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Discovery of Potent and Highly Selective A Adenosine Receptor Antagonist Chemotypes Abdelaziz El Maatougui, Jhonny Azuaje, Manuel González-Gomez, Gabriel Míguez, Abel Crespo, Carlos Carbajales, Luz Escalante, Xerardo Garcia-Mera, Hugo Gutiérrez-de-Terán, and Eddy Sotelo J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01586 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Journal of Medicinal Chemistry

Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes

Abdelaziz El Maatougui,1,2 Jhonny Azuaje,1,2 Manuel González-Gómez,1,2 Gabriel Miguez,1,2 Abel Crespo,1,2 Carlos Carbajales,1,2 Luz Escalante,1,2 Xerardo García-Mera,2 Hugo Gutiérrez-de-Terán3* and Eddy Sotelo1,2*

1

Centro Singular de Investigación en Química Biológica y Materiales Moleculares

(CIQUS), 2Departamento de Química Orgánica, Facultad de Farmacia. Universidade de Santiago de Compostela, E-15782. Santiago de Compostela, Spain. 3Department of Cell and Molecular Biology. Uppsala University, Uppsala SE-75124.

Key words: Biginelli reaction, homology modeling, ligand docking

*Corresponding authors: ES: +34 881815732, Fax: +34 881815704, e-mail: [email protected] HGT: +4618 471 5056, Fax: +46-(0)184715056, e-mail: [email protected]

ACS Paragon Plus Environment


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Abstract: Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series’ contain imidazole, 1,2,4-triazole or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled to identify a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behaviour of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.


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INTRODUCTION The endogenous purine nucleoside adenosine is an omnipresent intermediary metabolite released from almost all mammalian cells.1 Adenosine is structurally and metabolically related to the bioactive nucleotides2 [adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP)], RNA and the coenzymes A, NAD and FAD.2 In addition, extracellular adenosine functions as a signaling molecule that mediates a plethora of physiological effects through the stimulation of a family of G-protein-coupled receptors (GPCRs) named adenosine receptors (ARs).3 Human ARs, named A1, A2A, A2B and A3, exhibit high sequence homology4, and are usually clustered into A1 and A3 (49% sequence identity,) and A2A and A2B (59%). Notwithstanding, ARs also differ in their affinity for adenosine (0.3−0.7 M for A1, A2A and A3 vs. 24 M for A2B),5 the tissue localization, and the G-protein mediated downstream signaling pathways that they promote. Adenosine receptors have been implicated in several key physiological processes,1-2,6 and consequently the different ARs are considered attractive drug targets in diverse pathophysiological events related to modified adenosine levels.6-9

Since its discovery as a low-affinity receptor in the early 1990s,10 A2B adenosine receptor (A2BAR) has proven to be enigmatic in its function. Ubiquitously expressed in the body (e.g., lung, brain, gastrointestinal and urogenital tracts and cardiovascular and immune systems),10-14 its high similarity with A2A adenosine receptor (A2AAR), which exhibits significantly greater affinity for adenosine, led to the initial perception that A2BAR was not of substantial physiological relevance. However, recent evidence shows that A2BAR is transcriptionally regulated by factors implicated in inflammatory hypoxia and is involved in relevant biological process such as control of the vascular tone,



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cardiac myocite contractility, colonic motility, penile erection, glucose homeostasis, pulmonary inflammation, inflammatory response and pain.15 Curiously, the reason for the initial low interest in this receptor (namely its low affinity for adenosine) might make it an excellent therapeutic target that only becomes activated under pathological conditions (when adenosine levels increase to micromolar concentrations). Although abundant evidence exists regarding the pathophysiological implications of A2BAR in peripheral diseases,11-14 knowledge of its functions in the central nervous system remains poor.14-15

The interest in A2BAR in different therapeutic areas is currently increasing significantly,14-15 nonetheless the compendium of potent and selective ligands and molecular probes targeting the A2BAR remains scarce.16-17 Some promising A2BAR antagonists are currently under clinical trials in the treatment of different pathologies [e.g., diabetes18 and its related retinopathy,19 as well as asthma and chronic obstructive pulmonary disease (COPD)20]. The pursuit of A2BR antagonists has traditionally focused on the xanthine scaffold.7-9 The pharmacomodulation of naturally occurring xanthines (Figure 1, Cpds 1-2,)21 through the introduction of alkyl groups at positions 1 and/or 3 (Figure 1, Cpd 3),22 but most importantly of functionalized phenyl (Figure 1, Cpds 4-5)23-24 or heteroaryl (Figure 1, Cpds 6-8)25-27 moieties at position 8 of the heterocyclic core, has produced a plethora of selective xanthine A2BAR antagonists. Other xanthine-like compounds (Figure 1, Cpds 9-11)28-30 and some tricyclic derivatives (e.g. 12-13, Figure 1)31-32 have been also described. In contrast, very few examples of potent and selective non-xanthine A2BAR antagonists have been described (Figure 1, Cpds 15-16).33-35


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Figure 1. Structures of representative A2BAR antagonists.21-35 Xanthine congeners clearly predominate within the A2B antagonists as pharmacological tools and clinical candidates (Figure 1). Accordingly, there is a demand for the discovery of new families from unexplored diversity spaces. These



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scaffolds would provide novel topologies, physicochemical features and alternative binding modes. This might facilitate receptor specificity, with advantages as potential drugs or as pharmacological tools for the validation of emerging therapeutic applications for the A2BAR subtype (e.g., in the CNS).14,15 In this context, we recently carried out a preliminary exploration of the SARs of novel A2BAR antagonists based on the 3,4-dihydropyrimidin-2(1H)-one scaffold.35 In addition to its simple structure, excellent pharmacological profile and synthetic feasibility, to the best of our knowledge this chemotype provides the first examples of non-planar selective A2BAR antagonists. Taking advantage of the unique structural features of chemotype 16 (Figure 2), we decided to employ diverse medicinal chemistry design criteria to explore the diversity spaces around this attractive chemotype. We herein report here the preliminary results of this program and document three novel scaffolds that show highly appealing potency and selectivity antagonistic profiles toward A2BARs.

RESULTS AND DISCUSION

On the basis of the proposed binding mode and the SAR trends reported previously for chemotype 16,35 in this study we retained the most advantageous substituents at positions 4 (2-furyl, 3-furyl and 2-thienyl and 3-thienyl) and 5 (ethoxycarbonyl or isopropyloxycarbonyl). The previously stated criteria’s (binding mode and SAR) were also employed for scaffold prioritization among a set of 12 initial cores. The strategies employed for the design of the novel ligands are outlined in Figure 2. The selected scaffolds perfectly fit the binding pocket (Figure 2) while they contain structural features that affect the conformational, electronic, steric and lipo-/hydrophilic characteristics within the model scaffold (Figure 2, compounds 16). The first approach was the bioisosteric replacement of the NH group at position 3 of the 3,4-


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dihydropyrimidin-2(1H)-one (16) scaffold by a methylene group to generate the corresponding 3,4-dihydropyridin-2(1H)one bioisosteres 17a–h (Figure 2). The remarkable structural similarity between 3,4-dihydropyridin-2(1H)-ones 17 and the parent 3,4-dihydropyrimidin-2(1H)-ones (16) allow an evaluation of the contribution that the NH moiety at position 3 makes to the A2B receptor affinity.

As opposed to typically xanthine or purine-based heterocycles (Figure 1) the 3,4-dihydropyrimidin-2(1H)-ones (16) are the only A2BAR antagonist scaffold that does not consist on a highly planar central heterocycle, yet a promising affinity and selectivity profile was achieved. The binding mode proposed for this scaffold35 overlies with that of xanthines and purines in the A2BAR, and is dominated by interactions with hydrophobic residues and a key hydrogen bond with the conserved Asn2546.55. Taking this into consideration bicyclic (18–21) or tricyclic (22) derivatives were designed (Figure 2), thus providing planar regions fused to the central 3,4-dihydropyrimidin2(1H)one core, and the effect in affinity and selectivity was evaluated. The new derivatives (18–22) were designed by fusion of novel heterocyclic rings at either the positions 5,6- or 2,3- of the parent 3,4-dihydropyrimidin-2(1H)one core (Figure 2). Among the diverse scaffolds that could be created by this approach, in this study we explored the furo[3,4-d]pyrimidine-2,5-diones (18) and pyrrolo[3,4-d]pyrimidine-2,5diones (19) along with imidazo[1,2-a]pyrimidines (20), 1,2,4-triazolo[1,2-a]pyrimidines (21) and benzo[4,5]imidazo[1,2-a]pyrimidines (22). The comparative analysis of the biological activities of these compounds should provide valuable information regarding the groups that contribute most to the binding with the A2B receptor. The first heterobicyclic subset (18 and 19) retains the characteristic polar NH–CO–NH fragment of the central core but lacks the alkoxy residue of the ester function. In contrast, the second subclass (20–22) retains the ester moiety but does not contain one of the NH groups,



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while the carbonyl group at position 2 is substituted by the imine framework as part of the fused heterocycle (Figure 2).

Figure 2. Strategies employed for the design of the novel A2BAR antagonists, showing the initial docking of representative compounds for each scaffold on the A2BAR model.

Chemistry: In the same way as the 3,4-dihydropyrimidin-2(1H)-ones that inspired this work (16), all of the adenosine receptor antagonists described here (17–22) were assembled by employing multicomponent reactions (MCR)36 as the preparative tool, thus demonstrating the potential of the MCR-based approach for drug discovery. The synthetic strategy employed to obtain the target 3,4-dihydropyridin-2(1H)-ones 17a–h is depicted in Scheme 1. Following a previously described procedure,37 equimolar


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mixtures of Meldrum’s acid (23), a β-ketoester (24a–b), ammonium acetate and the corresponding pentagonal carbaldehyde (25a–d) in acetic acid were submitted to microwave irradiation (90 ºC) for 30–45 minutes. The heterocyclization reaction involves a Michael addition between two in situ generated intermediates [i.e., an enamine (from a β-ketoester and ammonia) and a Knoevenagel product (from Meldrum’s acid and carbaldehydes)]. Nucleophilic addition and subsequent elimination of acetone and CO2 provided the desired 3,4-dihydropyridin-2(1H)-ones 17a–h in moderate yields (50–75%).

Scheme 1. Multicomponent synthesis of the 3,4-dihydropyridin-2(1H)-ones 17a–h.

The bicyclic ligands 18 and 19 were readily prepared by the synthetic route shown in Scheme 2. The zinc chloride-catalysed Biginelli condensation of urea (26), ethyl 4chloroacetoacetate (27) and the pentagonal carbaldehydes selected for this study (25a– d) afforded a subset of 6-chloromethyl-3,4-dihydropyrimidin-2(1H)-ones (28),38 which were employed as precursors for subsequent annulation reactions (Scheme 2). The furo[3,4-d]pyrimidine-2,5-diones 18 were obtained by submitting 28 to an eliminative cyclization in dimethylformamide,38 whereas pyrrolo[3,4-d]pyrimidine-2,5-diones 19 were readily obtained by microwave heating of 28 with ethylamine.39



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Scheme 2. Three-component synthesis of the 3,4-dihydropyrimidin-2(1H)-ones 28 and their transformation into the bicyclic compounds 18a–d and 19a–d.

As for the previous target structures (17–19), the ligands bearing heterocycles fused to the 2,3-positions of the pyrimidine core (20–22) were assembled by a threecomponent Biginelli condensation (Scheme 3)40,41 that employs heterocyclic surrogates of the urea component [i.e., 2-aminoimidazole (29a), 2-amino-1,2,4-triazole (29b) or 2aminobenzimidazole (29c)] as key precursors in combination with the carbaldehydes (25a–d) and the corresponding alkyl acetoacetates (24a–b). Preliminary optimization of the synthesis showed that the synergic use of chloroacetic acid (as a catalyst) and microwave irradiation (as energy source) resulted in a striking increase in the yields of these compounds. An exhaustive description of the synthetic methods as well as the complete structural and spectroscopic data for all compounds is provided in the experimental part.


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Scheme 3. Three-component Biginelli synthesis of ligands 20a–h, 21a–h and 22a–h.

Biological Evaluation The affinities for the four (A1, A2A, A2B and A3) human adenosine receptor subtypes of the 8 reference 3,4-dihydropyrimidin-2(1H)-ones 16,35 and the 40 new ligands (17–22) were determined in vitro with radioligand binding assays, according to experimental protocols described elsewhere,42 and reported in Tables 1-5. In the previous35 and current study, all compounds were evaluated as racemic mixtures. In this sense, three of the compounds in the series (16b, 21b and 22b) are currently being separated to give pure enantiomers with the aim of determining the influence of the configuration at C4 on the A2BAR affinity for these structurally related chemotypes.

Human adenosine receptors expressed in transfected CHO (A1R), HeLa (A2AAR and A3AR) and HEK-293 (A2BR) cells were employed. (3H)-1,3-Dipropyl-8-



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cyclopentylxanthine ([3H]DPCPX) for A1AR and A2BAR, [3H]ZM241385 for A2AAR and [3H]NECA for A3AR were employed as radioligands in binding assays. The biological data (Tables 1–4) are expressed as Ki ± SEM (nM, n = 3) or percentage inhibition of specific binding at 1 µM (n = 2, average) for those compounds that did not fully displace radioligand binding. For the sake of comparison, the biological data obtained for the 3,4-dihydropyrimidin-2(1H)-one parent series (16) are also included (Table 1).

Table 1. Structure and affinity binding data for the 3,4-dihydropyrimidin-2(1H)-ones 16 at the human adenosine receptors.35

Compound

X

Y

R

Ki (nM) or % at 1 µM hA2Ab) hA2Bc) 41% 585.5 ± 61

16a

O

CH

Et

hA1a) 18%

16b

O

CH

i-Pr

20%

30%

40.80 ± 3.1

1%

16c

CH

O

Et

21%

39%

39.6 ± 2.9

1%

16d

CH

O

i-Pr

25%

26%

1486 ± 41

3%

16e

S

CH

Et

1%

33%

44%

1%

16f

S

CH

i-Pr

37%

19%

44%

14%

16g

CH

S

Et

22%

39%

23.60 ± 1.3

24%

16h

CH

S

i-Pr

26%

25%

56.70 ±2.9

1%

DPCPX

-

-

-

2.20 ± 0.2

157 ± 2.9

73.24 ± 2.0

1722 ± 11

ZM241385

-

-

-

683 ± 4.1

1.9 ± 0.1

65.7 ± 1.7

863 ± 4.0

a

hA3d) 2%

Displacement of specific [3H]DPCPX binding in human CHO cells expressed as Ki ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 µM (n = 2). b Displacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol binding in human HeLa cells expressed as Ki ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 µM (n = 2). c Displacement of specific [3H]DPCPX binding in human HEK-293 cells expressed as Ki ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 µM (n = 2). d Displacement of specific [3H]NECA binding in human HeLa cells expressed as Ki ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 µM (n = 2).


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Table 2. Structure and affinity binding data for the 3,4-dihydropyridin-2(1H)-ones 17a–h at the human adenosine receptors.

Compound

X

Y

R

Ki (nM) or % at 1 µM hA2Ab) hA2Bc) 13% 2536 ± 82

17a

O

CH

Et

hA1a) 22%

17b

O

CH

i-Pr

14%

14%

15%

31%

17c

CH

O

Et

9%

8%

14%

15%

17d

CH

O

i-Pr

23%

25%

20%

24%

17e

S

CH

Et

16%

11%

18%

21%

17f

S

CH

i-Pr

2%

1%

3%

22%

17g

CH

S

Et

1%

4%

12%

13%

17h

CH

S

i-Pr

4%

3%

21%

20%

DPCPX

-

-

-

2.20 ± 0.2

157 ± 2.9

73.24 ± 2.0

1722 ± 11

ZM241385

-

-

-

683 ± 4.1

1.9 ± 0.1

65.7 ± 1.7

863 ± 4.0

a

hA3d) 24%




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Table 3. Structure and affinity binding data for the furo[3,4-d]pyrimidine-2,5-diones 18a–d and pyrrolo[3,4-d]pyrimidine-2,5-diones 19a–d at the human adenosine receptors. Y X O H O

Compound

X

Y

N

Z N H

Z a)

Ki (nM) or % at 1 µM hA2Ab) hA2Bc) 2% 2%

hA3d) 3%

18a

O

CH

O

hA1 3%

18b

CH

O

O

9%

3%

9%

1%

18c

S

CH

O

1%

1%

10%

1%

18d

CH

S

O

13%

3%

8%

2%

19a

O

CH

N-Et

7%

7%

17%

20%

19b

CH

O

N-Et

16%

13%

35%

17%

19c

S

CH

N-Et

12%

2%

28%

21%

19d

CH

S

N-Et

27%

17%

18%

31%

DPCPX

-

-

-

2.20 ± 0.2

157 ± 2.9

73.24 ± 2.0

1722 ± 11

ZM241385

-

-

-

683 ± 4.1

1.9 ± 0.1

65.7 ± 1.7

863 ± 4.0

a



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Table 4. Structure and affinity binding data for the imidazo[1,2-a]pyrimidines 20a–h and the 1,2,4-triazolo[1,2-a]pyrimidines 21a–h at the human adenosine receptors. Y X O Q N N

Compound

Q

X

Y

O N H

R

Me

R

Ki (nM) or % at 1 µM hA2Ab) hA2Bc) 5% 108.0 ± 6

20a

CH

O

CH

Et

hA1a) 2%

20b

CH

O

CH

i-Pr

3%

4%

85.60 ± 4

2%

20c

CH

CH

O

Et

2%

1%

12%

3%

20d

CH

CH

O

i-Pr

1%

4%

276.9 ± 3

10%

20e

CH

S

CH

Et

2%

2%

8%

4611 ± 23

20f

CH

S

CH

i-Pr

3%

3%

728.1 ± 5

5%

20g

CH

CH

S

Et

1%

3%

10%

2%

20h

CH

CH

S

i-Pr

3%

1%

847.3 ± 4

1%

21a

N

O

CH

Et

15%

20%

3%

4%

21b (ISAM-148)

N

O

CH

i-Pr

2%

5%

55.60 ± 4

2%

21c

N

CH

O

Et

2%

1%

602.4 ± 10

4%

21d

N

CH

O

i-Pr

2%

2%

350.5 ± 8

20%

21e

N

S

CH

Et

1%

2%

15%

15%

21f

N

S

CH

i-Pr

1%

1%

1%

3%

21g

N

CH

S

Et

3%

21%

699.2 ± 5

2%

21h

N

CH

S

i-Pr

3%

2%

816.7 ± 7

5%

DPCPX

-

-

-

-

2.20 ± 0.2

157 ± 2.9

73.24 ± 2.0

1722 ± 11

ZM241385

-

-

-

-

683 ± 4.1

1.9 ± 0.1

65.7 ± 1.7

863 ± 4.0

a

hA3d) 6%




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Table 5. Structure and affinity binding data for the benzo[4,5]imidazo[1,2-a] pyrimidines 22a–h at the human adenosine receptors. Y X O N N

Compound

X

Y

O N H

R

R

Me

Ki (nM) or % at 1 µM hA2Ab) hA2Bc) 14% 12.03 ± 0.7

22a (ISAM-134)

O

CH

Et

hA1a) 5%

22b (ISAM-140)

O

CH

i-Pr

20%

25%

3.49 ± 0.2

2%

22c (ISAM-141)

CH

O

Et

7%

11%

20.60 ± 1.1

1%

22d (ISAM-142)

CH

O

i-Pr

12%

22%

11.40 ± 0.5

2%

22e

S

CH

Et

8%

16%

484.6 ± 3

1%

22f

S

CH

i-Pr

1%

17%

371.2 ± 5

3%

22g

CH

S

Et

3%

10%

29.71 ± 1.2

2%

22h

CH

S

i-Pr

11%

3%

29.34 ± 1.1

21%

DPCPX

-

-

-

2.20 ± 0.2

157 ± 2.9

73.24 ± 1.4

1722 ± 11

ZM241385

-

-

-

683 ± 4.1

1.9 ± 0.1

65.7 ± 1.1

863 ± 4.0

a

hA3d) 1%



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Functional Assay at Adenosine A2B Receptors

As part of the pharmacological characterization, two representative derivatives of the series’ under investigation (21b and 22b) were tested in cAMP assays in order to evaluate their ability to inhibit NECA-stimulated (100 nM) cAMP production (see supporting information). These experiments demonstrated that 21b and 22b inhibit cAMP accumulation and this finding validated the antagonistic behavior of these compounds at A2BAR. A comparison of their Ki and KB values reveals a good correlation between data obtained from binding (21b, Ki = 55.60 nM; 22b, Ki = 3.49 nM) and functional assays (KB = 265.50 nM and 27.00 nM, respectively).

Structure-Activity Relationship and Molecular Modeling

Examination of the binding data for the novel compounds (Tables 2–5) reveals some potent and highly selective ligands for the A2BAR, e.g. compounds 20b and 21b (Table 4) and compounds 22a, 22b, 22c, 22d, 22g and 22h (Table 5). The significant increase in affinity observed for tricyclic compounds (22), when compared to the parent series (compounds 16a–h, Table 1), demonstrates the benefits of a bicyclic heterocyclic framework fused at the 2,3-positions of the pyrimidine moiety. These and other structure-affinity relationships, along with some structural indicators of selectivity, will be discussed in a comprehensive manner by combination of three kinds of analyses. Thus, the most prominent features of the structure-activity relationships (SAR) are initially analyzed within each series of compounds. Where relevant, i.e., for the most successful series 20, 21 and 22, a comparative analysis of the binding affinities with those of the parent series 16 is provided. Finally, the global SAR data are evaluated in the context of a computational model of the A2BAR.



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The structural model of the ligand-receptor interactions presented here is based on our recent work explaining the binding mode of the parent series 16 to the A2BAR,35 as part of our adenosine receptor ligand design program.43 In the first stage all the designed compounds were docked to the A2BAR homology model. The conserved binding mode identified in our previous work for compound 16g (S)35 [therein denoted as 21a (S)] was achieved for the equivalent stereoisomer in most compounds, and in particular for all compounds with affinity for the A2BAR as confirmed in radioligand binding assays. It is worth noting that, due to the different substituents on each scaffold, the R/S nomenclature does not indicate the 3D equivalence of the stereoisomers and the isomers that are equivalent in 3D space were considered (Figure 3). We recently noted that the NH in position 1 of the parent 3,4-dihydropyrimidin-2(1H)-ones (16) could form a water-mediated hydrogen bond with Glu169EL2, a hypothesis that was validated by a molecular dynamics (MD) analysis of the A2B-16g(S) complex.43 A closer inspection of the binding mode shows that this possibility holds for all compounds with affinity for A2BAR (Figure 3). Moreover, a second potential position for a water molecule was identified in the region between the alkoxy-substituent and the furan/thienyl ring, which is actually equivalent to a conserved water molecule observed in the crystal structures of A2A with 4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5a][1,3,5]triazin-5-ylamino)ethyl)phenol (30).44,45 A second round of docking runs was therefore designed for the compounds that bind A2BAR in the nM range, where these potential water molecules were considered using the corresponding function in GOLD. This process resulted in a refined binding mode, which was observed for all compounds considered. These refined docking models will be used as a basis for the establishment of structure-activity relationships for the novel compounds in the next paragraphs.


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Figure 3. Initial binding mode proposed for the equivalent stereoisomer of the compounds with nM affinity for the A2BAR (see Tables 1–5). The two water molecules that mediate intra- and intermolecular contacts are represented in sticks. Dashed lines depict hydrogen bonds.

Inspection of the pharmacological data obtained for the 3,4-dihydropyridin2(1H)-ones 17a−h (Table 2) reveals that, regardless of the alkoxy residue in the ester moiety or the substituent at position 4, the bioisosteric NH/CH2 replacement at position 3 of the heterocycle completely cancelled the high affinities observed in the parent 3,4dihydropyrimidin-2(1H)-ones 16a−h (Table 1). Only one compound in this series showed affinity in the micromolar range towards the A2B receptor (compound 17a, Table 2, Ki = 2536 nM) and this is 5 times less potent that its parent analog (16a). These data are consistent with the drop in affinity observed after previous modifications of the nitrogen atom at position 3 (e.g., acetylation or aromatization of the heterocyclic core).35 However, the molecular models did not show any direct interaction by the NH group at position 3 of the parent 3,4-dihydropyrimidin-2(1H)-ones 16a−h and not even a water-mediated interaction was envisaged from the previous MD simulations of the A2B-16g(S) complex.43 This situation is consistent with the moderate-to-high affinity of compounds from series 20–22, which do not bear a hydrogen-bond donor on the equivalent position (Tables 4 and 5). The lack of affinity of the 3,4-dihydropyridin-



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2(1H)-ones 17a−h could therefore be due to their apparent inability to achieve the bioactive conformation. A direct comparison of the potent compound 16b with its analog 17b (Figure 4A) reveals that the conformation of the heterocycle in the 3,4dihydropyridin-2(1H)-ones (17) is less planar than in the parent 3,4-dihydropyrimidin2(1H)-ones (16). This change has an effect on the preferred conformation of the alkoxy moiety, which does not reach the equivalent bioactive conformation with the watermediated hydrogen bond described for the parent compound 16b.

Figure 4. (A) Biological alignment of compound 16b (purple) with 17b (cyan) and (B) with compounds 18b (cyan) and 19b (green). (C) Binding mode of compound 20b (cyan) superimposed to 16b (purple). (D) Binding mode of the most potent compound 22b. The Connolly surface is shown in gray for the residues in the binding site.


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The furo[3,4-d]pyrimidine-2,5-diones 18a–d and the pyrrolo[3,4-d]pyrimidine2,5-diones 19a–d (Table 3) were designed to explore the effect of fusing a heterocyle at the 5,6-positions of the pyrimidin-2(1H)-one core. The compounds in these series retain the carbonyl group at position 5, thus allowing the conserved polar interaction with Asn2546.55. The alkoxy residue in scaffold 16 is replaced by a conformationally constrained fused heterocyclic ring (i.e., furanone or pyrrolone), although the pyrrol-2ones 3 also retain a flexible ethyl chain on the nitrogen atom. However, both subsets are completely inactive at the four-adenosine receptors (Table 3), a finding that strongly supports a negative effect of heterocyclization on face d of the pyrimidin-2(1H)-one core. A molecular superposition of the bioactive conformation of selected examples (18b and 19b) with the pyrimidin-2(1H)-one 16b is presented in Figure 4B. It can be observed that the carbonyl group of the furan-2-one or pyrrol-2-one rings would displace the water molecule that links the furan and ester functions in the most potent compounds, and this probably influences the preferred conformation of the furan/thiophene ring at position 4, all of which could be the cause of the negligible binding to the A2BAR.

The bicyclic and tricyclic derivatives 20–22 were the most interesting compounds identified during this study. These series include a monocyclic (imidazole, series 20, or 1,2,4-triazole, series 21) or bicyclic (benzimidazole, series 22) ring fused to the parent pyrimidin-2(1H)-one core (16). This has two structural consequences when compared with the parent compound series: the loss of the NH group at position 3 and the bioisosteric replacement of the carbonyl group at position 2 by the imine framework of the heterocycle. At the same time, the alkoxy function is retained in all compounds in these series and both i-Pr and Et substitutions were explored. With the aim of



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facilitating the comparative analysis of the SAR within these compound series, the affinities for the A2BAR are represented graphically in Figure 5.

Figure 5. Effect of the nature of the heterocyclic ring at position 4 (2- or 3furyl/thienyl, indicated by different shading) and the alkoxy substituent at the ester group (i-Pr, blue colors, Et, brown colors) on the most potent series 20, 21 and 22. Data for the parent structures are also shown (series 16).

Pharmacological evaluation of the eight imidazo[1,2-a]pyrimidines (20a–h) highlighted ligands that exhibit weak to moderate affinity (Table 4), although the excellent selectivity profile observed for the parent pyrimidin-2(1H)-one series is maintained (see Table 4). The most promising derivative within this subset (20b, Ki = 85.60 nM) elicits a 2-fold reduced affinity with respect to its congener in the parent series (16b Ki = 40.80 nM). Interestingly, the opposite effect was observed for compound 20a (Ki = 108.0 nM), which exhibited an affinity that was five-times higher


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than that of 16a (Ki = 585.5 nM). From data presented in Table 4, it follows that compounds bearing isopropyl residues at the ester moiety systematically provide better affinity than their ethyl analogs (cf. pairs 20a/20b, 20c/20d, 20e/20f, 20g/20h). Another trend within this series is that the furan ring (2- or 3-substituted) is better tolerated than its thienyl congener.

The fusion of a 1,2,4-triazole ring at the 2,3-positions of the pyrimidin-2(1H)one scaffold (thus generating the 1,2,4-triazolo[1,2-a]pyrimidines 21) produces derivatives with attenuated affinity in comparison to the parent series (16), nevertheless the outstanding selectivity profile was maintained. The most striking compound (21b, Ki = 55.60 nM) shows the same potency range as its congener in the parent series (16b, Ki = 40.80 nM). In contrast with series 20, compounds bearing isopropyl residues at the ester moiety were better than its ethyl analogues only for furan containing compounds. It should be noted that, as observed for the parent series (16), the 1,2,4-triazolo[1,2a]pyrimidines bearing a 2-thienyl group (21e and 21f) are completely inactive (Table 4).

The binding mode of the bicyclic compounds 20a-h and 21a-h shows the expected molecular superposition with the rest of the series, thus confirming the equivalence of the different substituents discussed above (Figure 4C). Here, the atom denoted as ‘Q’ (Table 4) is perfectly superimposed with the hydrogen on the N at position 3 in the parent series (16). In these fused-ring series, where several compounds show A2BR affinity in the nanomolar range, this atom is either non-polar (CH, series 20) or a hydrogen bond acceptor (N, series 21). This finding confirms the modeling results



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obtained for compounds in series 16 and 17, i.e., that this position does not have any relevant interaction with the receptor.

The biological data obtained for the benzo[4,5]imidazo[1,2-a]pyrimidine subset (22a–h) unequivocally show the determinant influence on the binding profile of this series of introducing a third ring. The tricyclic derivatives 22a–h (Table 5) are the most attractive ligands reported here, with derivatives 22a, 22b and 22d combining outstanding affinity for the A2BAR (Ki = 12.03 nM, 3.49 nM and 11.40 nM, respectively) with remarkable selectivity (Ki ≥ 1000 nM) towards the remaining ARs (Table 5). Interestingly, the features that govern the SARs of compounds 22a–h follow the same trends identified in the previous series. Thus, compounds bearing furyl derivatives exhibit superior affinity than their thienyl analogs (cf. 22a/22d and 22e/22h) and i-Pr is always better tolerated than ethyl in the ester group (cf. 22a/22b, 22c/22d and 22e/22f). A comparison of the biological data for compounds in series 22 with their equivalent derivatives in the parent series 16 (Table 1) or the debenzo-analogs in series 20 (Table 4) reveals a remarkable increase in affinity of 2–1000 fold in all cases, except for the pair 16g/22g where an increase in affinity was not observed (see Figure 5). The structural reason for this enhanced affinity is envisaged in the computational models (see Figure 4D). It can be observed that the benzo moiety is perfectly accommodated in the aromatic cavity defined by residues Trp2476.48 and His2516.52 in TM6. The former residue is one of the most conserved amino acids in class-A GPCRs and it has been related with receptor activation,46 whereas His2516.52 is a smaller Ser in the A3AR, which could contribute to the marked selectivity against this receptor.

Finally, a better understanding of the SAR discussed here can be obtained by


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direct comparison of our models with the crystal structures of the most similar receptor, the A2AAR in complex with two different antagonist chemotypes. The most potent compound reported here (22b) is superimposed on the triazolotriazine 30 (PDB code 4EIY),45 a compound that also binds the A2BAR in the nM affinity range (Figure 6A). It can clearly be seen that the direct hydrogen bond of the exocyclic amine in 30 with Asn2546.55, which is also typical of other A2AAR antagonists,47 is replaced in our models by a water-mediated hydrogen-bond, whereas the second hydrogen bond with Asn2546.55 is achieved through the N in the heterotricyclic ring (analogous to the N in the bicyclic compounds 20 and 21 and the carbonyl in the rest of the series). More interesting is the perfect superposition of the phenyl ring of the tricyclic scaffold with the furan ring in 30, thus confirming the optimal interactions of this moiety with the cavity defined by Trp2476.48 and His2516.52. In Figure 6B compound 16b, which represents the parent scaffold, is superimposed on the prototypical AR antagonist caffeine, which is a micromolar binder on the ARs (PDB code 3RFM).48 The equivalence of the carbonyl group of scaffold 1 with O6 of caffeine is clear, along with the proximity of the oxygen of the 2-furyl substituent with O2 of caffeine, in the hydrated region of the binding cavity. This model also supports the lack of interaction of the N(H) at position 3, a group that we anticipate is needed to maintain the pseudoplanarity of the most successful scaffolds (16, 20–22). While the similarity between the binding pockets of the A2A and A2B receptors is quite high, one of the few aminoacid differences is the specific Val2506.51 in A2B, which is Leu in all other ARs.35,42,43 The different compound series display a characteristic V-shape between the core heterocycle and the furyl/thienyl ring, which can be perfectly accommodated by the smaller Val in A2B (see Fig. 4). As previously indicated for the parent series 16,42,43 this might provide rationale to the observed A2B specificity of these scaffolds.



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Figure 6. Superposition of representative compounds of these series onto AR antagonists crystallized with A2AAR: (A) the tricyclic compound 22b as docked on the A2B receptor (cyan carbons) is overlaid with the triazolotriazine antagonist 30 (gray carbons). Water molecules from this crystal structure are shown as spheres; in comparison to our predicted water molecules in A2BR (sticks) (B) Compound 16b of the parent monocyclic series (magenta) is superimposed onto caffeine (B, gray carbons).

Although the most potent ligand identified in the frame of this work [Compound 22b (Ki = 3.49 nM)] remains lightly less potent than the reference A2BAR antagonist N(4-Cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (MRS-1754),24 the herein documented lead compound (22b) exhibits superior selectivity profile (Table 5) and synthetic feasibility. It should be also noticed that 22b is obtained in just one synthetic step from commercial precursors (while the above mentioned reference compound requires at least 6 steps) in an environmentally friendly multicomponent process that produces water as side-product, thus documenting the methodological advantages of MCR in drug discovery.


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CONCLUSIONS Three novel families of A2BAR ligands have been prepared by exploiting the robustness and exploratory potential of the Biginelli reaction. The new scaffolds were identified in the context of the structural exploration of the successful 3,4dihydropyrimidin-2(1H)-one chemotype by fusion of imidazole, 1,2,4-triazole or benzimidazole rings to the 2,3-positions of the parent diazinone core. Excellent affinity towards the hA2BAR (Ki ≤ 25 nM) and remarkable selectivity profiles toward A1, A2A and A3 receptors were obtained for compounds 22a, 22b, 22c and 22d. The antagonistic behavior of two representative derivatives of these series was unequivocally confirmed by functional cAMP experiments. The design and the main SAR trends identified within the series were supported by a molecular modeling study based on a receptor-driven docking model. Further experiments are currently in progress in our laboratories to evaluate the A2BAR antagonistic effect of enantiomerically pure samples of ligands that are representative of the most successful scaffolds identified during this program. The biological profiles of these new derivatives will be published in due course.

EXPERIMENTAL SECTION

Chemistry. Unless otherwise indicated, all starting materials, reagents and solvents were purchased and used without further purification. After extraction from aqueous phases, the organic solvents were dried over anhydrous sodium sulphate. The reactions were monitored by thin-layer chromatography (TLC) on 2.5 mm Merck silica gel GF 254 strips, and the purified compounds each showed a single spot; unless stated otherwise, UV light and/or iodine vapour were used to detect compounds. The Biginelli reactions were performed in coated Kimble vials on a PLS (6×4) Organic Synthesiser



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with orbital stirring. Filtration and washing protocols for supported reagents were performed in a 12-channel vacuum manifold. The purity and identity of all tested compounds were established by a combination of HPLC, elemental analysis, mass spectrometry and NMR spectroscopy as described below. Purification of isolated products was carried out by column chromatography (Kieselgel 0.040–0.063 mm, E. Merck) or medium pressure liquid chromatography (MPLC) on a CombiFlash Companion (Teledyne ISCO) with RediSep pre-packed normal-phase silica gel (35–60 µm) columns followed by recrystallization. Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. The NMR spectra were recorded on Bruker AM300 and XM500 spectrometers. Chemical shifts are given as δ values against tetramethylsilane as internal standard and J values are given in Hz. Mass spectra were obtained on a Varian MAT-711 instrument. High-resolution mass spectra were obtained on an Autospec Micromass spectrometer. Analytical HPLC was performed on an Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 µm column with gradient elution using the mobile phases (A) H2O containing 0.1% CF3COOH and (B) MeCN and a flow rate of 1 mL/min. The purity of all tested compounds was determined to be >95%.

General procedure for the synthesis of alkyl 2-methyl-6-oxo-1,4,5,6tetrahydropyridine-3-carboxylates 17:37 A mixture of Meldrum´s Acid (7.5 mmol), the appropriate aldehyde 25a-d (5 mmol), the 1,3-dicarbonylic compound 24 (5 mmol) and ammonium acetate (20 mmol) in acetic acid (5 mL) was submitted to microwave irradiation at 90ºC for 30-45 minutes. After completion of the reaction, as indicated by TLC, the mixture was diluted with EtOAc and water and neutralized with 2N

NaOH. The phases were separated and the organic phase was dried over Na2SO4,


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filtered and concentrated to afford a yellow residue that was purified by column chromatography on silica gel.

(±) Ethyl 4-(furan-2-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (17a). Purified by column chromatography (dichloromethane – MeOH 7:3) and then recrystallized from EtOH to give 129 mg, 52%. Mp 120 – 122ºC. 1H-NMR (300 MHz, CDCl3), δ (ppm): 7.77 (brs, 1H), 7.30 – 7.25 (m, 1H), 6.23 (dd, J = 3.2, 1.9 Hz, 1H), 6.01 (dd, J = 3.2, 0.8 Hz, 1H), 4.34 (dd, J = 6.9, 2.5 Hz, 1H), 4.26 – 4.14 (m, 2H), 2.85 – 2.77 (m, 2H), 2.36 (s, 3H), 1.27 (t, J = 7.1, 3H). HRMS (CI+) m/z: calcd. for C13H16NO4 [M+H]+: 250.2780 found: 250.6870.

(±) Isopropyl 4-(furan-2-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydropyridine-3carboxylate (17b). Purified by column chromatography (dichloromethane – MeOH 7:3) and then recrystallized from EtOH to give 108 mg, 41%. Mp 125 – 126ºC. 1HNMR (300 MHz, CDCl3), δ (ppm): 8.32 (brs, 1H), 7.27 (s, 1H), 6.22 (dd, J = 3.2, 1.9 Hz, 1H), 6.00 (dd, J = 3.2, 0.8 Hz, 1H), 5.09 – 5.03 (m, 1H), 4.32 (dd, J = 6.6, 2.7 Hz, 1H), 2.84 – 2.77 (m, 2H), 2.34 (s, 3H), 1.27 (d, J = 6.2 Hz, 3H), 1.21 (d, J = 6.2 Hz, 3H). HRMS (CI+) m/z: cald. for C14H18NO4 [M+H]+: 264.2998 found: 264.8177.

(±)

Ethyl

4-(furan-3-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydropyridine-3-

carboxylate (17c). Purified by column chromatography (dichloromethane – MeOH 7:3) and then recrystallized from MeOH to give 122 mg, 49%. Mp 126 – 128ºC. 1H-NMR (300 MHz, CDCl3), δ (ppm): 7.91 (brs, 1H), 7.45 – 7.12 (m, 2H), 6.29 (d, J = 1.9 Hz, 1H), 4.27 – 4.13 (m, 3H), 2.80 – 2.70 (m, 2H), 2.33 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 171.6, 166.8, 145.9, 143.2, 138.8, 125.7, 109.6,



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107.5, 60.3, 36.5, 29.1, 19.0, 14.3. HRMS (CI+) m/z: calcd. for C13H16NO4 [M+H]+: 250.2780 found: 250.1107.

(±) Isopropyl 4-(furan-3-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydropyridine-3carboxylate (17d). Purified by column chromatography (dichloromethane – MeOH 7:3) and then recrystallized from EtOH to give 113 mg, 43%. Mp 126 – 127ºC. 1HNMR (300 MHz, CDCl3), δ (ppm): 8.04 (brs, 1H), 7.35 – 7.18 (m, 2H), 6.30 (s, 1H), 5.25 – 4.95 (m, 1H), 4.19 – 4.09 (m, 1H), 2.87 – 2.67 (m, 2H), 2.32 (s, 3H), 1.29 (d, J = 6.2 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H). HRMS (CI+) m/z: calcd. for C14H18NO4 [M+H]+: 264.2998 found: 264.1751.

(±)

Ethyl

2-methyl-6-oxo-4-(thiophen-2-yl)-1,4,5,6-tetrahydropyridine-3-

carboxylate (17e). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 132 mg, 50%. Mp 143 – 145ºC. 1H-NMR (300 MHz, CDCl3), δ (ppm): 7.36 (brs, 1H), 7.12 (d, J = 1.3 Hz, 1H), 6.91 – 6.80 (m, 2H), 4.53 (dd, J = 5.8, 3.4 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 2.90 – 2.86 (m, 2H), 2.36 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). HRMS (CI+) m/z: calcd. for C13H16NO3S [M+H]+: 266,3312, found: 266.1230.

(±) Isopropyl 2-methyl-6-oxo-4-(thiophen-2-yl)-1,4,5,6-tetrahydropyridine3-carboxylate (17f). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 120 mg, 43%. Mp 142 – 144ºC. 1HNMR (300 MHz, CDCl3), δ (ppm): 7.77 (brs, 1H), 7.12 (dd, J = 5.0, 1.3 Hz, 1H), 6.91 – 6.81 (m, 2H), 5.11 – 5.05 (m, 1H), 4.53 – 4.47 (m, 1H), 2.87 (dd, J = 4.7, 3.3 Hz, 2H), 2.35 (s, 3H), 1.29 (d, J = 6.3 Hz, 3H), 1.23 (d, J = 6.2 Hz, 3H).


13

C NMR (75 MHz,

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CDCl3), δ (ppm): 170.9, 166.0, 146.0, 145.5, 126.6, 123.9, 123.6, 108.2, 67.9, 37.7, 33.3, 22.1, 21.9, 19.1. HRMS (CI+) m/z: calcd. for C14H18NO3S [M+H]+: 280.3531 found: 280.0744.

(±)

Ethyl

2-methyl-6-oxo-4-(thiophen-3-yl)-1,4,5,6-tetrahydropyridine-3-

carboxylate (17g). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 156 mg, 59%. Mp 152 – 153ºC. 1H-NMR (300 MHz, CDCl3), δ (ppm): 7.78 (brs, 1H), 7.33 – 7.15 (m, 1H), 7.09 – 6.54 (m, 2H), 4.37 – 4.31 (m, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.11 – 2.52 (m, 2H), 2.35 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H).

13

C NMR (75 MHz, CDCl3), δ (ppm): 171.6, 166.8, 145.8, 142.4,

126.8, 126.0, 120.4, 107.8, 60.3, 37.0, 33.3, 19.1, 14.3. HRMS (EI) m/z: calcd. for C13H15NO3S [M]+: 265,3312, found: 265.3330.

(±) Isopropyl 2-methyl-6-oxo-4-(thiophen-3-yl)-1,4,5,6-tetrahydropyridine3-carboxylate (17h). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 170 mg, 61%. Mp 145 – 146ºC. 1HNMR (300 MHz, CDCl3), δ (ppm): 7.52 (brs, 1H), 7.30 – 7.19 (m, 2H), 7.05 – 6.93 (m, 1H), 5.16 – 4.97 (m, 1H), 4.36 – 4.29 (m, 1H), 2.85 – 2.78 (m, 2H), 2.34 (s, 3H), 1.28 (d, J = 6.2 Hz, 3H), 1.21 (d, J = 6.2 Hz, 3H).

13

C NMR (75 MHz, CDCl3), δ (ppm):

171.6, 166.3, 145.4, 142.5, 126.7, 125.9, 120.3, 108.2, 67.7, 36.9, 33.2, 22.0, 21.9, 19.1. HRMS (EI) m/z: calcd. for C14H17NO3S [M]+: 279,3582, found: 279.1210.

General procedure for the synthesis of 3,4-dihydrofuro[3,4-d]pyrimidine2,5(1H,7H)-diones 18a-d:38 A dissolution of the corresponding 6-chloromethyl-3,4,dihydropyrimidin-2(1H)-one 28a-d, (5 mmol) in DMF (8 mL) was stirred with orbital



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stirring at 150ºC for 24h. After completion of the reaction, as indicated by TLC, the solvent was removed in vacuum and the obtained oily residue was poured onto crushed ice. The solid separated was filtered under suction and then purified by column chromatography on silica gel.

(±) 4-(2-Furyl)-3,4-dihydrofuro[3,4-d]pyrimidine-2,5(1H,7H)-dione (18a). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 101 mg, 64%. Mp 227 – 278 ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 10.02 (s, 1H), 7.82 (s, 1H), 7.61 – 7.60 (m, 1H), 6.41 – 6.39 (m, 1H), 6.29 (d, J = 3.1 Hz, 1H), 5.30 (s, 1H), 4.84 (d, J = 3.3 Hz, 2H). HRMS (EI) m/z: calcd. for C10H8N2O4 [M]+: 220.0486, found: 220.0491.

(±) 4-(3-Furyl)-3,4-dihydrofuro[3,4-d]pyrimidine-2,5(1H,7H)-dione (18b). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 132 mg, 60%. Mp 276 – 277ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.92 (s, 1H), 7.71 (s, 1H), 7.59 (d, J = 1.7 Hz, 1H), 7.55 (s, 1H), 6.45 (d, J = 1.7 Hz, 1H), 5.21 (s, 1H), 4.80 (s, 2H). HRMS (EI) m/z: calcd. for C10H8N2O4 [M]+: 220.0487, found: 220.0489.

(±) 4-(2-Thienyl)-3,4-dihydrofuro[3,4-d]pyrimidine-2,5(1H,7H)-dione (18c). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 160 mg, 68%. Mp 281 – 282ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 10.05 (s, 1H), 7.97 (s, 1H), 7.45 (d, J = 4.9 Hz, 1H), 7.02 (d, J = 3.3 Hz, 1H), 6.97 (dd, J = 3.5 Hz, J = 4.9 Hz, 1H), 5.53 (s, 1H), 4.83 (d, J = 3.2 Hz, 2H). HRMS (EI) m/z: calcd. for C10H8N2O3S [M]+: 236.0262, found: 236.0264.


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(±) 4-(3-Thienyl)-3,4-dihydrofuro[3,4-d]pyrimidine-2,5(1H,7H)-dione (18d). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 146 mg, 62%. Mp 284 – 285ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.94 (s, 1H), 7.81 (s, 1H), 7.48 (d, J = 3.0 Hz, 1H), 7.31 (d, J = 3.0 Hz, 1H), 7.06 (s, 1H), 5.29 (s, 1H), 4.80 (s, 2H). HRMS (EI) m/z: calcd. for C10H8N2O3S [M]+: 236.0259, found: 236.0261.

General procedure for the synthesis of 3,4,6,7-tetrahydro-1H-pyrrolo[3,4d]pyrimidine-2,5-diones 19a-d:39 A mixture of the corresponding 6-chloromethyl-3,4,dihydropyrimidin-2(1H)-one 28a-d, and ethylamine (5 mmol) in MeOH in coated Kimble vials were stirred with orbital stirring at room temperature for 2 h. Then, the reaction mixture was stirred at 80ºC for 24 h. After completion of the reaction, as indicated by TLC, the solvent was removed and the oily residue obtained was poured onto crushed ice. The solid separated was filtered under suction, washed with ice-cold water (20 mL), dried in vacuum and then purified by column chromatography on silica gel.

(±) 6-Ethyl-4-(2-furyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5dione (19a). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 143 mg, 58%. Mp 246 – 247ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.58 (s, 1H), 7.56 (s, 1H), 7.51 (s, 1H), 6.36 (d, J = 1.5 Hz, 1H), 6.19 (s, 1H), 5.19 (s, 1H), 3.95 (s, 2H), 3.23 (d, J = 6.9 Hz, 2H), 1.01 (t, J = 7.0 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13N3O3 [M]+: 247.0958, found: 247.0961.



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(±) 6-Ethyl-4-(3-furyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5dione (19b). Purified by column chromatography (n-hexane – ethyl acetate 8:2) and then recrystallized from EtOH to give 158 mg, 64%. Mp 241 – 242ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.32 (s, 1H), 7.68 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 6.26 (s, 1H), 5.11 (s, 1H), 4.05 (d, J = 7.0 Hz, 2H), 3.71 (s, 2H), 1.15 (t, J = 6.9 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13N3O3 [M]+: 247.0960, found: 247.0959.

(±)

6-Ethyl-4-(2-thienyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-

2,5-dione (19c). Purified by column chromatography (n-hexane – ethyl acetate 8:2) and then recrystallized from EtOH to give 166 mg, 63%. Mp 240 – 241ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.65 (s, 1H), 7.69 (s, 1H), 7.38 (d, J = 4.8 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H), 6.94 (d, J = 3.6 Hz, 1H), 5.43 (s, 1H), 3.95 (s, 2H), 3.29 – 3.23 (m, 2H), 1.01 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13N3O2S [M]+: 263.0731, found: 263.0729.

(±)

6-Ethyl-4-(3-thienyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-

2,5-dione (19d). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 158 mg, 60%. Mp 234 – 235ºC. 1H-RMN (DMSO-d6 300 MHz), δ (ppm): 9.31 (s, 1H), 7.77 (s, 1H), 7.40 (s, 1H), 7.12 (s, 1H), 6.95 (s, 1H), 5.23 (s, 1H), 4.03 (s, 2H), 3.74 (s, 2H), 1.12 (t, J = 6.7 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13N3O2S [M]+: 263.0727, found: 263.0733.

General procedure for the Biginelli synthesis of derivatives 20, 21 and 22:41 A mixture of the urea surrogate 29a-d (7.5 mmol), the corresponding aldehyde 25a-d (5 mmol), the 1,3-dicarbonylic compound 24a-b (5 mmol) and a catalytic amount of 2-


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chloroacetic acid (0.05 mmol) in 3 mL of THF in coated Kimble vials was stirred with orbital stirring at 80ºC for 12h. After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 5-10 minutes. The solid separated was filtered under suction, washed with ice-cold water (20 mL), and then purified either by recrystallization from EtOH or column chromatography on silica gel.

(±) Ethyl 5-(furan-2-yl)-7-methyl-5,8-dihydroimidazo[1,2-a]pyrimidine-6carboxylate (20a). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 188 mg, 69%. Mp 193 – 196ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 12.29 (s, 1H), 7.28 (d, J = 8.9 Hz, 1H), 6.96 – 6.53 (m, 2H), 6.39 – 6.22 (m, 2H), 6.17 (d, J = 3.2 Hz, 1H), 4.39 – 3.83 (m, 2H), 2.56 (s, 3H), 1.39 – 0.91 (m, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 166.2, 154.6, 148.3, 142.1, 141.5, 124.7, 113.5, 110.2, 106.6, 92.1, 59.6, 51.6, 19.3, 14.3. HRMS (ESI) m/z: calcd. for C14H16N3O3 [M+H]+: 274,2924, found: 274.1189.

(±) Isopropyl 5-(furan-2-yl)-7-methyl-5,8-dihydroimidazo[1,2-a]pyrimidine6-carboxylate (20b). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 207 mg, 72%. Mp 181 – 182ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 1H NMR (300 MHz, CDCl3),δ12.48 (s, 1H), 7.29 (d, J = 2.1 Hz, 1H), 6.96 – 6.53 (m, 2H), 6.48 – 6.00 (m, 3H), 4.99 (p, J = 6.4 Hz, 1H), 2.56 (s, 3H), 1.23 (d, J = 6.1 Hz, 3H), 1.04 (d, J = 6.0 Hz, 3H).

13

C NMR (75 MHz,

CDCl3), δ (ppm): 165.7, 154.8, 148.1, 141.9, 141.6, 124.6, 113.4, 110.2, 106.6, 92.2, 77.0, 76.6, 66.7, 51.6, 22.1, 19.2. HRMS (ESI) m/z: calcd. for C15H18N3O3 [M+H]+: 288,3217, found: 288.1340.



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(±) Ethyl 5-(furan-3-yl)-7-methyl-5,8-dihydroimidazo[1,2-a]pyrimidine-6carboxylate (20c). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 142 mg, 52%. Mp 180 – 181ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 11.94 (brs, 1H), 7.45 – 7.10 (m, 3H), 6.76 (d, J = 1.6 Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 6.27 (s, 1H), 6.21 (dd, J = 1.9, 0.9 Hz, 1H), 4.16 (dt, J = 7.2, 3.6 Hz, 2H), 2.51 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z: calcd. for C14H15N3O3 [M]+: 273.2901, found: 273.0598.

(±) Isopropyl 5-(furan-3-yl)-7-methyl-5,8-dihydroimidazo[1,2-a]pyrimidine6-carboxylate (20d). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 161 mg, 56%. Mp 166 – 168ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 12.23 (brs, 1H), 7.57 – 7.11 (m, 2H), 6.75 (d, J = 1.6 Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 6.37 – 6.09 (m, 2H), 5.04 (p, J = 6.2 Hz, 1H), 2.51 (s, 3H), 1.26 (d, J = 6.2 Hz, 3H), 1.17 (d, J = 6.2 Hz, 3H).

13

C NMR (75 MHz,

CDCl3), δ (ppm): 165.8, 147.3, 143.2, 142.1, 139.1, 127.8, 124.7, 113.3, 108.8, 95.0, 66.9, 50.1, 22.2, 21.9, 19.3. HRMS (EI) m/z: calcd. for C15H17N3O3 [M]+: 287.3271, found: 287.3687.

(±) Ethyl 7-methyl-5-(thiophen-2-yl)-5,8-dihydroimidazo[1,2-a]pyrimidine6-carboxylate (20e). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 153 mg, 53%. Mp 166 – 169ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 12.34 (brs, 1H), 7.16 (dd, J = 4.6, 1.7 Hz, 1H), 7.02 – 6.35 (m, 5H), 4.37 – 3.88 (m, 2H), 2.55 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z: calcd. for C14H15N3O2S [M]+: 289.3508, found: 289.1982.


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(±)

Isopropyl

7-methyl-5-(thiophen-2-yl)-5,8-dihydroimidazo[1,2-a]pyri-

midine-6-carboxylate (20f). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 170 mg, 56%. Mp 198 – 199ºC. 1

H NMR (300 MHz, CDCl3), δ (ppm): 12.32 (brs, 1H), 7.16 (dt, J = 4.9, 1.1 Hz, 1H),

6.98 – 6.81 (m, 2H), 6.75 (dd, J = 1.7, 0.7 Hz, 1H), 6.64 (dd, J = 1.6, 0.7 Hz, 1H), 6.51 (s, 1H), 5.01 (p, J = 6.3 Hz, 1H), 2.55 (s, 3H), 1.24 (dd, J = 6.2, 0.7 Hz, 4H), 1.06 (dd, J = 6.2, 0.7 Hz, 3H). HRMS (EI) m/z: calcd. for C15H17N3O2S [M]+: 303.0896, found: 303.1597.

(±) Ethyl 7-methyl-5-(thiophen-3-yl)-5,8-dihydroimidazo[1,2-a]pyrimidine6-carboxylate (20g). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 150 mg, 52%. Mp 164 – 165ºC. 1H NMR (300 MHz, CDCl3), δ (ppm): 12.12 (brs, 1H), 7.19 (dd, J = 5.0, 3.0 Hz, 1H), 7.10 (dd, J = 3.0, 1.3 Hz, 1H), 6.92 (dd, J = 5.0, 1.4 Hz, 1H), 6.75 (d, J = 1.6 Hz, 1H), 6.56 (d, J = 1.6 Hz, 1H), 6.36 (s, 1H), 4.39 – 3.83 (m, 2H), 2.52 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z: calcd. for C14H15N3O2S [M]+: 289.0911, found: 289.0916.

(±)

Isopropyl

7-methyl-5-(thiophen-3-yl)-5,8-dihydroimidazo[1,2-a]pyri-

midine-6-carboxylate (20h). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 185 mg, 61%. Mp 172 – 174ºC. 1

H NMR (300 MHz, CDCl3), δ (ppm): 12.22 (brs, 1H), 7.19 (dd, J = 5.0, 3.0 Hz, 1H),

7.11 (dd, J = 3.0, 1.3 Hz, 1H), 6.94 (dd, J = 5.0, 1.4 Hz, 1H), 6.74 (d, J = 1.6 Hz, 1H), 6.55 (d, J = 1.6 Hz, 1H), 6.34 (s, 1H), 5.00 (p, J = 6.2 Hz, 1H), 2.53 (s, 3H), 1.24 (d, J = 6.2 Hz, 3H), 1.08 (d, J = 6.2 Hz, 3H). HRMS (EI) m/z: calcd. for C15H17N3O2S [M]+: 303.1029, found: 303.2319.



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(±) Ethyl 7-(furan-2-yl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21a). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 156 mg, 57%. Mp 179 – 181ºC. 1

H NMR (300 MHz, CDCl3), δ (ppm):11.66 (brs, 1H), 7.65 (s, 1H), 7.29 (s, 1H), 6.54

(s, 1H), 6.29 (s, 2H), 4.30 – 3.92 (m, 2H), 2.57 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H).

13

C

NMR (75 MHz, CDCl3), δ (ppm): 165.5, 152.8, 148.6, 147.7, 147.0, 142.5, 110.4, 107.8, 95.9, 60.1, 53.5, 19.1, 14.2. HRMS (ESI) m/z: calcd. for C13H15N4O3 [M+H]+: 275.2895, found: 275.3859.

(±) Isopropyl 7-(furan-2-yl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21b). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 196 mg, 68%. Mp 176 – 177ºC. 1

H NMR (300 MHz, CDCl3), δ (ppm): 11.66 (brs, 1H), 7.63 (s, 1H), 7.28 (s, 1H), 6.51

(s, 1H), 6.28 (d, J = 6.3 Hz, 2H), 4.99 (q, J = 6.1 Hz, 1H), 2.56 (s, 3H), 1.12 1.21 (d, J = 6.5 Hz, 3H), 1.03 (d, J = 6.3 Hz, 3H).

13

C NMR (75 MHz, CDCl3), δ (ppm): 164.9,

153.0, 148.6, 147.6, 146.7, 142.4, 110.4, 107.8, 96.1, 67.4, 53.6, 22.0, 21.6, 18.9. HRMS (ESI) m/z: calcd. for C14H17N4O3[M+H]+ : 289.2570, found: 289.3303.

(±) Ethyl 7-(furan-3-yl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21c). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 140 mg, 51%. Mp 200 – 202ºC. 1

H NMR (300 MHz, CDCl3), δ (ppm): 11.51 (brs, 1H), 7.65 (s, 1H), 7.37 (s, 1H), 7.30

(t, J = 1.7 Hz, 1H), 6.48 (s, 1H), 6.26 (s, 1H), 4.16 (qd, J = 7.1, 1.9 Hz, 2H), 2.54 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 165.6, 148.6, 147.8,


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146.2, 143.4, 139.9, 126.1, 108.9, 98.1, 60.2, 52.0, 19.0, 14.2. HRMS (EI) m/z: calcd. for C13H14N4O3 [M]+: 274.1189, found: 274.1892.

(±) Isopropyl 7-(furan-3-yl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21d). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 118 mg, 41%. Mp 175 – 176ºC. 1

H-RMN (300 MHz, CDCl3), δ (ppm): 11.47 (brs, 1H), 7.65 (s, 1H), 7.38 (s, 1H), 7.30

(q, J = 1.3, 0.8 Hz, 1H), 6.47 (s, 1H), 6.28 (s, 1H), 5.04 (td, J = 6.2, 0.8 Hz, 1H), 2.54 (s, 3H), 1.25 (d, J = 6.2 Hz, 3H), 1.13 (d, J = 6.2 Hz, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 165.1, 148.5, 147.8, 145.9, 143.3, 140.0, 126.2, 109.0, 98.2, 67.6, 52.0, 22.1, 21.7, 18.9. HRMS (EI) m/z: calcd. for C14H16N4O3 [M]+: 288.1264, found: 288.1791.

(±) Ethyl 5-methyl-7-(thiophen-2-yl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21e). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 151 mg, 52%. Mp 201 – 202ºC. 1

H-RMN (300 MHz, CDCl3), δ (ppm): δ 11.47 (brs, 1H), 7.66 (s, 1H), 7.20 (dd, J = 5.1,

1.3 Hz, 1H), 7.01 (dd, J = 3.6, 1.3 Hz, 1H), 6.91 (dd, J = 5.1, 3.5 Hz, 1H), 6.75 (s, 1H), 4.14 (qd, J = 7.1, 4.0 Hz, 2H), 2.58 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z: calcd. for C13H14N4O2S [M]+: 290.0812, found: 290.4972.

(±)

Isopropyl

5-methyl-7-(thiophen-2-yl)-4,7-dihydro-[1,2,4]triazolo[1,5-

a]pyrimidine-6-carboxylate

(21f).

Purified

by

column

chromatography

(dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 143 mg, 47%. Mp 163 – 166ºC. 1H-RMN (300 MHz, CDCl3), δ (ppm): 11.34 (brs, 1H), 7.65 (s, 1H), 7.20 (dd, J = 5.0, 1.3 Hz, 1H), 7.03 (dd, J = 3.6, 1.2 Hz, 1H), 6.91 (dd, J = 5.1, 3.5



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Hz, 1H), 6.73 (s, 1H), 5.00 (p, J = 6.2 Hz, 1H), 2.58 (s, 3H), 1.23 (d, J = 6.2 Hz, 3H), 1.03 (d, J = 6.2 Hz, 3H). HRMS (EI) m/z: calcd. for C14H16N4O2S [M]+: 304.1019, found: 304.2877.

(±) Ethyl 5-methyl-7-(thiophen-3-yl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (21g). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 119 mg, 41%. Mp 198 – 201ºC. 1

H-RMN (300 MHz, CDCl3), δ (ppm): 11.18 (brs, 1H), 7.58 (s, 1H), 7.29 – 7.04 (m,

2H), 6.93 (dd, J = 4.8, 1.6 Hz, 1H), 6.51 (s, 1H), 4.25 – 3.85 (m, 2H), 2.49 (s, 3H), 1.12 (t, J = 7.1 Hz, 3H).

13

C NMR (75 MHz, CDCl3), δ (ppm): 165.6, 148.5, 147.8, 146.2,

143.3, 139.9, 126.1, 108.9, 98.0, 77.0, 76.6, 60.2, 52.0, 18.9, 14.2. HRMS (EI) m/z: calcd. for C13H14N4O2S [M]+: 290.0892, found: 290.9271.

(±)

Isopropyl

5-methyl-7-(thiophen-3-yl)-4,7-dihydro-[1,2,4]triazolo[1,5-


(21h).

Purified

by

column

chromatography

(dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 118 mg, 39%. Mp 198 – 199ºC. 1H-RMN (300 MHz, CDCl3), δ (ppm): 10.86 (brs, 1H), 7.64 (d, J = 1.1 Hz, 1H), 7.39 – 7.11 (m, 2H), 7.01 (d, J = 4.8 Hz, 1H), 6.56 (s, 1H), 4.99 (p, J = 6.2 Hz, 1H), 2.55 (s, 3H), 1.23 (t, J = 6.5 Hz, 3H), 1.03 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 165.1, 148.5, 147.8, 145.9, 143.3, 140.0, 126.2, 109.0, 98.2, 67.6, 51.9, 22.1, 21.7, 18.9. HRMS (EI) m/z: calcd. for C14H16N4O2S [M]+: 304.1010, found: 304.8245.


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(±) Ethyl 4-(furan-2-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylate (22a). Purified by column chromatography (dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 178 mg, 55%. Mp 174 – 176ºC. 1

H-RMN (300 MHz, DMSO-d6) δ (ppm): 10.80 (brs, 1H), 7.60 – 7.21 (m, 3H), 7.18 –

6.87 (m, 2H), 6.55 (s, 1H), 6.43 (d, J = 3.2 Hz, 1H), 6.30 (dd, J = 3.2, 1.8 Hz, 1H), 4.33 – 3.76 (m, 2H), 2.44 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ (ppm): 165.5, 153.4, 148.1, 146.1, 142.9, 132.1, 122.3, 120.7, 117.3, 110.8, 110.1, 108.0, 95.0, 59.7, 49.7, 19.0, 14.6. HRMS (ESI) m/z: calcd. for C18H18N3O3 [M+H]+: 324.3582, found: 324.1343.

(±)

Isopropyl

4-(furan-2-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-


(22b).

Purified

by

column

chromatography

(dichloromethane – MeOH 9:1) and then recrystallized from EtOH to give 206 mg, 61%. Mp 253 – 255ºC. 1H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.78 (brs, 1H), 7.67 – 7.23 (m, 3H), 7.19 – 6.84 (m, 2H), 6.52 (s, 1H), 6.44 (d, J = 3.3 Hz, 1H), 6.37 – 6.23 (m, 1H), 4.86 (h, J = 6.3 Hz, 1H), 2.44 (s, 3H), 1.21 (d, J = 6.2 Hz, 3H), 1.05 (d, J = 6.1 Hz, 3H).

13

C NMR (75 MHz, DMSO-d6), δ (ppm): 165.0, 153.3, 148.0, 146.0, 143.0,

142.6, 132.0, 122.3, 120.7, 117.2, 110.8, 110.2, 108.2, 94.9, 67.0, 49.7, 22.3, 22.0, 19.1. HRMS (ESI) m/z: calcd. for C19H20N3O3 [M+H]+: 338.1488, found: 338.7927.

(±)

Ethyl

4-(furan-3-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyri-

midine-3-carboxylate (22c). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 233 mg, 72%. Mp 270 – 271ºC. 1

H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.70 (brs, 1H), 7.68 (s, 1H), 7.53 – 7.21 (m,

3H), 7.02 (dt, J = 19.3, 7.4 Hz, 2H), 6.44 (s, 1H), 6.16 (d, J = 2.3 Hz, 1H), 4.33 – 3.76



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(m, 2H), 2.40 (s, 3H), 1.17 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ (ppm): 165.6, 147.4, 146.3, 144.1, 142.8, 140.5, 131.9, 126.2, 122.2, 120.6, 117.2, 110.1, 109.2, 97.1, 59.8, 47.7, 19.0, 14.6. HRMS (ESI) m/z: calcd. for C18H18N3O3 [M+H]+: 324.3582 found: 324.9974.

(±)

Isopropyl

4-(furan-3-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-

a]pyrimidine-3-carboxylate (22d). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 239 mg, 71%. Mp 269 – 270ºC. 1H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.68 (brs, 1H), 7.71 (s, 1H), 7.53 – 7.18 (m, 3H), 7.01 (dddd, J = 16.7, 9.1, 7.4, 4.5 Hz, 2H), 6.41 (d, J = 2.6 Hz, 1H), 6.27 – 6.00 (m, 1H), 4.88 (p, J = 6.2 Hz, 1H), 2.40 (s, 3H), 1.21 (d, J = 6.2 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ (ppm): 165.1, 147.1, 146.3, 144.1, 142.8, 140.7, 132.0, 126.2, 122.2, 120.5, 117.2, 110.2, 109.3, 97.3 67.0, 47.7, 22.3, 22.0, 19.0. HRMS (ESI) m/z: calcd. for C19H20N3O3 [M+H]+: 338.3812 found: 338.4765.

(±)

Ethyl

2-methyl-4-(thiophen-2-yl)-1,4-dihydrobenzo[4,5]imidazo[1,2-

a]pyrimidine-3-carboxylate (22e). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 230 mg, 68%. Mp 262 – 264ºC. 1H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.85 (brs, 1H), 7.51 – 7.39 (m, 1H), 7.38 – 7.22 (m, 2H), 7.14 – 6.91 (m, 3H), 6.91 – 6.69 (m, 2H), 4.28 – 3.78 (m, 2H), 2.42 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ (ppm): 165.5, 147.4, 146.0, 145.5, 142.8, 131.8, 127.0, 126.1, 126.0, 122.4, 120.7, 117.3, 110.4, 98.2, 59.0, 51.1, 19.0, 14.6. HRMS (ESI) m/z: calcd. for C18H18N3O2S [M+H]+: 340.4112 found: 340.4765.


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(±) Isopropyl 2-methyl-4-(thiophen-2-yl)-1,4-dihydrobenzo[4,5]imidazo[1,2a]pyrimidine-3-carboxylate (22f). Purified by column chromatography (n-hexane – ethyl acetate 7:3) and then recrystallized from EtOH to give 233 mg, 66%. Mp 261 – 263ºC. 1H-RMN (DMSO, 300 MHz), δ (ppm): 10.81 (brs, 1H), 7.66 – 7.17 (m, 3H), 7.15 – 6.91 (m, 3H), 6.91 – 6.60 (m, 2H), 4.86 (q, J = 6.4 Hz, 1H), 2.42 (s, 3H), 1.21 (d, J = 6.2 Hz, 3H), 1.04 (d, J = 6.1 Hz, 3H).

13

C NMR (75 MHz, DMSO-d6), δ (ppm):

165.0, 147.1, 146.0, 145.6, 142.8, 131.8, 126.9, 126.3, 126.0, 122.4, 120.6, 117.3, 110.5, 98.4, 67.2, 51.2, 22.3, 21.0, 19.0. HRMS (ESI) m/z: calcd. for C19H20N3O2S [M+H]+: 354.4408 found: 354.6870.

(±)

Ethyl

2-methyl-4-(thiophen-3-yl)-1,4-dihydrobenzo[4,5]imidazo[1,2-

a]pyrimidine-3-carboxylate (22g). Purified by column chromatography (n-hexane – ethyl acetate 8:2) and then recrystallized from EtOH to give 230 mg, 68%. Mp 290 – 293ºC. 1H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.74 (brs, 1H), 7.57 – 7.17 (m, 4H), 7.16 – 6.73 (m, 3H), 6.53 (s, 1H), 4.26 – 3.78 (m, 2H), 2.41 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ (ppm): 165.6, 147.1, 146.1, 142.8, 132.0, 129.5, 127.1, 126.5, 123.3, 122.2, 120.6, 117.2, 110.2, 97.9, 59.8, 51.3, 19.0, 14.6. HRMS (ESI) m/z: calcd. for C18H18N3O2S [M+H]+: 340.4133 found: 340.9783.

(±) Isopropyl 2-methyl-4-(thiophen-3-yl)-1,4-dihydrobenzo[4,5]imidazo[1,2a]pyrimidine-3-carboxylate (22h). Purified by column chromatography (n-hexane – ethyl acetate 8:2) and then recrystallized from EtOH to give 236 mg, 67%. Mp 287 – 289ºC. 1H-RMN (300 MHz, DMSO-d6), δ (ppm): 10.70 (brs, 1H), 7.49 (dd, J = 2.9, 1.4 Hz, 1H), 7.43 – 7.20 (m, 3H), 7.14 – 6.76 (m, 3H), 6.51 (s, 1H), 4.85 (p, J = 6.3 Hz, 1H), 2.41 (s, 3H), 1.21 (d, J = 6.2 Hz, 3H), 1.02 (d, J = 6.2 Hz, 3H).


13

C NMR (75


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MHz, DMSO-d6), δ (ppm): 165.1, 149.4, 146.1, 142.9, 142.7, 132.0, 127.0, 126.6, 123.5, 122.2, 120.5, 117.2, 110.2, 98.0, 67.0, 51.4, 22.3, 21.9, 19.0. HRMS (ESI) m/z: calcd. for C19H20N3O2S [M+H]+: 354.4408 found: 354.7269.

General procedure for the Biginelli synthesis of 3,4-dihydropyrimidin-2(1H)ones 28a-d:38 A mixture of urea 26 (7.5 mmol), the corresponding pentagonal carbaldehyde 25a-d (5 mmol), ethyl 4-chloroacetoacetate 27 (5 mmol) and ZnCl2 (0.5 mmol) in 3 mL of THF in coated Kimble vials was stirred with orbital stirring at 80ºC for 12h. After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 5-10 minutes. The solid separated was filtered under suction, washed with ice-cold water (20 mL), dried in vacuum and then purified by column chromatography on silica gel.

(±) Ethyl 6-(chloromethyl)-4-(furan-2-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine5-carboxylate (28a): Purified by column chromatography (n-hexane – ethyl acetate 4:1) and then recrystallized from EtOH to give 1.079, 76%. Mp 168 – 169 ºC. 1H NMR (300 MHz, Chloroform-d) δ 7.56 (brs, 1H), 7.34 (s, 1H), 6.29 (dd, J = 4.9, 1.6 Hz, 1H), 6.16 (d, J = 2.8 Hz, 1H), 5.70 (brs, 1H), 5.52 (d, J = 2.7 Hz, 1H), 4.92 (d, J = 13.8 Hz, 1H), 4.81 (d, J = 13.2 Hz, 1H), 4.21 – 4.12 (m, 2H), 1.23 (t, J = 7.3 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13ClN2O4 [M]+: 284.0561, found: 284.0569.

(±)

Ethyl

6-(chloromethyl)-4-(furan-3-yl)-2-oxo-1,2,3,4-tetrahydropyri-

midine-5-carboxylate (28b): Purified by column chromatography (n-hexane – ethyl acetate 3:1) and then recrystallized from EtOH to give 980 mg, 69%. Mp 159 – 160 ºC.


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1

H NMR (300 MHz, DMSO-d6) δ 9.45 (brs, 1H), 7.74 (d, J = 3.6, 1H), 7.80 – 7.70 (m,

1H), 7.40 (dt, J = 1.6, 0.8 Hz, 1H), 6.34 (dd, J = 1.9, 0.9 Hz, 1H), 5.13 (d, J = 3.5 Hz, 1H), 4.76 (d, J = 10.6 Hz, 1H), 4.49 (d, J = 10.6 Hz, 1H), 4.31 – 3.78 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).

13

C NMR (75 MHz, DMSO-d6) δ 169.2, 157.9, 151.4, 148.9, 144.0,

133.2, 114.2, 106.7, 65.2, 51.0, 19.2. HRMS (EI) m/z: calcd. for C12H13ClN2O4 [M]+: 284.0563, found: 284.0570.

(±)

Ethyl

6-(chloromethyl)-2-oxo-4-(thiophen-2-yl)-1,2,3,4-tetrahydro-pyri-

midine-5-carboxylate (28c): Purified by column chromatography (n-hexane – ethyl acetate 3:1) and then recrystallized from EtOH to give 1.050 g, 70%. Mp 162 – 163 ºC. 1

H NMR (300 MHz, Chloroform-d) δ 8.26 (brs, 1H), 7.29 (s, 1H), 6.32 (dd, J = 4.7, 1.5

Hz, 1H), 6.24 (d, J = 2.6 Hz, 1H), 5.82 (brs, 1H), 5.59 (d, J = 3.4 Hz, 1H), 4.86 (d, J = 12.9 Hz, 1H), 4.72 (d, J = 12.9 Hz, 1H), 4.21 (qd, J = 7.4, 2.3 Hz, 2H), 1.26 (t, J = 7.3 Hz, 3H). HRMS (EI) m/z: calcd. for C12H13ClN2O3S [M]+: 300.0339, found: 300.0345.

(±)

Ethyl

6-(chloromethyl)-2-oxo-4-(thiophen-3-yl)-1,2,3,4-tetrahydropyri-

midine-5-carboxylate (28d): Purified by column chromatography (n-hexane – ethyl acetate 3:1) and then recrystallized from EtOH to give 1.065 g, 71%. Mp 158 – 159 ºC. 1

H NMR (300 MHz, Chloroform-d) δ 8.32 (brs, 1H), 7.26 (dd, J = 4.9, 3.1 Hz, 1H),

7.20 – 7.08 (m, 1H), 7.04 (dd, J = 5.0, 1.4 Hz, 1H), 6.50 (s, 1H), 5.54 (d, J = 3.2 Hz, 1H), 4.82 (d, J = 12.7 Hz, 1H), 4.68 (d, J = 12.7 Hz, 1H), 4.18 (qd, J = 7.2, 2.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, cdcl3) δ 164.4, 153.8, 143.5, 143.4, 126.8, 125.7, 121.7, 103.5, 60.8, 50.7, 39.5, 14.1. HRMS (EI) m/z: calcd. for C12H13ClN2O3S [M]+: 300.0338, found: 300.0342.



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Pharmacology. Radioligand binding competition assays were performed in vitro using A2A and A2B human receptors expressed in transfected HeLa (hA2A) and HEK-293 (hA2B) cells as described previously.35 A brief description is given below. Adenosine A1 receptor competition binding experiments were carried out in membranes from CHO-A1 cells labelled with 2 nM [3H]DPCPX. Non-specific binding was determined in the presence of 10 µM R-PIA. The reaction mixture was incubated at 25 °C for 60 min. Adenosine A2A receptor competition binding experiments were carried out in membranes from HeLa-A2A cells labelled with 3 nM [3H]ZM241385. Non-specific binding was determined in the presence of 50 µM NECA. The reaction mixture was incubated at 25 °C for 30 min. Adenosine A2B receptor competition binding experiments were carried out in membranes from HEK-293-A2B cells (Euroscreen, Gosselies, Belgium) labelled with 35 nM [3H]DPCPX. Non-specific binding was determined in the presence of 400 µM NECA. The reaction mixture was incubated at 25 °C for 30 min. Adenosine A3 receptor competition binding experiments were carried out in membranes from HeLa-A3 cells labelled with 30 nM [3H]NECA. Non-specific binding was determined in the presence of 100 µM R-PIA. The reaction mixture was incubated at 25 °C for 180 min. cAMP assays were performed at adenosine receptors transfected using a cAMP enzyme immunoassay kit (Amersham Biosciences). HEK293 cells were seeded (10000 cells/well) in 96-well culture plates and incubated at 37 ºC in an atmosphere with 5% CO2 in Eagle’s Medium Nutrient Mixture F-12 (EMEM F-12), containing 10% Foetal Calf Serum (FCS) and 1% L-glutamine. Cells were washed 3× with 200 µL assay medium (EMEM-F12 and 25 mM HEPES pH = 7.4) and pre-incubated with assay medium containing 30 µM rolipram and test compounds at 37 ºC for 15 min. 10 µM NECA was incubated for 15 min at 37 ºC (total incubation time 30 min). Reaction was stopped with lysis buffer supplied in the kit and the enzyme


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immunoassay was carried out for detection of intracellular cAMP at 450 nm in an Ultra Evolution detector (Tecan). Data Analysis: IC50 values were obtained by fitting the data with non-linear regression using Prism 2.1 software (GraphPad, San Diego, CA). For those compounds that showed either little affinity or poor solubility a percentage inhibition of specific binding is reported. Results are the mean of 3 experiments (n = 3) each performed in duplicate. The experimental details and dose-response curves obtained during the functional assays are described in the supporting information.

Molecular Modeling. Our computational strategy, which involves a mixture of the A2B receptor homology model, ligand-receptor docking and molecular dynamics relaxation protocols, was recently reviewed.43 Briefly, the modeling protocol included (i) Manually curated sequence alignment with the template A2AAR (PDB code 3EML), (ii) Generation and selection of homology models and loop refinement procedures with Modeler,49 (iii) assessment of Asn/Gln/His rotamers and side chain protonation states with the Molprobity web server (http://molprobity.biochem.duke.edu/) and (iv) tools from the Schrödinger Suite for energetic structural refinements.50 Both the R and the S stereoisomer for each ligand was built and optimized in the 3D using the Maestro graphical interface and the LigPrep utility from the Schrödinger suite.50 Each ligand was docked 20 times with default (high accuracy) genetic algorithm (GA) search parameters, using the scoring function Chemscore as implemented in GOLD51 and allowing full flexibility for the ligand, including flipping of amide bonds. The search sphere was centered on the side chain (CD1) of Ile7.39 and expanded with a radius of 15 Å, thus ensuring a generous enough search space comprising the antagonist binding site experimentally determined for adenosine receptors. The criterion for the selection of docking poses was based on a combination of the Chemscore ranking and the



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population (convergence) of the solutions according to a clustering criteria of 1 Å. For a second round of docking studies, we considered the presence of two potential water molecules in the binding cavity, with the algorithm implemented in GOLD. Here, each water is only included in the final docking pose if it produces an increase in the predicted scoring, and the hydrogen bond network is optimized (option trans_spin 0.5 in GOLD).

ASSOCIATED CONTENT Supporting Information. Experimental details, biological data and curves obtained during functional assays.

AUTHOR INFORMATION Corresponding Author(s) 1

Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and

2

Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de

Compostela, Santiago de Compostela 15782, Spain. Phone: +34-881815732, E-mail: [email protected]. 3

Department of Cell and Molecular Biology, Uppsala University, Uppsala SE-75124.

Phone: +46-(0)184715056, E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work has been developed in the frame of the Cost Action GLISTEN and financially supported by the Galician Government (Projects 09CSA016234PR and PS09/63).


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ABBREBIATIONS USED ADP, adenosine diphosphate; ATP, Adenosine-5'-triphosphate; AMP, Adenosine monophosphate; c-AMP, Cyclic adenosine monophosphate; NAD, Nicotinamide adenine dinucleotide; FAD, Flavin adenine dinucleotide; RNA, Ribonucleic acid; ARs, Adenosine receptors; GPCRs, G protein-coupled receptors; A2BR, human A2B adenosine receptors; A2AR, human A2A adenosine receptors; COPD, chronic and obstructive pulmonary disease; CNS, central nervous system; SAR, structure-activity relationships, MCR, Multicomponent reaction; MD, Molecular dynamics; CHO cells, Chinese Hamster Ovary cells; SEM, Standard error of the mean; PDB, Protein Data Bank; RMSD, Root Mean Square Deviation.

REFERENCES (1) Fredholm, B. B. Arslan, G. Halldner, L. Kull, B. Shulte, G. Wasserman, W. Structure and function of adenosine receptors and their genes. NaunynSchmiedeberg’s Arch. Pharmacol. 2000, 362, 364–374. (2) Murphree, L. J. Linden, J. Adenosine receptors. Encyclopaedia of Biol. Chem. 2004, 1, 34–39. (3) Fredholm, B. B. Ijzerman, A. P. Jacobson, K. A. Linden, J. Muller, C. E. Nomenclature and classification of adenosine receptors – An update. Pharmacol. Rev. 2011, 63, 1–34. (4) Moro, S. Deflorian, F. Bacilieri, M. Spalluto, G. Ligand-based homology modelling as attractive tool to inspect GPCR structural plasticity. Curr. Pharm. Des. 2006, 12, 2175–2185.



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Discovery of Potent and Highly Selective A2B ... - ACS Publications

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