-2-[(benzylamino)methyl]piperidine-1-carboxylate (10b).

According to general method 2, 10b was obtained as a yellow oil (0.453 g, 62%).

tert-butyl N-[(1S)-3-(benzylamino)-1-[(4-methoxyphenyl)methyl]-2-oxopropyl]carbamate (13b).

According to general method 2, 13b was obtained as a yellow oil (0.032 g, 22%).

Preparation of guanidine compounds 2-13.

General method 3.

To a solution of appropriate secondary amine (1 eq) in EtOH (1.6 mL/mmol of amine), NaHCO3 (3 eq) and BrCN (1.1 eq) were added. The mixture was stirred at room temperature for 5 h. The mixture was diluted with H2O and extracted twice with CH2Cl2. Combined organic layers were dried under MgSO4. The solution was filtered and evaporated in vacuo. No further purification was performed unless specified. The nitrile compound (1 eq) was dissolved in 3M HCl/dioxane (0.08 M) and stirred at room temperature overnight. Dioxane was evaporated in vacuo. Purification via flash chromatography was performed.

(10aS)-2-benzyl-1,5,10,10a-tetrahydroimidazo[1,5-b]isoquinolin-3-imine

monohydrochloride (2). According to general method 2, 2 was obtained (chromatography, CH2Cl2/methanol 94/6) as a white powder (0.13g, 46%).

Figure 2 .

 2 Figure 2.

  Scheme 2.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

  1 H NMR (CD3OD, 300 MHz), δ (ppm): 7.41-7.36 (m, 5H); 7.26-7.18 (m, 4H); 4.72-4.54 (m, 4H); 4.11-4.01 (m, 1H); 3.61 [AB part of ABX system (t, J = 9.0 Hz, 1H) and (t, J = 6.0 Hz, 1H), Δυ = 144 Hz]; 2.95 [AB part of ABX system (dd, J = 18.0 and 9.0 Hz, 1H) and (dd, J = 15.0 and 9.0 Hz, 1H), Δυ = 72 Hz].

4. 4

 4 .3. (10aS)-2-[2-(2-pyridyl)ethyl]-1,5,10,10a-tetrahydroimidazo[1,5-b]isoquinolin-3-imine dihydrochloride(3). According to general method 2, 3 was obtained (chromatography, CH2Cl2/methanol 90/10) as a white powder (0.10 g, 67%).1 H NMR (CD3OD, 300 MHz), δ (ppm): 8.84 (d, J = 6.0 Hz, 1H); 8.64 (t, J = 9.0 Hz, 1H); 8.23 (d, J = 6.0 Hz, 1H); 8.04 (t, J = 9.0 Hz, 1H); 7.23 (s, 4H); 4.67 (AB system, J = 15.0 Hz, Δυ = 75 Hz, 2H]; 4.15-3.91 (m, 4H); 3.63 [AB part of ABX system (t, J = 9.0 Hz, 1H) Δυ = 114 Hz]; 3.56 (t, J = 8.0 Hz, 2H); 2.99 [AB part of ABX system (d, J = 15.0 Hz, 1H] and (t, J = 9.0 Hz, 1H), Δυ = 51 Hz).

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2. A2AR's crystal structure with (i) high-affinity antagonist ZM241385, (ii) Tic-H 1 and (iii) Tic-guanidine 2. Hydrogen bonds with Glu 169 and Asn 253 missing with Tic-H (ii) are being restored with the guanidine function (iii).

Figure 3 .

 3 Figure 3. Pharmacomodulations of compounds 2-6 of the Tic-guanidine core using various amines in the reductive amination step.

Figure 4 .

 4 Figure 4. Opening of the central Tic-guanidine core.

Figure 5 .

 5 Figure 5. Thermal ellipsoid drawing 47 (30% probability) of the asymmetric unit of compound 2 (left) and 3 (right) showing the molecular structure and the labeling scheme.

Figure 6 .

 6 Figure 6. Thermal ellipsoid drawing 47 (30% probability) of the asymmetric unit of compound 7-9 and 11 showing the molecular structure and the labeling scheme.

  Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

  .28-5.17 (d, J = 12.1 Hz, 1H), 5.00-4.82 (unresolved X part of ABX system, m, 1H); 3.67 (s, 3H); 3.16 (s, 3H); 2.87 [AB part of ABX system (dd, J = 12.2 and 6.3 Hz, 1H) and (dd, J = 12.1 and 6.6 Hz, 1H), Δυ = 60 Hz]; 1.38 (s, 9H).13 C NMR (CDCl3, 75 MHz), δ (ppm): 172.0, 163.5, 160.2 (JC-F = 234 Hz); 155.1, 132.3, 131.0, 130.9, 115.3, 115.0, 79.6, 61.6, 51.5, 38.1,

	32.0, 28.3. MS (ESI + ): m/z = 327.1 [M,H] + found; C16H24FN2O4 calculated m/z =
	327.2[M,H] + .
	4.2.6.	tert-butyl	(2S)-2-[methoxy(methyl)carbamoyl]piperidine-1-carboxylate	(10a).
	According to general method 1, 10a was obtained as a yellow oil (0.986 g, 83%). 1 H NMR
	(CDCl3, 300 MHz), δ (ppm): 5.18-4.87 (m, 1H); 4.10-3.84 (m, 1H); 3.76 (s, 3H); 3.58-3.39
	(m, 1H); 3.18 (s, 3H); 2.01 (d, J = 15.0 Hz, 1H); 1.75-1.57 (m, 3H); 1.44 (s, 9H); 1.34-1.20
	(m, 2H). 13 C NMR (CDCl3, 75 MHz), δ (ppm): 173.5, 156.2, 79.7, 61.3, 50.8, 42.3, 32.2, 29.7,
	28.5, 26.5, 19.6. MS (ESI + ): m/z = 273.1 [M,H] + found; C13H25N2O4 calculated m/z =
	273.2[M,H] + .

1

H NMR (CDCl3, 300 MHz), δ (ppm): 7.18-7.09 (m, 2H); 7.02-6.90 (m, 2H); 5

  dd, J = 12.3 and 3.1 Hz, 1H) and (dd, J = 12.0 and 6.6 Hz, 1H), Δυ = 81.2 Hz); 2.73-2.69 (m, 1H); 1.76-1.53 (m, 6H); 1.44 (s, 9H).13 C NMR (CDCl3, 75 MHz), δ (ppm): 155.1, 140.4, 128.3, 128.0, 126.9, 124.8, 79.4, 53.4, 49.9, 48.0, 39.3, 28.5, 26.7, 25.4, 19.3. MS (ESI + ): m/z = 305.0 [M,H] + found; C18H28N2O2 calculated m/z = 305.2 [M,H] + .

	4.3.11. tert-butyl N-[2-(benzylamino)-1-methyl-ethyl]carbamate (11b). According to general
	method 2, 11b was obtained as a yellow oil (0.286 g, 19%). Analyses similar to literature
	description. 55
	4.3.12. tert-butyl N-[(1S)-1-[(benzylamino)methyl]-2-(4-tert-butoxyphenyl)ethyl]carbamate
	(12b). According to general method 2, 12b was obtained as a yellow oil (0.252 g, 58%). 1 H
	NMR (CDCl3, 300 MHz), δ (ppm): 7.33-7.20 (m, 5H); 7.14-6.98 (m, 2H); 6.91-6.82 (m, 2H);
	4.98 (br s, 1H); 3.94-3.84 (unresolved X part of ABX system, m, 1H); 3.77 (AB system, J =
	13.1 Hz, Δυ = 26.8 Hz, 2H); 3.12-2.91 (unresolved AB part of ABX system, m, 1H); 2.90-
	2.69 (unresolved AB part of ABX system, m, 3H); 1.41 and 1.32 (2s, 19H). 13 C NMR (CDCl3,
	75 MHz), δ (ppm): 155.8, 153.7, 139.0, 132.8, 129.7, 128.5, 127.2, 124.1, 79.2, 78.2 53.2,
	51.0, 38.5, 28.8, 28.4. MS (ESI

1 H NMR (CDCl3, 300 MHz), δ (ppm): 7.37-7.20 (m, 5H); 4.45-4.32 (unresolved X part of ABX system, m, 1H); 4.02-3.87 (m, 1H); 3.83 (AB system, d, J = 12.0 Hz, Δυ = 24.2 Hz, 1H); 2.79 [AB part of ABX system(+ ): m/z = 413.1 [M,H] + found; C25H37N2O3 calculated m/z = 413.3 [M,H] + .

Table 1 .

 1 Cells were seeded at 2,000 cells per well onto 96-well plates in DMEM medium. Cells were starved for 24 hours to obtain synchronous cultures, and were then incubated in culture medium that contained various concentrations of test compounds, each dissolved in less than 0.1% DMSO. After 72 hours of incubation, cell growth was estimated by the colorimetric MTT (thiazolyl blue tetrazolium bromide) assay.(France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER). This work was supported by Lille 2 University, ANR « Adoratau », PRES Univ Lille Nord de France. Valeria Moas Heloire is the recipient of a fellowship from Lille 2 University. a Yield of the cyclization step. b A2aR's agonist CGS-21680 used as a reference (Ki = 0.503 µM). Displacement of specific [ 3 H]-ZM 241385 binding in membranes obtained from hA2a receptor stably expressed in HEK293 cells. c Cytotoxicity assays on SY5Y cells. c Ki > 450 µM.

	13 C NMR

127.9, 127.

4

, 126.7, 53.8, 51.1, 48.5, 39.9. HRMS (ESI + ): m/z = 266.16529 [M,H] + found; C17H20N3 calculated m/z = 266.16517 [M,H] + . Mp: 196.1°C. αD (20°C, CH3OH) = -

Table 1 .

 1

	series A

Acknowledgments

We express our thanks to Mariam Lamarti, Phillip James Brennan and Alice Cao for their contribution in organic synthesis and Amélie Barczyk for some in vitro cytotoxicity experiments. The 300 MHz NMR facilities were funded by the Région Nord-Pas de Calais


Rajâa Boulahjar

Angela Rincon Arias

Raphaël Bolteau

Nicolas Renault

Mathilde Coevoet

Amelie Barczyk

Romain Duroux

Saïd Yous

Patricia Melnyk

Laurence Agouridas

Rajaa Boulahjar

Amélie Barczyk

Rajaa Boulahjar

,2a]pyridines, a promising privileged structure

Keywords: privileged structure, amino-imidazopyridine, A2A receptor, palladium-catalyzed coupling reaction

come

Introduction

Among the various imidazopyridine isomers, [1] imidazo [1,2a]pyridines have gained much attention in the field of biological research [2] for their applications as antiprotozoal, [3] antimicrobial, [4] antiinflammatory [5] and antitumor agents, [6][7][8] as well as their potential in the field of neurological and neurodegenerative disorders. [START_REF] Lefin | Imidazo[1,2-α]pyridines possess adenosine A 1 receptor affinity for the potential treatment of cognition in neurological disorders[END_REF]10] If the synthesis of substituted imidazo [1,2a]pyridines is well described and still leads to original chemical pathways, [START_REF] Bagdi | Synthesis of imidazo[1,2-a]pyridines: a decade update[END_REF][START_REF] Herath | Fully Automated Continuous Flow Synthesis of Highly Functionalized Imidazo [1,2-a] Heterocycles[END_REF][13] studies describing access methods to 8amino substituted imidazo [1,2a]pyridines are less common. [2,[START_REF] Herath | Fully Automated Continuous Flow Synthesis of Highly Functionalized Imidazo [1,2-a] Heterocycles[END_REF][START_REF] Trapani | Novel 2-Phenylimidazo [1,2-a] pyridine Derivatives as Potent and Selective Ligands for Peripheral Benzodiazepine Receptors : Synthesis , Binding Affinity , and in Vivo Studies[END_REF][START_REF] Dwyer | Versatile templates for the development of novel kinase inhibitors: Discovery of novel CDK inhibitors[END_REF][START_REF] Liscio | Synthesis , Crystallographic Studies and Preliminary Biological Appraisal of New Substituted Triazolo [4,3-b] pyridazin-8-amine Derivatives as Tankyrase Inhibitors[END_REF][START_REF] Shukla | Antibacterial activities of Groebke-Blackburn-Bienaymé-derived imidazo[1,2-a]pyridin-3-amines[END_REF][18][START_REF] Vu | Imidazopyridine and related analogs as sirtuin modulators[END_REF][START_REF] Chiesa | Tricyclic imidazopyridines and intermediates for the synthesis thereof[END_REF] This class of heterocyclic amines constitutes a promising privileged structure as they are composed of a central scaffold that can be decorated by at least 3 substituants to explore diverse interactions. By integrating an exocyclic amine, they also offer the possibility to generate additional hydrogen bond that might be required for the interaction with their target. It constitutes an interesting challenge as it can find many applications in medicinal chemistry. [START_REF] Evans | Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists[END_REF][START_REF] Welsch | Privileged scaffolds for library design and drug discovery[END_REF] In this way, we aim to prepare 2,6-disubstituted-8-amino imidazo [1,2a]pyridines, a substitution pattern rarely found in the litterature, [START_REF] Herath | Fully Automated Continuous Flow Synthesis of Highly Functionalized Imidazo [1,2-a] Heterocycles[END_REF][START_REF] Shuwen | Heterocyclic compounds and methods of use thereof for treatment of hepatitis C[END_REF][START_REF] Bamford | Derivatives of imidazo (1,2-a) pyridine useful as medicaments for treating gastrointestinal diseases[END_REF] and to evaluate the effects of these modulations and the importance of the exocyclic amine on the affinity towards the adenosine receptor A2AR, a very promising target in the field of neurodegenerative diseases. [START_REF] Ruiz | Adenosine A2A Receptor as a Drug Discovery Target[END_REF] This protein constitutes a target of interest in our group and promising antagonists compounds have been recently published. [START_REF] Duroux | Antagonists of the adenosine A2A receptor based on a 2arylbenzoxazole scaffold: Investigation of the C5-and C7-positions to enhance affinity[END_REF][START_REF] Duroux | Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A 2A receptor[END_REF]. This receptor is widely expressed in the central nervous system. Expressed at different levels (neurons, astrocytes, microglial cells), [START_REF] Vu | Imidazopyridine and related analogs as sirtuin modulators[END_REF][START_REF] Rebola | Adenosine A1 and A2A receptors are co-expressed in pyramidal neurons and co-localized in glutamatergic nerve terminals of the rat hippocampus[END_REF][START_REF] Blum | Adenosine receptors and Huntington ' s disease Adenosine receptors and Huntington's disease : implications for pathogenesis and therapeutics[END_REF][START_REF] Chen | Adenosine A2A receptors and brain injury: Broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation[END_REF][START_REF] Milne | Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor[END_REF][START_REF] Fredholm | International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors[END_REF][START_REF] Fredholm | Aspects of the general biology of adenosine A2A signaling[END_REF][START_REF] Laurent | A2A adenosine receptor deletion is protective in a mouse model of Tauopathy[END_REF] it acts at various levels of regulation. [START_REF] Popoli | Functions, dysfunctions and possible therapeutic relevance of adenosine A2A receptors in Huntington's disease[END_REF][START_REF] Matos | Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes[END_REF][START_REF] Ribeiro | Modulation and metamodulation of synapses by adenosine[END_REF][START_REF] Orr | Adenosine A2A receptor mediates microglial process retraction[END_REF][START_REF] Minghetti | Effects of the adenosine A2A receptor antagonist SCH 58621 on cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor availability in a rat model of striatal neurodegeneration[END_REF] Therefore, A2ARs are viewed as promising targets in various neurodegenerative diseases, mainly Parkinson's (PD) and Alzheimer's (AD) diseases. [START_REF] Gomes | Adenosine receptors and brain diseases: Neuroprotection and neurodegeneration[END_REF][START_REF] Popoli | A Critical Evaluation of Adenosine A2A Receptors as Potentially "Druggable" Targets in Huntingtons Disease[END_REF][START_REF] Preti | History and Perspectives of A2A Adenosine Receptor Antagonists as Potential Therapeutic Agents[END_REF] In fact, many A2AR's antagonists showed very promising results in cognitive impairments in both pathologies [START_REF] Preti | History and Perspectives of A2A Adenosine Receptor Antagonists as Potential Therapeutic Agents[END_REF] through motor and neuroprotective effects. Some compounds reached clinical phases (Istradefylline, Preladenant,Tozadenant) [START_REF] Shook | Adenosine A2A receptor antagonists and Parkinson's disease[END_REF][START_REF] Hauser | Tozadenant (SYN115) in patients with Parkinson's disease who have motor fluctuations on levodopa: A phase 2b, double-blind, randomised trial[END_REF] in the case of PD. In the case of AD, it is now well understood that A2AR's blockade leads to improvement of spatial memory related to the decrease of Aβ amyloid burden, Tau hyper phosphorylation and neurotoxicity. [START_REF] Dall'igna | Caffeine and adenosine A2a receptor antagonists prevent β-amyloid (25-35)-induced cognitive deficits in mice[END_REF][START_REF] Lunven | Disruption of Fibers from the Tau Model AcPHF6 by Naturally Occurring Aurones and Synthetic Analogues[END_REF] Docking studies, based on the co-crystallized structure of A2AR with the high affinity antagonist ZM-241385 [START_REF] Jaakola | The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist[END_REF] confirmed the importance of the exocyclic amine. Additionally, these studies combined to results recently obtained by our group on benzoxazole derivatives [START_REF] Duroux | Antagonists of the adenosine A2A receptor based on a 2arylbenzoxazole scaffold: Investigation of the C5-and C7-positions to enhance affinity[END_REF][START_REF] Duroux | Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A 2A receptor[END_REF] led us to explore various substitutions on position 6 of 8-amino-imidazo [1,2a]pyridines and therefore different chemical pathways to access these compounds. We describe here the functionalization of the position 6 using palladium catalyzed reaction and N-hydroxysuccinimidyl activated ester. [START_REF] Barré | Palladium-Catalyzed Carbonylation of (Hetero)Aryl, Alkenyl and Allyl Halides by Means of N-Hydroxysuccinimidyl Formate as CO Surrogate[END_REF] Figure 1.

Results and discussion

Modeling studies

Structure-based studies were performed using docking equipements into the orthosteric binding site of most recent crystal structure of A2AR bound to the high affinity and selective antagonist ZM-241385. [START_REF] Liu | Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions[END_REF] Expecting the appropriate binding mode of de novo designed molecules, in silico process of docking was validated comparing position and interactions of reference ZM-241385 in both crystallized and docking structures (Figure 2). From this protocol, docking of amino-imidazopyridine 16 showed a position similar to ZM-241385, including hydrogen bonds with Glu 169 and Asn 253 .

Chemical synthesis

Aryl coupling

In order to first confirm the importance of the exocyclic amine of 8-amino imidazo [1,2a]pyridines, non aminated imidazopyridine analogs were prepared (Scheme 1). As commonly described in the literature, substituents on position 2 were chosen due to their capacity to interact with Trp 246 in the bottom of the binding site. [START_REF] Jaakola | The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist[END_REF] Central imidazopyridine core (4-7) was obtained via reaction of brominated ketone (1- Functionalization of position 6 of 2-aryl-8-amino imidazo [1,2a]pyridines via Suzuki coupling has never been described, neither with N-protected amine nor with the free exocyclic amine. As described in Figure 1, Suzuki coupling was afforded from brominated intermediates 12, 13 and 15. These compounds were obtained as described in the litterature [START_REF] Elman | Process for preparing a substituted imidazopyridine compound[END_REF] by cyclization of appropriate brominated ketone (1)(2)(3) with commercially available 2,3-diamino-5-bromopyridine in cyclohexanone (Scheme 2). Cyclization in cyclohexanone at 130°C afforded corresponding heterocycles 12-15 with yields from 43 to 90%.

Suzuki coupling was conducted in the same conditions as depicted in Scheme Sonogashira coupling constitutes an interesting way to substitute 6-position with an alkyne, starting from non-protected 8-amino-imidazopyridine. If conversion was complete as shown by LC-MS analysis, yield was drastically impacted due to solubility issues of compound 24 during purification step.

Amide linker

Studies in our group identified benzoxazole as a promising scaffold for A2AR's antagonists. [START_REF] Duroux | Antagonists of the adenosine A2A receptor based on a 2arylbenzoxazole scaffold: Investigation of the C5-and C7-positions to enhance affinity[END_REF][START_REF] Duroux | Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A 2A receptor[END_REF] More precisely, amide substitution gave interesting results. Therefore, we aim to synthesize aminoimidazopyridines substituted on position 6 with an amide moiety (Scheme 6) using the Nhydroxysuccinimidyl activated ester method. [START_REF] Barré | Palladium-Catalyzed Carbonylation of (Hetero)Aryl, Alkenyl and Allyl Halides by Means of N-Hydroxysuccinimidyl Formate as CO Surrogate[END_REF] Compound 13 first needed to be protected as conversion of unprotected compound to activated ester did not work. The amine group of substituted imidazopyridine 13 was protected using Boc2O and a large excess of NaHMDS. Then, Nhydroxysuccinimidyl ester 26 is prepared to obtain desired amides 27 to 33. This intermediate is formed from brominated derivative 25 and N-hydroxysuccinimidyl formate by a palladium-catalyzed carbonylative process. [START_REF] Barré | Palladium-Catalyzed Carbonylation of (Hetero)Aryl, Alkenyl and Allyl Halides by Means of N-Hydroxysuccinimidyl Formate as CO Surrogate[END_REF] The reaction was performed under a nitrogen atmosphere in a sealed tube in the presence of Pd(OAc)2, Xantphos and triethylamine at 60°C in THF. The use of very dry NHS formate (NHSF) and a very small scale (less than 1 mmol in our hands) is required. NHS formate was prepared in good yield from hydroxysuccinimide and formic acid according to a procedure described in the litterature. [START_REF] Barré | Palladium-Catalyzed Carbonylation of (Hetero)Aryl, Alkenyl and Allyl Halides by Means of N-Hydroxysuccinimidyl Formate as CO Surrogate[END_REF] Compound 26 was obtained with 40% yield. Unfortunately, all our efforts to optimize the yield of this step were unsuccesfull: time of reaction had no impact on the conversion and use of compound 13 in its unprotected form, either as its acid or basic form, did not lead to the desired ester, only starting material was recovered. On another side, the use of the di-Boc-protected compound just gave degradation products. Finally, the resulting NHS ester To optimize the reaction for carbonylation of halide derivative 25, we evaluated replacement of bromide by iodide. The reaction was performed under the same conditions as described previously (Scheme 6)

to afford iodinated compound 41. To our delight, unprotected iodide compound 22 was totally converted into NHS ester as monitored by LC-MS (Scheme 7). However, this method suffers from several drawbacks such as the insolubility of corresponding ester and the formation of byproducts, making the isolation of pure NHS esters and then pure final amides difficult. Similarly, the use of N-Boc derivative 41 afforded the corresponding ester but also, many degradation products. Therefore, we chose to keep the brominated imidazopyridines synthesis pathway (Scheme 6) in which isolation of the products was much easier.

Scheme 7. (i) NHSF, Xantphos, Pd(OAc)2, Et3N, THF, 60°C, 18h.

Biological evaluation of 8-amino-imidazopyridine derivatives

Affinity of our compounds was evaluated on human A2A receptor membranes stably expressed in HEK293 cells, by a competitive radioligand displacement assay using [ we tried to replace it with 3,4-dimethoxyphenyl. As shown with compound 17, this was successfull as this latter compound exhibited a higher affinity. Therefore, we decided to keep 3,4-dimethoxyphenyle on position 2 for the second series of compounds (Table 2). As previously identified in our group for benzoxazole scaffolds [START_REF] Duroux | Antagonists of the adenosine A2A receptor based on a 2arylbenzoxazole scaffold: Investigation of the C5-and C7-positions to enhance affinity[END_REF][START_REF] Duroux | Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A 2A receptor[END_REF], amide substitution gave very promising results. Therefore, affinity of 6amide substituted amino-imidazopyridine compounds was evaluated ( with their standard deviation. Ki (inhibition constant at 50%) was calculated from two independent expriments. b CC50: cytotoxic concentration ie compound concentration causing 50% of SY5Y cell death after 24 h treatment.

Unfortunately, unlike results obtained for benzoxazole series, no affinity for 6-amide substituted aminoimidazopyridines 34-40 was observed and amino-imidazopyridines substituted with small aromatic moieties (9,16 and 17, Table 1) remained the most promising compounds able to bind A2AR.

Conclusion

This work describes new pathways to functionalize position 6 of 2-aryl-8-amino-imidazopyridine.

Presented compounds exhibited moderate affinity towards A2AR, however the low toxicity of most of them described in Tables 1 and2 offers numerous possibilities to test them as ligands of interest in other fields. This scaffold therefore constitutes a promising easily approachable privileged structure [START_REF] Evans | Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists[END_REF][START_REF] Welsch | Privileged scaffolds for library design and drug discovery[END_REF] as an anchor point to orientate substituents in various positions enabling various pharmacomodulations. In addition, the exocyclic amine, rarely found on this type of imidazopyridine compound, brings interesting assets. General method A for the cyclization of imidazo [1,2a] General method C for Suzuki coupling. To a solution of appropriate 6-bromo-imidazopyridine derivative (1 eq) in DMF (2.5 mL/mmol of brominated derivative), boronic acid derivative (1.9 eq) and aqueous [START_REF] Ruiz | Adenosine A2A Receptor as a Drug Discovery Target[END_REF]. Into an oven dried flask and under nitrogen atmosphere were placed 6-bromo-2-(3,4dimethoxyphenyl)imidazo[1,2-a]pyridin-8-amine hydrobromide 13 (3 g, 6.99 mmol) in dry THF (50 mL). NaHMDS (600 %, 1 M, 41.9 mL) was added at -10°C and the mixture was stirred at RT for 3h.

Experimental section

General information

Tert-Butyl N-[6-bromo-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyridin-8-yl]carbamate
Then, a solution of Boc2O (4.58 g, 3 eq) dissolved in dry THF (40 mL) was added and the reaction mixture was stirred at RT for 3h. The mixture was quenched by saturated solution of ammonium chloride (35 mL) and extracted 3 times by EtOAc, dried over MgSO4, filtered and the solvent was evaporated.

Flash chromatography (Cy/CH2Cl2, 50:50 to CH2Cl2) afforded compound 25 as brown crystals (2.2 g, 70%). 1

2,5-dioxopyrrolidin-1-yl 8-{[(tert-butoxy)carbonyl]amino}-2-(3,4-dimethoxyphenyl)imidazo [1,2-a]
pyridine-6-carboxylate [START_REF] Duroux | Antagonists of the adenosine A2A receptor based on a 2arylbenzoxazole scaffold: Investigation of the C5-and C7-positions to enhance affinity[END_REF]. In a dried sealed tube were placed tert-butyl N-[6-bromo-2-(3,4dimethoxyphenyl)imidazo[1,2-a]pyridin-8-yl]carbamate 25 (300 mg, 0.669 mmol), 2,5dioxopyrrolidin-1-yl formate (191 mg, 2 eq), Xantphos (9.68 mg, 0.025 eq), Pd(OAc)2 (4.51 mg, 0.03 eq). The vial was evacuated and then purged three times with nitrogen. Then 4 mL of dry and degazed THF were added. The mixture was stirred at 60°C and NEt3 (0.112 mL, 1.2 eq) was quickly added via a syringe. Fast gas evolution was observed and the reaction was left stirring for 18h at 60°C. The reaction mixture was diluted with EtOAc, filtered through celite and evaporated to dryness. The residue was taken up in dichloromethane, washed with water and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. Flash chromatography (CH2Cl2 to CH2Cl2/EtOAc, 9:1) afforded compound 26 as yellow solid (137 mg, 40%). 1 H NMR (CDCl3, 300 MHz) δ(ppm):

Tert-Butyl N-[2-(3,4-dimethoxyphenyl)-6-{[3-(morpholin-4-yl)propyl]carbamoyl}imidazo[1,2-a]
pyridin-8-yl]carbamate [START_REF] Blum | Adenosine receptors and Huntington ' s disease Adenosine receptors and Huntington's disease : implications for pathogenesis and therapeutics[END_REF]. Compound 29 was obtained following the general procedure D from the activated ester (70 mg, 0.14 mmol) and the N-(3-Aminopropyl)morpholine (1.0 eq, 0.14 mmol) after purification by flash chromatography (CH2Cl2 to CH2Cl2/MeOH, 96:4) as a beige solid (70 mg, 95%).

Docking process

Molecular docking was performed into 4EIY pdb structure [START_REF] Liu | Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions[END_REF] using Gold suite v5.2 within the Hermes v1.6 GUI (CCDC©). Thus after adding hydrogen atoms and removing water molecules, docking was performed in a 10 Å radius pocket around the co-crystallized ZM-241385. Early termination of 3 docking solutions within 1.5 Å was set up in order to highlight ligands converging toward a few binding modes, using GoldScore scoring function in a 100% automatic searching process. The best docking pose per ligand was selected as the best scored pose of the most populated pose cluster.

16 Figure 2 .

 162 Figure 2. (i) A2AR's crystal structure with high-affinity antagonist ZM-241385[49], (ii) predicted position of ZM-241385 in the docking model, (iii) predicted position of compound 16. Distances are mentioned for each receptor-ligand hydrogen bond. Illustrations were performed with UCSF Chimera v1.12.[START_REF] Pettersen | UCSF Chimera -A visualization system for exploratory research and analysis[END_REF]

  , J = 8.4 Hz, 1H), 7.47 (dd, J = 8.3, 2.0 Hz, 1H),

26 was reacted with various amines under mild conditions before amine deprotection to provide different substituted imidazopyridines as hydrochloride salts 35-40. Compound 34 required the use of dioxane and

	Scheme 6. (i) NaHMDS, Boc2O, THF, r.t., 6h, 70%; (ii) NHSF, Xantphos, Pd(OAc)2, Et3N, THF,
	60°C, 18h, 40%; (iii) Amine, CH2Cl2, RT or dioxane, 50°C, 18h, 51-95%; (iv) Dioxane HCl(4M),
	60°C, 2h, 42-70%.
	heating at 50°C for 18h as
	mild conditions in dichloromethane were unsuccessful. Stoichiometry of chlorhydrate salts after
	HCl/dioxane exposure was determined using a silver nitrate titration in the presence of potassium
	chromate.

Table 1 .

 1 3 H]-ZM-241385 (Table1).[START_REF] Lopes | Cross talk between A(1) and A(2A) adenosine receptors in the hippocampus and cortex of young adult and old rats[END_REF]

	Cpd	R'	R	X	Ki (µM) a	CC50 (µM) b
	6 18	/	Fur	H NH2	>10 >10	>100 >100
	11 20	Ph	Fur	H NH2	>10 >10	0.42 0.5
	10 19	Fur	Fur	H NH2	1.63±0.24 >10	3 2.27
	8 16	Fur	Ph	H NH2	>10 0.418±0.019	>100 26
	9 17	Fur	Ph(OMe)2	H NH2	0.238±0.033 0.117±0.049	30 36.4
	23		Ph(OMe)2	NH2	>10	66.7
	24		Ph(OMe)2	NH2	>10	50.4

a Displacement of specific [ 3 H]-ZM-241385 binding to hA2A receptors stably expressed in HEK293 cells. A 10µM screening was first completed to select compounds of interest. Results are given with their standard deviation. Ki (inhibition constant at 50%) was calculated from two independent experiments. b CC50: cytotoxic concentration ie compound concentration causing 50% of SY5Y cell death after 24 h treatment.

From table 1, it appears that exocyclic amine systematically improves imidazopyridines A2AR's affinities. Substitution on position 6 is required as shown by compounds 6 and 18. For aminated compounds, furane on position 2 leads to a better affinity than phenyl moiety (20 and 19 vs entry 16), which is in accordance with the fact that A2AR's ligands commonly present a furane moiety, able to interact with Trp 246 in the bottom of the binding pocket. Due to well known stability issues of the furane,

Table 2

 2 ).

	Cpd	R	Ki (µM) a CC50 (µM) b
	34		>10	>100
	35		>10	26.9
	36		>10	>100
	37		>10	>100
	38		>10	49.6
	39		>10	>100
	40		>10	31.8

Table 2 .

 2

a Displacement of specific [ 3 H]-ZM-241385 binding to hA2A receptors stably expressed in HEK293 cells. A 10µM screening was first completed to select compounds of interest. Results are given

  a flow rate of 0.2 mL/min for 35 min using the following conditions: a gradient run from 100% of buffer A over1 min, then to 100% of buffer B over the next 30 min. The melting point analyses were performed on Barnstead Electrothermal Melting Point Series IA9200 and are uncorrected. The mixture was stirred at 60°C for 18 h. The reaction was filtered, 10 mL of water were added and the mixture was extracted twice with EtOAc. Organic layer was then washed once

	Preparative HPLC were performed using a Varian PRoStar system using an OmniSphere 10 column C18
	250 mm × 41.4 mm Dynamax from Varian, Inc. A gradient starting from 20% CH3CN/80% H2O/0.1%
	formic acid and reaching 100% CH3CN/0.1% formic acid at a flow rate of 80 mL/min was used.
	4.2. Protocols
	2-bromo-3',4'-dimethoxyacetophenone (2). A solution of 3',4'-dimethoxyacetophenone (600 mg, 3.33
	mmol) in CHCl3 and EtOAc (6 mL, 1/1) was stirred for 10 minutes at RT. CuBr2 (1,487 g, 2 eq) was
	added in one portion.
	Thermo Scientific Exactive spectrometer. The purity of final compounds was verified by two types of
	high pressure liquid chromatography (HPLC) columns: C18 Interchrom UPTISPHERE and C4
	Interchrom UPTISPHERE. Analytical HPLC was performed on a Shimadzu LC-2010AHT system

Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise noted. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2mm). Purification of products was carried out by either column chromatography or thick layer chromatography. Column chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1 H and 75 MHz for 13 C). Chemical shifts are expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position (δ in ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = double doublet, br = broad and m = multiplet), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of 2D experiments (COSY, HSQC and HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionization and a UV detector (diode array). HRMS-ESI spectra were recorded on a equipped with a UV detector set at 254 nm and 215 nm. Compounds were dissolved in 50 µL methanol and 950 µL buffer A, and injected into the system. The following eluent systems were used: buffer A (H2O/TFA, 100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention times (HPLC tR) were obtained at with NaCl saturated solution, dried over MgSO4 and evaporated in vacuo. Flash chromatography (pure CH2Cl2) afforded compound 2 as a white solid (517.3 mg, 60%).

1

H NMR (CDCl3, 300MHz), δ(ppm):

3.96 (s, 3H), 3.98 (s, 3H), 4.42 (s, 1H), 6.91 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.63 (dd, J = 8.4 and 2.1 Hz, 1H).

[START_REF] Moreno | Simple Route to New Phenanthroand Phenanthroid-Fused Thiazoles by a PIFA-Mediated ( Hetero ) biaryl Coupling Reaction[END_REF]

2-bromo-acetylfuran

(3)

. A solution of 2-acetylfurane (182 µL, 1.82 mmol) in CHCl3 and EtOAc (3.3 mL, 1/1) was stirred for 10 minutes at RT. CuBr2 (811 mg, 2 eq) was added in one portion. The mixture was stirred at 60°C for 18 h. The reaction was filtered, 10 mL of water were added and the mixture was extracted twice with EtOAc. Organic layer was then washed once with NaCl saturated solution, dried over MgSO4 and evaporated in vacuo. Flash chromatography (pure CH2Cl2) afforded compound 3 as a dark yellow solid (199.4 mg, 58%).

1

H NMR (CDCl3, 300MHz), δ(ppm): 4.33 (s, 2H), 6.59-6.64 (m, 1H), 7.35 (d, J = 2.9 Hz, 1H), 7.65 (s, 1H).

[START_REF] Amador | Enantioselective Photocatalytic [3 + 2] Cycloadditions of Aryl Cyclopropyl Ketones[END_REF]

  The mixture was stirred at 70°C overnight. The solvent was evaporated in vacuum and the crude product was dissolved in CH2Cl2, washed twice with NaOH 1M solution, once with water and dried over MgSO4. The solvent was evaporated in vacuo. Compound was purified, if necessary, by flash chromatography. 1H).13 C NMR (CD3OD, 75 MHz), δ(ppm): 107.0, 108.3, 118.1, 125.6, 126.1, 128.1, 128.3, 128.8, 133.2, 144.1, 146.7. MS (ESI + ): m/z = 272.9/274.9 [M,H] + found; C13H9N2Br calculated m/z = 273.0/275.0 [M,H] + .

	(6-bromo-2-phenyl-imidazo[1,2-a]pyridin-8-yl)ammonium bromide (12). According to general
	procedure B, a solution of 0.250 g of 2,3-diamino-5-bromopyridine (1.33 mmol) in cyclohexanone and
	0.318 g of 2-bromoacetophenone (1.6 mmol) were used. Compound 12 was obtained as a light brown
	powder (440 mg, 90%). 1 H NMR (CD3OD, 300 MHz), δ(ppm): 7.16 (d, J = 1.5 Hz, 1H), 7.55-7.68 (m,
	6-bromo-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyridine (5). According to general procedure A, a 3H); 7.86-7.93 (m, 2H), 8.34 (d, J = 1.5 Hz, 1H), 8.45 (s, 1H). 13 C NMR (CD3OD, 75 MHz), δ(ppm):
	solution of 192 mg of 2-bromo-1-(3,4-dimethoxyphenyl)ethan-1-one (0.783 mmol) in ethanol and 135 111.4, 112.8, 113.5, 116.4, 125.9, 129.4, 130.6, 132.0, 133.9, 136.0. MS (ESI + ): m/z = 287.9/289.9
	mg of 2-amino-5-bromopyridine (0.783 mmol) were used. Flash chromatography (CH2Cl2/MeOH, 95:5) [M,H] + found; C13H10N3Br calculated m/z = 288.0/290.0 [M,H] + .
	afforded compound 5 as a gold yellow solid (100 mg, 57%). 1 H NMR (CD3OD, 300 MHz), δ(ppm):
	3.89 (s, 3H), 3.94 (s, 3H), 7.03 (d, J = 8.4 Hz, 1H), 7.38-7.55 (m, 4H), 8.14 (s, 1H), 8.68 (q, J = 0.9 Hz, 6-bromo-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyridin-8-amine hydrobromide (13). According to
	1H). 13 C NMR (CD3OD, 75 MHz), δ(ppm): 55.0, 106.7, 108.7, 109.5, 111.7, 116.2, 118.6, 126.7, 127.1, general procedure B, a solution of 464.4 mg of 2,3-diamino-5-bromopyridine (2.47 mmol) in
	127.6, 128.6, 145.0, 149.5. MS (ESI + ): m/z = 332.9/334.9 [M,H] + found; C15H13BrN2O2 calculated m/z cyclohexanone and 800 mg of 2-bromo-1-(3,4-dimethoxyphenyl)ethanone (3.01 mmol) were used.
	= 333.0/335.0 [M,H] + . Compound 13 was obtained as a light brown powder (959 mg, 90%). 1 H NMR (dmso-d6, 300 MHz),
	δ(ppm): 4.07 (s, 3H), 4.12 (s, 3H), 7.15 (s br, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.74 (dd, J = 8.5 and 1.5
	Hz, 1H), 7.77 (d, J = 1.5 Hz, 1H), 8.57 (s br, 1H), 8.72 (s br, 1H). 13 C NMR (dmso-d6, 75 MHz), δ(ppm):
	56.2, 56.5, 110.2, 111.1, 111.61, 112.1, 112.6, 115.8, 122.7, 132.3, 134.8, 136.5, 149.7, 150.9. MS
	6.88 (d, J = 3.3Hz, 1H), 7.44 (m, 2H), 7.58 (dd, J =1.3 and 0.8 Hz, 1H), 8.02 (s, 1H), 8.68 (dd, J = 1.8 and 0.9 Hz, 1H). 13 C NMR (CD3OD, 75 MHz), δ(ppm): 106.8, 106.9, 108.7, 111.2, 116.3, 126.8, 129.1, (ESI [6-bromo-2-(2-furyl)imidazo[1,2-a]pyridin-8-yl]ammonium bromide (15). According to general
	137.6, 142.5, 143.8, 148.5. MS (ESI + ): m/z = 262.9/264.9 [M,H] + found; C11H7BrN2O calculated m/z = procedure B, a solution of 414.5 mg of 2,3-diamino-5-bromopyridine (2.20 mmol) in cyclohexanone
	263.0/265.0 [M,H] + . and 500 mg of 2-bromo-1-(2-furanyl)-ethanone (2.64 mmol) were used. Compound 15 was obtained as
	a light brown powder (560 mg, 71%). 1 H NMR (CD3OD, 300 MHz), δ(ppm): 6.73 (dd, J = 3.5 and 1.8
	6-bromo-2-phenyl-imidazo[1,2-a]pyridine (4). According to general procedure A, a solution of 0.217 g General method B for the cyclization of 8-amino imidazo[1,2a]pyridines. To a solution of appropriate Hz, 1H), 7.13-7.18 (m, 2H), 7.83 (dd, J = 1.8 and 0.6 Hz, 1H), 8.31-8.36 (m, 2H). 13 C NMR (CD3OD,
	diaminopyridine derivative (1eq) in cyclohexanone (1 mL/mmol of ketone), 2-bromoacetyle or 2-75 MHz), δ(ppm): 110.0, 110.9, 112.1, 112.8, 113.7, 116.5, 127.3, 131.1, 133.8, 141.2, 145.2. MS of 2-amino-5-bromopyridine (1.26 mmol) in ethanol and 0.250 g of 2-bromoacetophenone (1.26 mmol) iodoacetyle derivative (1.2 eq) was added in one portion. The reaction was heated to 130°C overnight. (ESI + ): m/z = 277.9/279.9 [M,H] + found; C11H8N3OBr calculated m/z = 277.9/279.9 [M,H] + .

pyridines. To a solution of appropriate bromo acetyle derivative (1eq) in ethanol (4 mL/mmol), 2-aminopyridine or 2-amino-5-bromopyridine (1eq) was added in one portion. were used. Compound 4 was isolated without any further purification as a light brown powder (173 mg, 45%).

1

H NMR (CD3OD, 300 MHz), δ(ppm): 7.23 (dd, J = 9.6 and 1.8 Hz, 1H), 7.31-7.37 (m, 1H), 7.45 (t, J =7.1 Hz, 2H), 7.53 (d, J = 9.6 Hz, 1H), 7.81 (s, 1H), 7.91-7.97 (s, 1H), 8.25 (d, J = 0.7 Hz, 2-(furan-2-yl)imidazo[1,2-a]pyridine (6). According to general procedure A, a solution of 389 mg of 2bromo-1-(2-furyl)ethanone (2.06 mmol) in ethanol and 194 mg of 2-aminopyridine (2.06 mmol) were used. Flash chromatography (CH2Cl2-MeOH, 97:3) afforded compound 6 as a brown powder (110 mg, 29%). %). 1 H NMR (CDCl3, 300 MHz), δ(ppm): 6.50 (m, 1H), 6.76 (dt, J = 6.8 et 1.0 Hz, 1H), 6.88 (m, 1H), 7.15 (m, 1H), 7.46 (m, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.78 (s, 1H), 8.06 (d, J = 9.2 Hz, 1H). 13 C NMR (CDCl3, 75 MHz), δ(ppm): 106.7, 107.9, 111.6, 112.6, 117.4, 125.0, 125.7, 138.0, 142.1, 145.6, 149.6. [59] HRMS (ESI + ): m/z = 185.07147 [M,H] + found; C11H9N2O calculated m/z = 185.07094 [M,H] + . HPLC: C4 column: tR = 7.8 min, purity 96% C18 column: tR = 12.0 min, purity 98%.

6-bromo-2-(2-furyl)imidazo[1,2-a]pyridine

(7)

. According to general procedure A, a solution of 100 mg of 2-bromo-1-(2-furyl)ethanone (0.529 mmol) in ethanol and 91.5 mg of 2-amino-5-bromopyridine (0.529 mmol) were used. Flash chromatography (CH2Cl2/EtOAc, 95:5) afforded compound 7 as a light brown solid (79 mg, 57%).

1

H NMR (CD3OD, 300 MHz), δ(ppm): 6.56 (dd, J= 3.4 and 1.8 Hz, 1H), TBME was added to the mixture. The precipitate was filtered and washed with TBME. The product was then dried further in vacuo. No further purification was performed. + ): m/z = 348.0/350.0 [M,H] + found; C15H15N3O2Br calculated m/z = 348.0/350.0 [M,H] + .

2-(furan-2-yl)imidazo[1,2-a]pyridin-8-amine hydrochloride

[START_REF] Trapani | Novel 2-Phenylimidazo [1,2-a] pyridine Derivatives as Potent and Selective Ligands for Peripheral Benzodiazepine Receptors : Synthesis , Binding Affinity , and in Vivo Studies[END_REF]18)

. According to general procedure B, a solution of 205 mg of 2,3-diamino-5-bromopyridine (1.88 mmol) in cyclohexanone and 425 mg of 2bromo-1-(2-furanyl)-ethanone (2.25 mmol) were used. Product 14 was chorhydrated. Compound 18 was obtained as a light brown powder (161.7 mg, 43%).

1

H NMR (CD3OD, 300 MHz), δ(ppm): 6.71 (dd, J = 3.5 and 1.9 Hz, 1H), 7.07 (d, J = 9.1 Hz, 1H), 7.19 (d, J = 6.3 Hz, 1H), 7.26 (dd, J = 9.0 and 6.1 Hz, 1H), 7.80 (d, J = 1.5 Hz, 1H), 8.10 (d, J = 6 Hz, 1H), 8.36 (s, 1H). 13 C NMR (CD3OD, 75 MHz), δ(ppm): 110.0, 110.8, 111.6, 112.1, 116.8, 118.7, 126.9, 133.0, 133.3, 141.5, 145.0. HRMS (ESI + ): m/z = 200.08184 [M,H] + found; C11H10N3O calculated m/z = 200.08237 [M,H] + . Mp: 240.7°C. HPLC: C4 column: tR = 7.7 min, purity 99% C18 column: tR = 12.5 min, purity 99%.

  The mixture was heated under microwave at 100 °C for 30 min. The solvents were removed under reduced pressure. Water was added and the compound was extracted twice with CH2Cl2. The organic phase was then washed with brine, dried over MgSO4 and evaporated under reduced pressure.

	2,6-bis(2-furyl)imidazo[1,2-a]pyridin-8-amine (19). According to general procedure C, a solution of 60 NMR (dmso-d6, 75 MHz), δ(ppm): 56.2, 56.4, 83.3, 110.1, 110.5, 112.7, 116.8, 119.5, 120.5, 132.6,
	mg of 6-bromo-2-(furanyl)imidazo[1,2-a]pyridine (0.167 mmol) in DMF, 28 mg of 2-furanboronic acid 134.7, 136.0, 149.7, 150.9. MS (ESI + ): m/z = 396.0 [M,H] + found; C15H15IN3O2 calculated m/z = 396.0
	(0.251 mmol), 0.638 mL of K3PO4 2M solution and 19.3 mg of Pd(PPh3)4 (0.017 mmol) were used. [M,H] + .
	Preparative HPLC purification (pH 9, acetonitrile/water) afforded compound 19 as a beige solid (37 mg,
	9%). 1 H NMR (CDCl3, 300 MHz), δ(ppm): 6.54 (dd, J = 3.4 and 1.9 Hz, 1H), 6.58 (dd, J = 3.4 and 1.8 2-(3,4-dimethoxyphenyl)-6-(phenylethynyl)imidazo[1,2-a]pyridin-8-amine (23). A mixture of 2-(3,4-
	Hz, 1H), 6.75 (dd, J = 3.4 and 0.5 Hz, 1H), 6.85 (d, J = 1.4 Hz, 1H), 6.88 (dd, J = 3.4 and 0.6 Hz, 1H), dimethoxyphenyl)-6-iodoimidazo[1,2-a]pyridin-8-amine hydroiodide (0.3 g, 0.573 mmol) in DMF
	7.57 (dd, J = 1.3 and 0.7 Hz, 1H), 7.61 (dd, J = 1.8 and 0.7 Hz, 1H), 8.01 (s, 1H), 8.18 (d, J = 1.4 Hz, (2.29 mL) with Bis(triphenylphosphine)palladium(II) chloride (2 %, 8.05 mg, 0.0115 mmol) and Et3N
	1H). 13 C NMR (CDCl3, 75 MHz), δ(ppm): 101.2, 105.7, 106.7, 109.8, 110.9, 111.3, 111.4, 119.8, 134.5, (2.29 mL) was degassed for 15 min. CuI (21.8 mg, 0.115 mmol) and phenylacetylene (69.3 μL, 1.1 eq)
	135.4, 137.5, 142.4, 142.5, 147.8, 150.8. HRMS (ESI + ): m/z = 266.09240 [M,H] + found; C15H12N3O2 calculated m/z = 266.09298 [M,H] + . Mp: 124.7°C. HPLC: C4 column: tR = 8.3 min, purity 95% C18 column: tR = 17.4 min, purity 99%. were added. Flash chromatography (CH2Cl2-EtOAc, 80 :20) afforded compound 23 as a brown solid (201 mg, 95%).
	2-(2-furyl)-6-phenyl-imidazo[1,2-a]pyridin-8-amine (20). According to general procedure C, a solution 1 H NMR (CDCl3, 300MHz), δ(ppm): 3.93 (s, 3H), 4.02 (s, 3H), 4.71 (s (br), 2H), 6.45 (s, 1H), 6.90 (d,
	of 150 mg of 6-bromo-2-(furyl)imidazo[1,2-a]pyridine (0.418 mmol) in DMF, 76.4 mg of J = 9.9 Hz, 1H), 7.36 (m, 4H), 7.53 (m, 2H), 7.56 (d, J = 2.0 Hz, 1H), 7.67 (s, 1H), 7.82 (s, 1H). 13 C
	phenylboronic acid (0.627 mmol), 1.60 mL of K3PO4 2M solution and 48.3 mg of Pd(PPh3)4 (0.042 NMR (CDCl3, 75MHz), δ(ppm): 55.9, 56.1, 86.3, 89.6, 105.2, 108.6, 109.3, 110.1, 111.1, 118.3, 118.7,
	δ(ppm): 103.6, 105.8, 109.5, 111.1, 112.8, 126.4, 127.3, 128.5, 128.7, 135.7, 137.8, 138.5, 142.0, 149.0. 2-(3,4-dimethoxyphenyl)-6-phenethylimidazo[1,2-a]pyridin-8-amine (24). 2-(3,4-dimethoxyphenyl)-6-
	HRMS (ESI + ): m/z = 276.11314 [M,H] + found; C17H14N3O calculated m/z = 276.1137 [M,H] + . Mp: (2-phenylethynyl)imidazo[1,2-a]pyridin-8-amine (50 mg, 0.135 mmol), ammonium formate 341 mg, 4
	150.0°C. HPLC: C4 column: tR = 8.3 min, purity 99% C18 column: tR = 18.7 min, purity 99%. eq) and Pd(OH)2 (19 mg, 1 eq) were dissolved in MeOH (2.03 mL) and heated under reflux for 2.5 h at
	65 °C. The mixture was filtered through celite and washed with methanol. After evaporation of the
	2-iodo-3',4'-dimethoxyacetophenone (21). 3',4'-dimethoxyacetophenone (2g, 11.1 mmol) was dissolved filtrate under reduced pressure, flash chromatography (CH2Cl2-MeOH, 97:3) afforded compound 24 as
	in MeOH (22 mL). I2 (2.82 g, 1 eq) and CuO (0.882 g, 1 eq) were added. The mixture was heated at a beige solid (10.1 mg, 20%). 1 H NMR (CDCl3, 300 MHz), δ(ppm): 2.72-2.97 (m, 4H), 3.92 (s, 3H),
	65°C for 3 hours. Mixture was filtered on celite with EtOAc, washed with saturated NaHCO3 and brine, 3.99 (s, 3H), 4.69 (s (broad), 1H), 6.24 (s, 1H), 6.91 (d, J = 8.4 Hz, 1H), 7.13-7.33 (m, 6H), 7.41 (dd, J
	dried on MgSO4 and evaporated in vacuo. The crude solid was purified on flash chromatography (pure = 8.4 and 1.9 Hz, 1H), 7.58 (m, 2H). 13 C NMR (CDCl3, 75 MHz), δ(ppm): 34.9, 37.0, 55.9, 56.0, 105.2,
	DCM) to afford compound as a brown solid (3 g, 88%). Flash chromatography (pure CH2Cl2) afforded 108.4, 109.3, 111.3, 113.7, 118.3, 126.1, 126.7, 127.3, 128.4, 128.5, 134.9, 138.4, 141.1, 143.6, 148.7,
	compound 21 as a white solid (517.3 mg, 60%). 1 H NMR (CDCl3, 300MHz), δ(ppm): 3.94 (s, 3H), 3.97 149.2. HRMS (ESI + ): m/z = 374.18630 [M,H] + found; C23H24N3O2 calculated m/z = 374.18683 [M,H] + .
	(s, 3H), 4.33 (s, 1H), 6.90 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 8.4 and 2.1 Hz, Mp: 164°C. HPLC: C4 column: tR = 15.2 min, purity 99% C18 column: tR = 27.2 min, purity 99%.
	1H). [60]
	6-iodo-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyridin-8-amine hydroiodide (22). According to general
	procedure B, a solution of 1 g of 2,3-diamino-5-iodopyridine (4.26 mmol) in cyclohexanone and 1.63 g
	13 C

mmol) were used. Preparative HPLC purification (pH 9, acetonitrile/water) afforded compound 20 as a beige solid (34 mg, 30%).

1

H NMR (CD3OD, 300 MHz), δ(ppm): 6.53 (dd, J = 3.4 and 1.8 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 6.81 (dd, J = 3.5 and 0.6 Hz, 1H), 7.28-7.33 (m, 1H), 7.35-7.41 (m, 2H), 7.50-7.52 (m, 1H), 7.53-7.54 (m, 2H), 7.88 (s, 1H), 7.92 (d, J = 1.5 Hz, 1H). 13 C NMR (CD3OD, 75 MHz), of 2-iodo-1-(3,4-dimethoxyphenyl)ethanone (5.32 mmol) were used. Compound 22 was obtained as a light brown powder (1.19 g, 53%). 1 H NMR (dmso-d6, 300 MHz), δ(ppm): 3.83 (s, 3H), 3.88 (s, 3H), 7.06 (s, 1H), 7.17 (dd, J = 8.1, 1H), 7.44-7.49 (m, 2H), 8.36 (d, J = 1.7 Hz, 1H), 8.45 (s br, 1H). 122.9, 126.3, 128.4, 128.5, 131.5, 135.0, 138.5, 144.8, 149.0, 149.2. HRMS (ESI + ): m/z = 370.15500 [M,H] + found; C23H20N3O2 calculated m/z = 370.15553 [M,H] + . Mp: 190°C. HPLC: C4 column: tR = 15.3 min, purity 99% C18 column: tR = 28.6 min, purity 99%.

  H NMR (CDCl3, 300 MHz) δ(ppm): 1.57 (s, 9H), 3.93 (s, 3H), 4.00 (s, 3H), 6.93 (d, J = 8.4 Hz, 1H), 7.42 (dd, J = 8.3, 2.0 Hz, 1H), 7.53 (s, 1H), 7.70 (s, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.96 (d, J = 4.4

	Hz, 2H). MS (ESI + ): m/z = 450.2 [M,H] + found; C20H23N3O4Br calculated m/z = 450.2 [M,H] + .

  , J = 1.6 Hz, 1H). MS (ESI + ): m/z = 511.2 Amine (1.0 or 1.5 eq) was added and the mixture was stirred for 2h at RT. The reaction mixture was evaporated to dryness, taken up in CH2Cl2 and washed with water. The aqueous layer was extracted 5 times with CH2Cl2. The organics layers were washed with brine and dried over MgSO4. The solvent was evaporated under vacuum and the crude product was purified by flash chromatography to give

	mL). expected products.
	tert-butyl	N-[2-(3,4-dimethoxyphenyl)-6-{[2-(4-hydroxyphenyl)ethyl]carbamoyl}imidazo	[1,2-
	a]pyridin-8-yl]carbamate (28). Compound 28 was obtained following the general procedure D from the
	activated ester 26 (110 mg, 0.21 mmol) and the tyramine (1.5 eq, 0.32 mmol) after stirring for 6h and
	purification by flash chromatography (CH2Cl2/MeOH, 98:2) as a white solid (76 mg, 66%). 1 H NMR
	(CDCl3, 300 MHz) δ(ppm): 1.52 (s, 9H), 2.82 (t, J = 7.2 Hz, 2H), 3.62 (dd, J = 13.2, 6.8 Hz, 2H), 3.92
	(d, J = 6.3 Hz, 6H), 6.68 (t, J = 5.6 Hz, 1H), 6.80 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.3 Hz, 1H), 7.05 (d,
	J = 8.2 Hz, 2H), 7.38-7.46 (m, 1H), 7.51 (d, J = 1.4 Hz, 1H), 7.76 (s, 1H), 7.93 (s, 1H), 7.96 (s, 1H),
	8.14 (s, 1H), 8.44 (s, 1H). MS (ESI + ): m/z = 533.2 [M,H] + found; C29H33N4O6 calculated m/z = 533.2
	[M,H] + .
	[M,H] + found; C25H27N4O8 calculated m/z = 511.2 [M,H] + .
	Tert-Butyl-N-[2-(3,4-dimethoxyphenyl)-6-({4-[4-(2-methoxyethoxy)phenyl]piperazin-1-
	yl}carbonyl)imidazo[1,2-a]pyridin-8-yl]carbamate	(27).	2,5-dioxopyrrolidin-1-yl-8-{[(tert-
	butoxy)carbonyl]amino}-2-(3,4-dimethoxyphenyl)imidazo [1,2-a]pyridine-6-carboxylate 26 (65 mg,
	0.127 mmol) was dissolved in dry dioxane (2 mL). 1-[4-(2-methoxyethoxy)phenyl]piperazine (30.1 mg,
	1 eq) was added and the mixture was heated at 50°C for 18h. The reaction mixture was evaporated to
	dryness, taken up in CH2Cl2 and washed with water. The aqueous layer was extracted 5 times with
	CH2Cl2. The organics layers were washed with brine and dried over MgSO4. The solvent was evaporated
	under vacuum and the crude product was purified by flash chromatography (CH2Cl2 to CH2Cl2/MeOH,
	98:2) to give the expected product 27 as white solid (53 mg, 66 %). 1 H NMR (CDCl3, 300 MHz) δ(ppm):
	1.57 (s, 9H), 3.12 (s, 4H), 3.45 (s, 3H), 3.74 (dd, J = 5.5, 3.9 Hz, 2H), 3.83 (s, 4H), 3.94 (s, 3H), 4.01
	(s, 3H), 4.08 (dd, J = 5.5, 4.0 Hz, 2H), 6.87-6.92 (m, 4H), 6.95 (d, J = 8.4 Hz, 1H), 7.46 (dd, J = 8.3, 1.9
	Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.80 (s, 1H), 7.87 (s, 1H), 7.98 (s, 1H), 8.04 (d, J = 1.4 Hz, 1H). MS
	(ESI + ): m/z = 632.3 [M,H] + found; C34H42N5O7 calculated m/z = 632.3 [M,H] + .
	General	method	D.	2,5-dioxopyrrolidin-1-yl-8-{[(tert-butoxy)carbonyl]amino}-2-(3,4-
	dimethoxyphenyl)imidazo [1,2-a]pyridine-6-carboxylate 26 (1.0 eq) was dissolved in dry CH2Cl2 (2

  , 72.3, 109.8, 110.8, 112.4, 113.3, 116.5, 116.6, 120.2, 120.5, 120.6, 128.5, 134.3, 135.1, 138.3, 145.7, 151.3, 152.8, 155.8, 167.9. HRMS (ESI + ): m/z = 532.25686 [M,H] + found; C29H34N5O5 calculated m/z = 532.25545 [M,H] + . Mp: 204.0°C. HPLC: C4 column: tR = 15.1 min, purity 99% C18 column: tR = 21.5 min, purity 97%. Compound 35 was obtained following the general procedure E from the intermediate 28 (0.14 mmol) as a beige solid (38 mg, 57%). 1 H NMR (dmso-d6, 300 MHz) δ(ppm): 2.73 (t, J = 6.7 Hz, 2H), 3.40-3.50 (m, 2H), 3.83 (s, 3H), 3.90 (s, 3H), 6.68 (d, J = 7.9 Hz, 2H), 6.28-7.27 (m, 2H), 7.03 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 7.18 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 8.45 (s, 1H), 8.69 (s, 1H), 8.82 (s, 1H), 9.12 (s, 1H). 13 C NMR (dmso-d6, 75 MHz) δ(ppm): 34.1, 41.3, 55.7, 56.1, 107.5, 110.0, 111.0, 112.1, 115.1, 115.5, 118.8, 119.3, 126.3, 129.3, 129.5, 132.7, 133.2, 135.9, 149.2, 150.3, 155.7, 163.3. HRMS (ESI + ): m/z = 433.18829 [M,H] + found; C24H25N4O4 calculated m/z = 433.18703 [M,H] + . Mp: 246.0°C. HPLC: C4 column: tR = 13.7 min, purity 99% C18 column: tR = 22.0 min, purity 98%. Compound 36 was obtained following the procedure E from the intermediate 29 (0.13 mmol) as a beige solid (40 mg, 60%).1 H NMR (dmso-d6, 300 MHz) δ(ppm): 1.89-2.05 (m, 2H), 2.95-3.19 (m, 4 H), 3.30-3.52 (m, 4H), 3.73-4.01 (m, 10H), 7.14 (d, J = 8.6 Hz, 1H), 6.46-7.59 (m, 2H), 7.20 (s, 1H), 7.70 (dd, J = 8.4, 2.0 Hz, 1H), 7.87 (d, J = 1.9 Hz, 1H), 8.56 (d, J = 1.0 Hz, 1H), 8.70 (s, 1H), 9.01 (s, 1H), 10.89 (s, 1H). 13 C NMR (dmso-d6, 75 MHz) δ(ppm): 23.2, 36.6, 51.0, 53.9, 55.7, 56.2, 63.2, 107.4, 110.0, 110.9, 112.2, 115.7, 119.0, 119.3, 125.9, 133.0, 133.3, 136.3, 149.2, 150.3, 163.8. HRMS (ESI + ): m/z = 440.23098 [M,H] + found; C23H30N5O4 calculated m/z = 440.22923 [M,H] + . Mp: 216.0°C. HPLC: C4 column: tR = 13.5 min, purity 99% C18 column: tR = 19.1 min, purity Compound 37 was obtained following the general procedure E from the intermediate 30 (0.04 mmol) as a dark beige solid (14 mg, 60%). 1 H NMR (dmso-d6, 300 MHz) δ(ppm): 1.93-2.08 (m, 1H), 3.18-3.24 (m, 1H), 3.27-3.50 (m, 8H), 3.63-3.73 (m, 2H), 3.83 (s, 3H), 3.90 (s, 3H), 7.13 (d, J = 8.6 Hz, 1H), 6.90-7.41 (m, 2H), 7.21 (s, 1H), 7.71 (dd, J = 8.4, 1.9 Hz, 1H), 7.88 (d, J = 1.7Hz, 1H), 8.57 (s, 1H), 8.71 (s, 1H), 9.00 (t, J = 5.2 Hz, 1H), 9.75 (s, 2H).13 C NMR (dmso-d6, 75 MHz) δ(ppm): 23.3, 39.5, 47.7, 53.6, 55.7, 56.2, 107.4, 110.0, 110.9, 112.2, 115.7, 118.9, 119.3, 126.0, 132.9, 133.3, 136.1, 149.2, 150.3, 163.7. HRMS (ESI + ): m/z = 439.24652 [M,H] + found; C23H31N6O3 calculated m/z = 439.24522 [M,H] + . Mp: 220.0°C. HPLC: C4 column: tR = 5.5 min, purity 98% (254 nm). C18 column: tR = 18.4 min, purity 96%. 0.1% DMSO. After 72 h of incubation, cell growth was estimated by the colorimetric MTT (thiazolyl blue tetrazolium bromide) assay.

	activated ester 26 (70 mg, 0.14 mmol) and the 3-(piperidin-1-yl)propan-1-amine (1.0 eq, 0.14 mmol)
	after purification by flash chromatography (CH2Cl2 to CH2Cl2/MeOH 96/4) as a white solid (50 mg,
	68%). 1 H NMR (CDCl3, 300 MHz) δ(ppm): 1.20-1.30 (m, 2H), 1.55 (s, 9H), 1.70-1.86 (m, 4H), 1.91-
	2.08 (m, 2H), 2.60-2.89 (m, 6H), 3.50-3.60 (m, 2H), 3.92 (s, 3H), 3.98 (s, 3H), 6.92 (d, J = 8.4 Hz, 1H),
	7.44 (dd, J = 8.3, 1.7 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.85 (s, 1H), 8.01 (s, 1H), 8.11 (s, 1H), 8.41 (s, 8-amino-2-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenethyl)imidazo[1,2-a]pyridine-6-carboxamide
	1 H NMR (CDCl3, 300 MHz) δ(ppm): 1.55 (s, 9H), 1.80 (s, 2H), 2.51 (d, J = 13.7 Hz, 8H), 3.58 (s, 2H), 3.72 (s, 4H), 3.92 (s, 3H), 3.98 (s, 3H), 6.93 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 6.8 Hz, 1H), 7.54 (s, 1H), 7.81 (d, J = 2.3 Hz, 1H), 7.87 (s, 1H), 8.06 (s, 2H), 8.52 (s, 1H). MS (ESI + ): m/z = 540.3 [M,H] + found; C28H37N5O6 calculated m/z = 540.3 [M,H] + . Tert-Butyl N-[2-(3,4-dimethoxyphenyl)-6-{[3-(piperazin-1-yl)propyl]carbamoyl}imidazo[1,2-a] 1H), 8.64 (s, 1H). MS (ESI + ): m/z = 538.4 [M,H] + found; C29H40N5O5 calculated m/z = 538.4 [M,H] + . Tert-Butyl N-[2-(3,4-dimethoxyphenyl)-6-{[3-(4-methylpiperazin-1-yl)propyl]carbamoyl}imidazo [1,2-a]pyridin-8-yl]carbamate (32). Compound 32 was obtained following the general procedure D from the activated ester 26 (80 mg, 0.16 mmol) and the 3-(4-methylpiperazin-1-yl)propan-1-amine (1.0 eq, 0.16 mmol) after purification by flash chromatography (CH2Cl2 to CH2Cl2/MeOH, 96:4) as a white solid (52 mg, 60%). 1 H NMR (CDCl3, 300 MHz) δ(ppm): 1.52 (s, 9H), 1.75-1.87 (m, 2H), 2.27 (s, 3H), 2.44-2.79 (m, 10H), 3.54 (s, 2H), 3.90 (s, 3H), 3.94 (s, 3H), 6.90 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 1.5 Hz, 1H), 7.81 (s, 1H), 7.98-8.19 (m, 3H), 8.50 (s, 1H). MS (ESI + ): m/z = 553.4 [M,H] + found; C29H41N6O5 calculated m/z = 553.4 [M,H] + . methoxyethoxy)phenyl]piperazin-1-yl}ethan-1-amine (1.0 eq, 0.14 mmol) after purification by flash chromatography (CH2Cl2 to CH2Cl2/MeOH, 96:4) as a beige solid (60 mg, 65 %). 1 H NMR (CDCl3, 300 MHz) δ(ppm): 1.48 (s, 9H), 2.73 (s, 6H), 3.20 (s, 4H), 3.45 (dd, J = 3.4, 2.2 Hz, 3H), 3.61 (s, 2H), 3.68-3.77 (m, 2H), 3.93 (s, 3H), 3.99 (s, 3H), 4.07 (s, 2H), 6.90 (dt, J = 16.4, 5.5 Hz, 5H), 7.23-7.32 (m, 1H), 7.46 (dd, J = 8.2, 1.8 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.98 (s, 1H), 8.12 (s, 1H), 8.54 (s, 1H). MS (ESI + ): m/z = 675.5 [M,H] + found; C36H47N6O7 calculated m/z = 675.5 [M,H] + . hydrochloride (35). 96%.
	pyridin-8-yl]carbamate (30). Compound 30 was obtained following the general procedure D from the
	activated ester 26 (43 mg, 0.08 mmol) and the 3-(piperazin-1-yl)propan-1-amine (1.0 eq, 0.08 mmol) General method E. Intermediates 5 to 11 (0.10 mmol) was dissolved in dry dioxane HCl (4M) (2 mL) 8-amino-2-(3,4-dimethoxyphenyl)-N-(3-(piperazin-1-yl)propyl)imidazo[1,2-a]pyridine-6-carboxamide
	after purification by flash chromatography (CH2Cl2 (1% TEA) to CH2Cl2/MeOH, 9:1+1%TEA) as a and heated at 60°C for 2h. Reaction was monitored by LCMS. The mixture was evaporated to dryness, trihydrochloride (37).
	white solid (23 mg, 51%). 1 H NMR (CDCl3, 300 MHz) δ(ppm): 1.41-1.48 (m, 1H), 1.56 (s, 9H), 1.81 dried several hours and washed by CH2Cl2 to afford expected products.
	(dd, J = 12.4, 6.2 Hz, 2H), 2.49-2.56 (m, 6H), 2.84-3.02 (m, 4H), 3.56 (dd, J = 11.4, 5.8 Hz, 2H), 3.92
	(s, 3H), 3.99 (s, 3H), 6.93 (d, J = 8.4 Hz, 1H), 7.46 (dd, J = 8.3, 1.9 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H), (8-amino-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyridin-6-yl)(4-(4-(2-methoxyethoxy)phenyl)
	piperazin-1-yl)methanone hydrochloride (34). Compound 34 was obtained following the general 7.83 (s, 2H), 8.03 (s, 1H), 8.05 (d, J = 1.1 Hz, 1H), 8.52 (d, J = 1.4 Hz, 1H). MS (ESI + ): m/z = 539.4 [M,H] + found; C28H39N6O5 calculated m/z = 539.4 [M,H] + . procedure E from the intermediate 27 (0.13 mmol) as a grey solid (30 mg, 63%). 1 H NMR (MeOD, 300
	MHz) δ(ppm): 3.15 (s, 4H), 3.32 (s, 4H), 3.42 (s, 3H), 3.70-3.74 (m, 2H), 3.92 (s, 3H), 3.97 (s, 3H),

Tert-Butyl

N-[2-

(3,4

-dimethoxyphenyl)-6-{

[3-(piperidin-1-yl)

propyl]carbamoyl}imidazo

[1,2-a]

pyridin-8-yl]carbamate

[START_REF] Milne | Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor[END_REF]

. Compound 31 was obtained following the general procedure D from the

Tert-Butyl N-[2-(3,4-dimethoxyphenyl)-6-[(2-{4-[4-(2-methoxyethoxy)phenyl]piperazin-1-yl}ethyl) carbamoyl]imidazo[1,2-a]pyridin-8-yl]carbamate

[START_REF] Fredholm | Aspects of the general biology of adenosine A2A signaling[END_REF]

. Compound 33 was obtained following the general procedure D from the activated ester 26 (70 mg, 0.14 mmol) and the 2-{4-[4-(2-4.04-4.09 (m, 2H), 6.85-6.95 (m, 3H), 6.97-7.04 (m, 2H), 7.15 (d, J = 8.7 Hz, 1H), 7.45-7.52 (m, 2H), 8.20 (d, J = 1.2 Hz, 1H), 8.38 (s, 1H). 13 C NMR (MeOD, 75 MHz) δ(ppm): 49.0, 52.6, 56.6, 56.8, 59.2, 68.88-amino-2-(3,4-dimethoxyphenyl)-N-(3-morpholinopropyl)imidazo[1,2-a]pyridine-6-carboxamide dihydrochloride (36).

Acknowledgments

We express our thanks to Daniel Ryan Parle, Joshua Sharp, Andrew Montgomery, Mickaël Belaud-Rotureau, Valeria Moas-Heloire and Noémie Deguine for their contribution in organic synthesis and Séverine Ravez for in vitro binding experiments. The 300 MHz NMR facilities were funded by the Région Nord-Pas de Calais (France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER). We thank the Lille University Binding facility headed by Christophe Furman. This work was supported by Lille 2 University, ANR « Adoratau », PRES Univ Lille Nord de France. Romain Duroux and Raphaël Bolteau are recipients of a fellowship from Lille 2 University.

of Boc2O (3.0 eq, 497 mg) dissolved in dry THF (4 mL) was added and the reaction mixture was stirred at room temperature for 3h. The mixture was quenched by saturated solution of ammonium chloride (25 mL)

Displacement binding assays

Radioligands were obtained from the following source: [ 3 H]-ZM241385 from American Radiolabeled Chemicals (ART 0884: 1 mCi/ml / 50 Ci/mmol). For A2A receptor binding evaluation, the stock solution of compounds was prepared in DMSO (10mM). The final concentration of DMSO was no more than 3% for radioligand binding assay. Competition binding curves of the A2A receptor antagonist [ 3 H]-ZM-241385 by the designed A2A antagonists described above, were performed as before [START_REF] Lopes | Cross talk between A(1) and A(2A) adenosine receptors in the hippocampus and cortex of young adult and old rats[END_REF] Displacement reference curves were performed with ZM-241385 (Bio-Techne Europe, Lille, Ref: 1036) from 0 to 24 nM with a Ki of 4.2 nM in accordance with the literature. For Ki determination, a 10 µM screening first enabled us to select promising compounds. Ki of selected compounds (ie 9, 10, 16 and 17) were measured using the following range of concentrations: from 6.10 -9 M to 3.6.10 -4 M.

Cell culture and cytotoxicity assay

The human neuroblastoma cell line (SY5Y) was cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco, Waltham, MA) supplemented with 2mM L-glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin, 1mM non-essential amino acids and 10% (v/v) heat-inactivated foetal bovine serum (Sigma-Aldrich, Saint-Louis, MO), and grown at 37 °C in a humidified incubator with 5% CO2. Cells were seeded at 10000 cells per well onto 96-well plates in DMEM medium. Cells were incubated in a culture medium that contained various concentrations of test compounds, each dissolved in less than