New potent and selective A1 adenosine receptor antagonists as potential tools for the treatment of gastrointestinal diseases

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New potent and selective A adenosine receptor antagonists as

1

potential tools for the treatment of gastrointestinal diseases

1

,¥

1,¥

1

Catia Lambertucci, Gabriella Marucci, Diego Dal Ben, Michela Buccioni, Andrea Spinaci,

2

,1

Sonja Kachler, Karl-Norbert Klotz, and Rosaria Volpini*

1

School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032

2

Camerino (MC), Italy; Institut für Pharmakologie und Toxikologie, Universität Würzburg,

Versbacher Str. 9, 97078 Würzburg, Germany.

AUTHOR INFORMATION

Corresponding Author

*E-mail: rosaria.volpini@unicam.it. Phone: (+39)737402278

Author Contributions

¥

These authors contributed equally.

HIGHLIGHTS

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Adenine derivatives designed and synthesized as A adenosine receptor antagonists

1

Biological evaluation shows good A affinity and selectivity for some derivatives

1

Molecular modeling studies performed to analyze the compound affinities

Compounds restore the agonist-prevented normal motility function on mouse ileum

ABSTRACT

The synthesis of 9-alkyl substituted adenine derivatives presenting aromatic groups and cycloalkyl

6

rings in 8- and N -position, respectively, is reported. The compounds were tested with radioligand

binding studies showing, in some cases, a low nanomolar A adenosine receptor affinity and a very

1

good selectivity versus the other adenosine receptor subtypes. Functional assays at human

adenosine receptors and at a mouse ileum tissue preparation clearly demonstrate the antagonist

profile of these molecules, with inhibitory potency at nanomolar level. A molecular modelling

study, consisting in docking analysis at the recently reported A adenosine receptor crystal

1

6

structure, was performed for the interpretation of the obtained pharmacological results. The N -

cyclopentyl-9-methyl-8-phenyladenine (17), resulting the most active derivative of the series (K =

i

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2

.8 nM and IC = 14 nM), was also very efficacious in counteracting the effect of the agonist

5

0

CCPA on mouse ileum contractility. This new compound represents a tool for the development of

new agents for the treatment of intestinal diseases as constipation and postoperative ileus.

KEYWORDS

Adenosine; adenosine receptors; adenosine receptor antagonists; A adenosine receptor; purinergic

1

receptors, mouse ileum.

ABBREVIATIONS

AcOH, acetic acid; Ado, adenosine; AR, Adenosine receptor; cAMP, cyclic adenosine

6

monophosphate; CCPA, 2-chloro-N -cyclopentyladenosine; CHO, Chinese Hamster ovary; CPA,

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N -cyclopentyladenosine; DPCPX, dipropylcyclopentylxanthine; EL, extracellular loop; GPCR, G

protein-coupled receptor; NECA, 5’-N-ethylcarboxamidoadenosine; r.t., room temperature; THF,

tetrahydrofuran; TLC, thin-layer chromatography; TM, transmembrane.

Introduction

The G protein-coupled receptors activated by extracellular adenosine (Ado), called Adenosine

receptors (ARs), are widely expressed in the organism as four subtypes, namely A , A , A , and

1

2A

2B

A ARs [1,2]. Due to the involvement of these membrane proteins at different levels in a number of

3

physiological functions and pathological conditions, the development of selective ligands (agonists,

antagonists, inverse agonists, and allosteric modulators) for AR subtypes represents a hot topic in

medicinal chemistry. In particular, while A AR agonists are of interest for their cardioprotective,

1

cerebroprotective, and antinociceptive properties or for the treatment of diabetes (partial agonists)

[

3-5], antagonists of this AR subtype are evaluated for a possible therapeutic application as diuretics

and antihypertensives, or for their beneficial role in heart failure, allergy, and asthma modulation

6-17]. It has been widely reported that A ARs are involved in the modulation of the intestinal

[

1

motility. In particular, the activation of this receptor leads to an inhibition of the propulsive motor

activity of ileus. Conversely, blocking of the same receptor by antagonists restores the normal

motility function without any induction of diarrhea. These data suggest the administration of A AR

1

antagonists as a potential therapeutic strategy for various functional motility syndromes in the

colon, including constipation and postoperative ileus [18-22].

A prototypical scaffold used to obtain A AR antagonists is the xanthine moiety, with the

1

introduction of two alkyl chains to the N1 and N3 nitrogen atoms and a bulky and generally

hydrophobic group in 8-position [23]. This plot led to the development of A AR antagonists like

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DPCPX, BG 9719, BG 9928, and KW 3902 (1-1c, respectively, Chart 1) that represent reference

molecules for pharmacological studies at the A AR and/or have been evaluated in various clinical

1

trials [24]. Among them, BG 9719 (1a, also named naxifylline or CVT 124) was developed by

Biogen Inc. for the treatment of congestive heart failure with concomitant renal impairment, where

it protected against decline in renal function. The molecule is endowed with an affinity at the

human (h) A AR of 10.3 nM and selectivity versus the other AR subtypes [25]. Unfortunately, it

1

was withdrawn after phase 2 clinical trials because it lowered seizure threshold [8,26]. The same

company similarly evaluated the oral drug BG 9928 [27] (1b, also named tonapofylline) in two

large pivotal Phase 2 trials (POSEIDON and TRIDENT-1) aiming at improving natriuresis and

volume loss in high-risk patients with acute decompensated heart failure. Results from these studies

showed lack of statistical difference between treated and controlled groups in regards to body

weight change, improved dyspnea, worsened renal function, and length of hospital stay, alongside

inducing a 1% risk of seizure [16,28]. This outcome, in conjunction with the clinical failure of

another A AR antagonist KW 3902 [29] (1c, also named rolofylline and MK7418) in a Phase 3 trial

1

by NovoCardia Inc. and Merck & Co. Inc., aroused concerns about the clinical safety and efficacy

of A antagonists in the treatment of a broad range of patients with heart failure [30]. On the other

1

hand, A AR antagonists were developed also based on various non-xanthine scaffolds [31] such as

1

purines [32], imidazopurinones [33], pyrazolopyrimidines [34], pyrazolopyridines [35],

naphthyridines [36], pyrimidoindoles [37], thiazoles [38], pyrimidines [39], pyrazoles [40],

benzimidazolquinolines [41], and pyrazolopyrimidopyridazones [42] as examples. Even in these

cases, the compounds present one or more bulky and hydrophobic groups that (like for the above

cited xanthine derivatives) appear to generally provide affinity for the A AR and selectivity versus

1

the other AR subtypes.

In the late ‘90s, Solvay Pharmaceuticals discontinued the Phase 2 development of a promising

potent and highly selective A AR antagonist based on the pyrrolopyrimidine scaffold, SLV320 [43]

1

(2, Chart 1, also named derenofylline; K hA AR = 1 nM), for the treatment of acute heart failure

i 1

[

24]. In the last years, some investigations at rat cortex and striatum preparations on the activity of

6

adenine derivatives bearing alkyl or cycloalkyl groups in the N ,9-positions and on the activity at

human ARs of 8-arylsubstituted 9-methyladenines were reported [32,44,45]. Among these

molecules N-0861 (3, Chart 1) represents a prototypical example [46]. More recently, we reported

the design, synthesis, and biological evaluation of AR antagonists based on the adenine moiety and

presenting different substituents in 2-, 8-, and 9-position that conferred particular AR subtype

selectivity [47-52]. Interestingly, among these derivatives, the compound 9-ethyl-8-phenyladenine

(

4, Chart 1) resulted an human A AR (hA AR) ligand endowed with nanomolar affinity (K = 31

1

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nM) at this receptor subtype [50,53,54]. Starting from these observations and with the aim of

developing potent and selective A AR antagonists, we designed and synthesized 8-phenyladenine

1

derivatives presenting a small alkyl (methyl, ethyl, or propyl) group in the 9-position and different

6

cycloalkyl (c-butyl, c-pentyl, 3-tetrahydrofuryl, and c-hexyl) substituents in the N -position.

PLEASE INSERT CHART 1 HERE

Binding studies and functional assays at Chinese hamster ovary (CHO) cells transfected with the

human recombinant ARs demonstrated that the newly synthesized derivatives behave as selective

6

A AR ligands. Among them, the N -cyclopentyl-9-methyl-8-phenyladenine (17, Scheme 1, Table 1)

1

showed the highest A AR affinity (K = 2.8 nM) and a good selectivity versus the other AR

1

i

6

subtypes. This compound was hence selected as prototype to develop a second series of N -

cyclopentyl-9-methyladenine derivatives presenting in the 8-position a substituted phenyl ring or a

3

-pyridyl group, with the aim to evaluate the effect of this functionalization on the A AR affinity

1

and selectivity. The affinities of the compounds were analyzed with the aid of molecular modeling

studies performed at the recently published crystal structure of the human A AR.

1

Furthermore, a set of compounds selected for their good affinity/selectivity profile or chemical-

physical properties was tested at functional studies on CHO cells stably transfected with hA ARs to

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evaluate their antagonist activity. Based on the observation that the A AR activation affects the

1

intestinal motility, the same molecules were also tested at functional studies at mouse ileum

preparations to evaluate their ability to counteract the inhibitory effect of A AR activation on the

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tissue motility. Finally, an in-silico analysis allowed a prediction of their pharmacokinetic profile.

Results and Discussion

Chemistry

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The N ,8,9-trisubstituted adenine derivatives 12-25 were synthesized starting from commercially

available 5-amino-4,6-dichloropyrimidine (5, Scheme 1).

Reaction of 5 with the suitable amine in the presence of triethylamine led to the substitution of one

chlorine atom obtaining the mono-substituted intermediates 6-8 in good yield.

PLEASE INSERT SCHEME 1 HERE

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In a two-step procedure, these intermediates were reacted with benzaldehyde under acidic catalysis

to form the imines, which by ring closure, performed by reaction with iron (III) chloride in ethanol,

furnished intermediates 9-11. Compounds 7 and 10 were previously reported [50]. The 9-alkyl-6-

chloro-8-phenyl-9H-purines 9-11 were subjected to amination at the 6-position to afford the desired

di- and tri-substituted adenine derivatives 12, 13 and 14-25, respectively.

6

The synthesis of 8-aryl-N -cyclopentyl-9-methyladenines 37-47 (Scheme 2) was undertaken

following a similar synthetic procedure. Various arylaldehydes were reacted with intermediate 6 in

an acidic medium leading to the respective imines. After azeotropic distillation of the residue with

toluene, the imines were subjected to ring closure in the presence of iron (III) chloride to obtain the

8-aryl-6-chloro-9-methyl-9H-purines 26-36. Replacement of the 6-chlorine atom by reaction with

cyclopentylamine afforded the final products 37-47.

PLEASE INSERT SCHEME 2 HERE

Binding studies

The newly synthesized compounds 12-25 and 37-47 were evaluated at the human recombinant ARs,

stably transfected into CHO cells, utilizing radioligand binding studies (A , A , and A ARs) or

1

2A

3

adenylyl cyclase activity assay (A AR). Receptor binding affinity was determined using

2

B

3

6

3

[

H]CCPA (2-chloro-N -cyclopentylAdo) as radioligand for A AR, whereas [ H]NECA (5’-N-

1

ethylcarboxamidoAdo) was used for the A₂_Aand A AR subtypes. In the case of A AR, K values

3

2B

i

were calculated from IC₅₀values determined by inhibition of NECA-stimulated adenylyl cyclase

activity [55]. The results of binding (A , A , and A ARs) and adenylyl cyclase assays (A AR)

1

2A

3

2B

are reported in Table 1 and they are discussed along with molecular modeling studies.

PLEASE INSERT TABLE 1 HERE

Structure-activity relationships and molecular modeling

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To depict the structural features at the base of the different binding affinities of these derivatives,

molecular modeling analyses consisting in docking studies at the recently reported crystal structure

of the human A AR (covalently bound to the xanthine-based ligand DU172, endowed with a pK

1

i

affinity data of 7.4 for this AR; pdb code: 5UEN; 3.2-Å resolution [56]) were performed and are

here described. The crystal structure was refined within MOE [57] software by removing the

covalently bound ligand DU172 and the engineered segment bRIL and by restoring the IL3 missing

loop and the wild type receptor sequence that was changed by the N159A mutation inserted in the

X-ray structure. Docking analyses were performed with CCDC Gold [58] and Autodock [59,60]

software. The obtained highest score and most frequent docking conformations present the purine

6

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scaffold positioned between the Phe171 (EL2) and the Leu250

sidechains (Figure 1A), in an

arrangement almost corresponding to the one of the purine moiety in the adenosine- and NECA-

bound A AR crystal structures [61].

2

A

6

.55

The polar hydrogen of the 6-amine points towards Asn254 and together with the N7 atom makes

a double H-bond interaction with the amide group of the sidechain of this amino acid. About the

activity of the molecules, we previously reported that 9-ethyl-8-phenyladenine (4) is endowed with

nanomolar affinity at A AR (K = 31 nM) and good selectivity versus the A and A AR subtypes,

1

i

2B

3

while the selectivity the A ARs was modest (4: A /A = 7; A /A = 97; A /A = 186, Table 1).

2

A

2A

1

2B

1

3

1

Hence, we evaluated the impact of the length of the 9-alkyl substituent on the A AR affinity and

1

selectivity, by introducing a methyl or a propyl chain (12 and 13, respectively, Table 1) in this

position. The replacement of the 9-ethyl group with a propyl chain decreased A AR affinity and

1

selectivity (13: K A AR = 101 nM) while the 9-substitution with a shorter methyl group led to a

i

1

slight improvement of affinity and a significant increase in A AR selectivity (12: K A AR = 23

1

i

1

nM; A /A = 17; A /A = 339; A /A = 609).

2

A

1

2B

1

3

1

6

The results of binding studies show that, among the compounds presenting the same N -cycloalkyl

substituent, the 9-methyl derivatives appear again as the most active ligands and the trend of affinity

9-methyl > 9-ethyl > 9-propyl is maintained. Interestingly, such analogous trend is observed even

considering the selectivity versus the other AR subtypes (Table 1, compare 14-16, 17-19, 20-22,

3

.32

and 23-25). Docking conformations make the 9-alkyl group being in proximity to the Val87

residue and pointing toward its side chain.

PLEASE INSERT FIGURE 1 HERE

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The volume of the 9-substituent appears critical as large 9-substituents could take to a steric clash

3

.32

with the Val87

residue. Hence, a small methyl group appears the most suitable substituent for

this purine position as it is well inserted in the available space and avoids any hindrance problem

with the surrounding protein residues. On the contrary, the presence of a longer propyl chain in the

9-position makes the molecule getting slightly displaced respect to the corresponding 9-methyl

substituted compounds (Figure 1B). This helps to interpret the different A AR affinity based on the

1

9-substituent.

6

The introduction of a cycloalkyl group in the N -position generally takes to an improvement of the

A and A and a decrease of the A and A₂_BAR affinities respect to the corresponding compounds

1

3

2A

with the unsubstituted 6-amine. Considering the three 9-methyl, 9-ethyl, and 9-propyl substituted

6

series, the cyclopentyl ring in the N -position generally provides the best A AR affinity among the

1

various inserted cycloalkyl groups. In detail, considering the 9-methyl substituted derivative series,

compound 20 shows low nanomolar affinity and the best selectivity profile versus the A₂_Aand A₃

AR subtypes (20: K A AR = 8.0 nM; A /A = 229; A /A = 1,663; A /A = 783), but compound

i

1

2A

1

2B

1

3

1

7 presents the highest A AR affinity (i.e. compare 17 with the other 9-methyl-8-phenyl derivatives

1

2, 14, 20, and 23) and a good selectivity versus the other AR subtypes (17: K A AR = 2.8 nM;

i

1

A /A = 178; A /A = 3,039; A /A = 302).

2

A

1

2B

1

3

1

PLEASE INSERT FIGURE 2 HERE

6

According to docking results, the N -cyclic moiety gets positioned almost in correspondence of the

cyclohexyl group of the co-crystallized compound DU172, in an external region between the side

6

.51

6.54

chains of residues belonging to the EL2 (Glu172 and Met177), TM6 (Leu250 , Leu253 , and

6

.58

7.35

Thr257 ), and TM7 (Thr270 ) domains (Figure 2A). This group occupies also the same position

6

that is expected to be occupied by the N -cyclopentyl group of the agonists CPA (N -

cyclopentylAdo) and CCPA, in the case these compounds get an analogue arrangement of Ado and

NECA at the A AR [62]. The presence of this group makes the Glu172 residue being oriented

2

A

toward the positively charged Lys173 residue in proximity. This amino acid makes a sort of

electrostatic bridge between Glu172 and Glu170, providing a stabilizing interaction network for the

two carboxylated residues. This may explain why the insertion of cycloalkyl groups is well

tolerated at Ado derivatives (i.e. the above cited reference agonists CPA and CCPA) but not at the

A AR. This receptor presents a glutamate residue (Glu169) corresponding to the Glu172 of the

2

A

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A AR, but the orientation of this residue is generally toward the free amine groups of ligands that

1

6

.55

are positioned in proximity of the Asn253

residue, as observed at several A AR crystal

2A

structures reported to date [62-65]. The orientation of this A AR residue gets changed only in the

2

A

case of the presence of H-bond donor groups within the ligand to work as counterpart of the H-bond

acceptor Glu169, as observed at some ligand-bound X-ray structures of the same receptor [66,67].

As a second step of development, we further modified compound 17 to have a second series of

6

derivatives consisting in N -cyclopentyl-9-methyladenines presenting substituted aryl groups in the

8-position, with the aim to evaluate the effect on the biological activity given by the insertion of

substituents on the 8-phenyl ring or by substituting this aryl moiety with a pyridyl group. It may be

observed that the introduction of small substituents in the ortho-position of the 8-phenyl ring leads

to a significant drop of A AR affinity respect to the unsubstituted analogue 17 (compounds 38 and

1

4

1, K = 55 and 37 nM, respectively).

i

The 8-phenyl group is inserted in a hydrophobic sub-cavity given by residues belonging to the TM3

3

.33

3.36

5.38

5.42

6.48

6.51

(

Leu88

and Thr91 ), TM5 (Met180

and Asn184 ), and TM6 (Trp247 , Leu250 , and

6

.52

His251 ) domains (Figure 2B). The presence of small groups in the ortho-position of the 8-phenyl

ring (38 and 41) makes the compounds to displace from the above described docking arrangement

probably due to a steric clash between the ortho-substituent itself and the 9-methyl group or the

3

.32

6.48

6.51

6.55

sidechains of amino acids in proximity (Val87 , Phe171, Trp247 , Leu250 , Asn254 ).

These molecules adopt a different orientation within the cavity and get more externally located at

the binding cavity. Such displacement could explain the significant decrease of the A AR affinity

1

respect to the unsubstituted compound 17. The replacement of the 8-phenyl ring with a 3-pyridine

(37) takes to a general drop of AR affinity. The substitution at the meta-position of the 8-phenyl

ring provides the most active compounds of this second series with A AR affinity at the low

1

nanomolar range (39 and 42, K = 6.2 and 6.5 nM, respectively). On the other hand, these

i

modifications improve the affinity at the other AR subtypes leading to compounds presenting an

unsuitable selectivity profile, lower than the unsubstituted analogue 17.

PLEASE INSERT FIGURE 3 HERE

According to docking results, the meta-substituents get located in small sub-pockets given by the

3

.32

3.33

5.38

sidechains of residues belonging to TM3 and TM5 segments (Val87 , Leu88 , Met180 ) or

6

.48

6.51

6.52

TM6 (Trp247 , Leu250 , His251 ). The introduction of these substituents is hence well

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tolerated even if it does not take to an improvement of A AR affinity (Figure 3A). The modification

1

in the para-position of the 8-phenyl ring of 17 is tolerated only with the introduction of small

moieties like a methyl group or a fluorine atom affording compounds 40 and 44, respectively. The

latter compound presents a good affinity for A AR and the best selectivity profile versus the A AR

1

2A

among the compounds of both series and is also inactive at the A AR (44: K A AR = 6.2 nM;

2

B

i

1

A /A = 387; A /A > 4,839; A /A = 184). The introduction of a hydroxyl group in the para-

2

A

1

2B

1

3

1

position (46) is slightly detrimental for the affinity for the A AR but not A and A AR subtypes

1

2A

3

and hence it takes to a decrease of the A AR selectivity. The insertion of a carboxyl group (47)

1

takes to an almost complete loss of the affinity at all the ARs. Docking results show that the para-

substituent gest located in a small pocket closely related to the one of the meta-substituents (see

3

.32

3.33

5.38

6.48

6.51

6.52

above) and given again by Val87 , Leu88 , Met180 , Trp247 , Leu250 , and His251

Figure 3B). Considering the described binding mode of the compounds, this cavity allows the

(

insertion of small groups with a not significantly polar profile. The observation that compounds

containing a para-substituent with these properties (i.e. 40 and 44) present higher A AR affinity

1

respect to the other para-substituted compounds (see Table 1) provides a confirm of the reliability

of the docking results.

Functional studies

A subset of synthesized adenine derivatives, selected for their good A AR affinity/selectivity profile

1

(compounds 17, 39, 40, 44) or chemical-physical properties (compound 46, bearing a more

hydrophilic 8-substituent respect to the other selected molecules) was tested in GloSensor cAMP

Assay to evaluate their functional profile at the human A AR. This assay allows to monitor GPCR

1

activity through change in the intracellular cAMP concentration by using a mutant form of Photinus

pyralis luciferase into which a cAMP-binding protein moiety has been inserted [68]. Results are

showed in Figure 4 and Table 2 as concentration-response curves and IC₅₀values, respectively.

First, it was evaluated the full A AR agonist CCPA and it was constructed its concentration-

1

response curve (Figure 4A) in which the maximum effect in reducing luminescence signa, which is

correlated to the inhibition of AC, was detected at a concentration of 1 µM.

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Then, the new compounds were evaluated for their ability (Figure 4B) in reverting the effect

induced by 0.1 µM CCPA, which is the dose that produce the 80% of maximum effect. The

selective A AR antagonist DPCPX was tested as reference compound. The results of these

1

experiments showed that all the selected compounds, as well as DPCPX, could completely revert

the inhibition of luminescence induced by CCPA so behaving as antagonists of the human A AR. In

1

Table 2 are reported the calculated IC values of the tested compounds. It is worthwhile to note

5

0

that, although DPCPX resulted the most potent (IC₅₀= 2.9 nM), all the new adenine derivatives

showed IC values in the nanomolar range, presenting a fairly analogue trend respect to the binding

5

0

affinity data (Table 2). These experiments confirm compound 17 as the most potent (IC = 14 nM).

5

0

The same set of compounds was also tested in a functional assay at mouse ileum, whose amplitude

of the rhythmic spontaneous contraction is reduced by the activation of A ARs [51,69]. Hence, it

1

was evaluated the ability of the new compounds to counteract the reduction of mouse ileum

contractility induced by the full agonist CCPA. Figure 5A shows the ability of increased

concentration of CCPA to decrease the spontaneous contraction of the mouse ileum. From the

concentration-response curves of the CCPA alone (as “control” curve) and in the presence of

DPCPX or the new compounds 17, 39, 40, 44, and 46 at 1 µM (data not shown) the K values were

b

calculated (Table 2). The results confirm the antagonist profile of the new compounds as they could

shift the CCPA concentration-response curve at higher concentration, while they were unable to

affect both amplitude and frequency of the rhythmic spontaneous contraction of ileum in the

absence of the agonist (data not shown). In Figure 5A is presented the curve of rhythmic

spontaneous contractions of mouse ileum affected by increasing concentrations of CCPA, while, in

Figure 5B, is represented (as an example) the ability of the most potent compound 17 (at the

concentration of 1 µM) to preserve the mouse ileum contractility in the presence of CCPA. This

new derivative, as well as all the other tested, could partly restore the spontaneous contraction of the

intestine inhibited by CCPA.

PLEASE INSERT FIGURE 5 HERE

The inhibition of rhythmic spontaneous contractions of mouse ileum induced by increased

concentration of CCPA alone or in the presence of the A AR antagonist 17 at the concentration of 1

1

µM was transformed in concentration-response curve reported in Figure 6.

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PLEASE INSERT FIGURE 6 HERE

-6

The curves of Figure 6 highlight that 1 µM (10 M) concentration of CCPA takes to a tissue

response of about 20%, that is restored to about 75% by same concentration of 17, while the effect

-7

of 0.1 µM (10 M) concentration of CCPA led to tissue response of about 65% and this effect is

almost completely reverted (100% of spontaneous contractions) by 1 µM of compound 17.

At the end of experiment the wash out of tissue was performed. After 30 min, the tissue completely

recovered and the initial twitch was reached, showing that compound 17 is a reversible antagonist.

Moreover, the parallel right shift of the concentration-response curve (in red) represents the ability

of compound 17 to competitively counteract the CCPA agonist effect. It is worthwhile to note that

the K values, obtained with this functional assay at mouse ileum, are again in good agreement with

b

the K values from radioligand binding assay at human A AR, as in the case of cAMP functional

i

1

assay. An exception given by compound 40 presenting a high nanomolar K data in this experiment.

b

In all cases, compound 17 results the most potent derivative with a K data of 17 nM.

b

PLEASE INSERT TABLE 2 HERE

In-silico pharmacokinetic evaluation

Given the significant presence of hydrophobic moieties within the molecular structure of the

developed compounds, a concern could be related to the pharmacokinetic profile of these

molecules. Furthermore, as reported in the introduction, a drawback of the therapeutic employment

of A AR antagonists is the occurrence of side effects, in particular the ones related to the activity of

1

these molecules within the CNS [70,71]. Clinical evaluation of some A AR antagonists was

1

discontinued even due to the increased seizure risk or decreased seizure threshold consequent to the

compound administration [8,16,26,28,72]. We hence performed an in-silico analysis of the above

described set of selected compounds (17, 39, 40, 44, 46), aimed at obtaining a prediction of their

pharmacokinetic profile. Several parameters were considered in this task, from the classical

descriptors LogP/SLogP (Log of octanol-water partition coefficient) and LogS (Log of aqueous

solubility) to pharmacokinetic parameters estimated through more sophisticated approaches. LogP,

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SLogP and LogS data were calculated within MOE, while a set of estimated absorption,

distribution,

and

excretion

data

were

obtained

from

the

pkCSM

(http://biosig.unimelb.edu.au/pkcsm/) [73], SwissADME (http://www.swissadme.ch/) [74], and

cbligand-BBB (http://www.cbligand.org/BBB/) [75] webservices. Reference A AR antagonists

1

SLV320, DPCPX, and KW 3902 were considered for comparison. Data are reported in Table 3.

PLEASE INSERT TABLE 3 HERE

Results show that the selected compounds are endowed with a hydrophobic profile that may be

observed from LogP and LogS data. The introduction of a polar group (i.e. the hydroxy group in

cpd 46) takes to a slight decrease of the hydrophobicity. Given their chemical-physical profile, the

compounds appear readily absorbed at intestinal level. On the same bases, almost all the selected

compounds appear endowed with a good ability to cross the BBB and enter the CNS. As observed

for the LogP data, cpd 46 presents a predicted different behavior respect to its analogues as it is

predicted to not cross the BBB in two out of three analyses. Considering the same parameter,

compound SLV320 is predicted to cross the BBB in two out of three studies. This molecule

presents an analogue chemical-physical profile to 46 and clinical evaluations of this molecule did

not report an increase of seizure risk [17,76]. Taken together these results, a further development of

this structural class of compounds as A AR antagonists should be aimed at achieving a well-

1

balanced profile between pharmacodynamic and pharmacokinetic properties. This would lead to a

compound endowed with good affinity and selectivity at the A AR and suitable pharmacokinetic

1

properties to not give side effects at the CNS.

Conclusions

6

The design and synthesis of N ,8,9-trisubstituted adenine derivatives presenting aromatic and

cycloalkyl groups, to mimic the chemical-physical properties of known A AR antagonists, was

1

undertaken with the aim at developing new potent and selective ligands of this AR subtype. A first

series of derivatives was hence synthesized starting from the 8-phenyladenine and inserting a small

6

alkyl chain of various length and cycloalkyl groups in the 9- and N -position, respectively. Results

of radioligand binding studies showed that the obtained compounds are endowed with high affinity

for the A AR, in some cases at low nanomolar level. The introduction of a methyl group and a

1

ACCEPTED MANUSCRIPT

6

cyclopentyl ring in the 9- and the N -position, respectively (17), provided the best combination of

A AR affinity and selectivity. A second step of the work was the development of a further series of

1

derivatives starting from compound 17 and making small modifications at the 8-aromatic moiety.

The affinity for the A AR was not improved but the selectivity versus the A AR was increased.

1

2A

Molecular modelling studies provided an interpretation of the pharmacological data and the possible

binding mode of the synthesized compounds at the recently published crystal structure of the

hA AR.

1

A selected set of these new derivatives was then used for functional assays at the human A AR

1

showing, as expected, an antagonist profile with IC values at nanomolar level. The same

5

0

molecules showed also to be efficacious as nanomolar antagonists in a functional assay aimed at

analyzing their ability to counteract the inhibition of the rhythmic contraction of mouse ileum tissue

given by A AR activation. In-silico pharmacokinetic evaluation suggests that the modulation of the

1

chemical-physical profile of the molecules could take to a modification of their ability to enter CNS

and to potentially give side effects at this level.

The presented data confirm the 2-6-9-trisubstituted adenine as a very promising scaffold for the

6

development of potent and selective A AR antagonists. The N -cyclopentyl-9-methyl-8-

1

phenyladenine (17), resulting the most active derivative of the series, could hence be used as a

starting point for the development of new safe therapeutic agents for the treatment of intestinal

diseases like constipation and postoperative ileus.

Experimental Section

Chemistry

1

Melting points were determined with a Büchi apparatus and are uncorrected. H NMR spectra were

1

13

obtained with Varian Gemini 200 or VX 300 MHz spectrometers ( H NMR); C NMR spectra

were obtained with a Bruker Ascend 500 MHz spectrometer; values are in ppm, J values are in

δ

Hz. All exchangeable protons were confirmed by the addition of D O. Mass spectra were recorded

2

on an HP 1100-MSD series instrument. Thin-layer chromatography (TLC) was carried out on pre-

coated TLC plates with silica gel 60 F₂₅₄(Fluka). For column chromatography, silica gel 60

(

Merck) was used. Elemental analyses were determined on Fisons Instruments Model EA 1108

CHNS-O model analyzer and are within 0.4% of theoretical values. Purity of the compounds is ≥

8% according to elemental analysis data.

9

4

General procedure for the synthesis of N -alkyl-4,5-diamino-6-chloropyrimidine 6-8. A

solution of 5-amino-4,6-dichloropyrimidine (5, 1.0 mmol), triethylamine (1.0 mmol), ethanol (4.3

ACCEPTED MANUSCRIPT

mL), and the appropriate amine (2.5 mmol) was heated in a steel vial for the time specified for each

compound. After the removal of volatiles in vacuo, the crude was purified as described for each

compound.

4

,5-Diamino-6-chloro-N -methylpyrimidine (6). The reaction mixture containing methylamine

was heated at 55 °C for 3 h. Column chromatography on silica gel, eluting with CHCl -cC H -

3

6

12

CH OH (70:20:10) and recrystallization from EtOAc afforded 6 as a white solid (148 mg, 93%);

3

1

mp: 165–67 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 2.89 (d, J = 4.4 Hz, 3H, CH ), 4.96 (br s, 2H,

3

NH ), 6.86 (br q, J = 4.4 Hz, 1H, NH), 7.75 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

2

5

7

4

,5-Diamino-6-chloro-N -ethylpyrimidine (7). The reaction mixture containing ethylamine was

heated at 150 °C for 27 h. Column chromatography on silica gel, eluting with CHCl -CH OH

3

(99.5:0.5) and recrystallization from EtOAc afforded 7 as a white solid (161 mg, 93%); mp: 142–

1

44 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 1.16 (t, J=7.2 Hz, 3H, CH ), 3.39 (m, 2H, CH ), 5.01

3

2

(

br s, 2H, NH ), 6.55 (t, J = 4.9 Hz, 1H, NH), 7.72 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

2

6

9

4

,5-Diamino-6-chloro-N -propylpyrimidine (8). The reaction mixture containing propylamine

was heated at 80 °C for 8 h. Column chromatography on silica gel, eluting with CHCl -cC H -

3

6

12

CH OH (70:28:2) and recrystallization from dichloromethane afforded 8 as a yellow solid (179 mg,

3

1

9

6%); mp: 112–115 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 0.91 (t, J = 7.5 Hz, 3H, CH ), 1.56

3

(

m, 2H, CH CH CH ), 3.30 (m, 2H, N-CH ), 5.02 (br s, 2H, NH ), 6.79 (m, 1H, NH), 7.72 (s, 1H,

2 2 3 2 2

H-2); Anal (C H ClN ) C, H, N.

7

11

4

General procedure for the synthesis of 9-alkyl-6-chloro-8-phenyl-9H-purine 9-11. A mixture of

4

the N -alkyl-4,5-diamino-6-chloropyrimidines 6-8 (1 mmol), benzaldehyde (1.2 mmol), acetic acid

(2.3 mmol) in methanol (4.3 mL) was stirred at room temperature for 16 h. The reaction mixture

was concentrated to dryness under reduced pressure. The residue was azeotropically distilled with

toluene (2 x 4 mL) and used directly in the next step. To the suspension in ethanol (6 mL) of the

crude imine, a solution of FeCl (1 mmol) in ethanol (3 mL) was added. The mixture was stirred at

3

80 °C for 24-48 h. The solvent was removed and the 9-alkyl-6-chloro-8-phenyl-9H-purines were

obtained as pure compounds after silica gel chromatography or crystallization.

6-Chloro-9-methyl-8-phenyl-9H-purine (9). The desired product 9 was obtained from 6;

1

crystallization from ethanol afforded 9 as a white solid (118 mg, 48%); mp: 166–168 °C; H NMR

(

400 MHz, [D ]DMSO): δ = 3.95 (s, 3H, CH ), 7.65 (m, 3H, H-Ph), 7.98 (m, 2H, H-Ph), 8.82 (s,

6

3

1

H, H-2); Anal (C H ClN ) C, H, N.

12 9 4

6-Chloro-9-ethyl-8-phenyl-9H-purine (10). The desired product 10 was obtained from 7; silica

gel flash column chromatography eluting with cC H -EtOAc (70:30) furnished 10 as a white solid

6

12

1

(

132 mg, 51%); mp: 115–117 °C; H NMR (400 MHz, [D ]DMSO): δ = 1.36 (t, J = 7.2 Hz, 3H,

6

ACCEPTED MANUSCRIPT

CH ), 4.40 (q, J = 7.2 Hz, 2H, CH ), 7.66 (m, 3H, H-Ph), 7.88 (m, 2H, H-Ph), 8.82 (s, 1H, H-2);

3

2

Anal (C H ClN ) C, H, N.

1

3

11

4

6-Chloro-9-propyl-8-phenyl-9H-purine (11). The desired product 11 was obtained from 8; silica

gel flash column chromatography eluting with cC H -EtOAc (90:10) furnished 11 as a white solid

6

12

1

(

218 mg, 80%); mp: 92–94 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 0.74 (t, J = 7.5 Hz, 3H, CH₃),

1

.72 (m, 2H, CH CH CH ), 4.37 (t, J = 7.5 Hz, 2H, N-CH ), 7.66 (m, 3H, H-Ph), 7.89 (m, 2H, H-

2

3

2

Ph), 8.83 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

1

4

13

4

General procedure for the synthesis of 9-alkyl-6-amino-8-phenyl-9H-purine 12 and 13. 9-

Alkyl-6-chloro-8-phenyl-9H-purines 9 and 11 (1 mmol) were, in turn, added to ammonia condensed

in a steel vial, cooled with liquid nitrogen. The reaction mixture was left to warm at room

temperature and then heated at the temperature and for the time described for each compound, after

that ammonia was allowed to evaporate at room temperature.

6-Amino-9-methyl-8-phenyl-9H-purine (12). Compound 12 was obtained from 9 by reaction at

70 °C for 16 h. After crystallization from methanol, 12 was obtained after crystallization from

1

methanol as a white solid (187 mg, 83%); mp: 230–232 °C; H NMR (400 MHz, [D

6]DMSO): δ =

3

.81 (s, 3H, CH ), 7.31 (br s, 2H, NH ), 7.58 (m, 3H, H-Ph), 7.88 (m, 2H, H-Ph), 8.19 (s, 1H, H-2);

3 2

Anal (C H N ) C, H, N.

1

2

11

5

6-Amino-9-propyl-8-phenyl-9H-purine (13). Compound 13 was obtained from 11 by reaction at

8

0 °C for 12 h. After silica gel flash column chromatography eluting with CHCl -cC H -CH OH

3

6

12

3

(60:39:1) and crystallization from methanol as a white solid (193 mg, 76%); mp: 229–232 °C;

1

H NMR (400 MHz, [D ]DMSO): δ = 0.71 (t, J=7.4 Hz, 3H, CH ), 1.65 (m, 2H, CH CH CH ), 4.21

6

3 2 2 3

(

t, J = 7.4 Hz, 2H, N-CH ), 7.29 (br s, 2H, NH ), 7.58 (m, 3H, H-Ph), 7.78 (m, 2H, H-Ph), 8.18 (s,

2

1

H, H-2); Anal (C H N ) C, H, N.

1

4

15

5

General procedure for the synthesis of 9-alkyl-6-(cyclobutylamino)-8-phenyl-9H-purine 14-16.

-Alkyl-6-chloro-8-phenyl-9H-purines 9-11 (1 mmol) were solubilized in 12 mL of dry THF. To

9

the solution and cyclobutylamine hydrochloride (2 mmol) and triethylamine (12 mmol) were added

and the reaction mixture left to stir at 80 °C for 60-70 h. After in vacuo evaporation of volatiles, the

reaction mixture was chromatographed on silica gel column eluting with the suitable solvent.

6-(Cyclobutylamino)-9-methyl-8-phenyl-9H-purine (14). Compound 14 was obtained from 9.

Flash column chromatography eluting with CHCl -cC H -CH OH (70:28:2) and crystallization

3

6

12

3

1

from acetonitrile afforded 14 as a white solid (146 mg, 52%); mp: 159–161 °C; H NMR

(

400 MHz, [D

6

]DMSO): δ = 1.68 (m, 2H, H-cyclobutyl), 2.20 (m, 4H, H-cyclobutyl), 3.81 (s, 3H,

6

CH ), 4.75 (m, 1H, N -CH), 7.59 (m, 3H, H-Ph), 7.88 (m, 2H, H-Ph), 8.10 (m, 1H, NH), 8.24 (s,

3

1

H, H-2); Anal (C H N ) C, H, N.

16 17 5

ACCEPTED MANUSCRIPT

6-(Cyclobutylamino)-9-ethyl-8-phenyl-9H-purine (15). Compound 15 was obtained from 10.

Flash column chromatography eluting with CHCl -cC H -CH OH (70:28:2) and crystallization

3

6

12

3

from dichloromethane/petroleum ether afforded 15 as a white solid (191 mg, 65%); mp: 135–138

1

°

C; H NMR (400 MHz, [D

6

]DMSO): δ = 1.28 (t, J = 7.1 Hz, 3H, CH ), 1.65 (m, 2H, H-

3

6

cyclobutyl), 2.21 (m, 4H, H-cyclobutyl), 4.25 (q, J = 7.0 Hz, 2H, CH ), 4.72 (m, 1H, N -CH), 7.56

2

(

m, 3H, H-Ph), 7.78 (m, 2H, H-Ph), 8.09 (m, 1H, NH), 8.23 (s, 1H, H-2); Anal (C H N ) C, H, N.

17 19 5

6-(Cyclobutylamino)-9-propyl-8-phenyl-9H-purine (16). Compound 16 was obtained from 11.

Flash column chromatography eluting with CHCl -cC H -CH OH (70:28:2) and crystallization

3

6

12

3

1

from acetonitrile afforded 16 as a white solid (176 mg, 57%); mp: 79–81 °C; H NMR (400 MHz,

[

(

D ]DMSO): δ = 0.71 (t, J = 7.5 Hz, 3H, CH ), 1.66 (m, 4H, H-cyclobutyl and CH CH ), 2.09-2.25

6

3 2 3

6

m, 4H, H-cyclobutyl), 4.22 (q, J=7.0 Hz, 2H, N-CH ), 4.75 (m, 1H, N -CH), 7.58 (m, 3H, H-Ph),

2

7

.79 (m, 2H, H-Ph), 8.10 (m, 1H, NH), 8.24 (s, 1H, H-2); Anal (C H N ) C, H, N.

18 21 5

General procedure for the synthesis of 9-alkyl-6-(cyclopentylamino)-8-phenyl-9H-purine 17-

9. 9-Alkyl-6-chloro-8-phenyl-9H-purines 9-11 (1 mmol) were, in turn, added to cyclopentylamine

10 mL). The reaction mixtures were stirred at room temperature for 5 h. After that,

1

(

cyclopentylamine in excess was removed under vacuum and the pure products were obtained after

silica gel chromatography of the crude residue by eluting with the suitable solvent.

6-(Cyclopentylamino)-9-methyl-8-phenyl-9H-purine (17). Compound 17 was obtained from 9.

The crude residue was chromatographed on silica gel flash chromatography eluting with CHCl₃-

cC H -CH OH (70:28:2). 17 was obtained as a white solid after crystallization from EtOAc (132

6

12

3

1

mg, 45%); mp: 143–145 °C; H NMR (400 MHz, [D ]DMSO): δ = 1.63 (m, 6H, H-cyclopentyl),

6

1

.96 (m, 2H, H-cyclopentyl), 3.80 (s, 3H, N-CH ), 4.57 (m, 1H, N -CH), 7.57 (m, 3H, H-Ph), 7.73

3

1

3

(

d, J = 7.8 Hz, 1H, NH), 7.87 (m, 2H, H-Ph), 8.24 (s, 1H, H-2); C NMR (400 MHz, [D ]DMSO):

6

δ = 154.6, 153.0, 151.8, 150.1, 130.5, 130.4, 129.6, 129.4, 119.9, 52.2, 32.8, 31.0, 24.2.; Anal

(

C H N ) C, H, N.

17 19 5

6-(Cyclopentylamino)-9-ethyl-8-phenyl-9H-purine (18). Compound 18 was obtained from 10 as

a white solid by crystallization from the crude residue using dichloromethane/petroleum ether (169

1

mg, 55%); mp: 108–110 °C; H NMR (400 MHz, [D ]DMSO): δ = 1.28 (t, J=7.1 Hz, 3H, CH ),

6

3

1

.62 (m, 6H, H-cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 4.25 (q, J = 7.4 Hz, 2H, N-CH ), 4.57

2

6

(

m, 1H, N -CH), 7.59 (m, 3H, H-Ph), 7.75 (m, 3H, H-Ph and NH), 8.24 (s, 1H, H-2); Anal

(

C H N ) C, H, N.

1

8

21

5

6-(Cyclopentylamino)-9-propyl-8-phenyl-9H-purine (19). Compound 19 was obtained from 11.

The crude residue was chromatographed on silica gel flash chromatography eluting with EtOAc-

cC H (60:40). 19 was obtained as a light brown solid after crystallization from acetonitrile (296

6

12

ACCEPTED MANUSCRIPT

1

mg, 92%); mp: 105–117 °C; H NMR (400 MHz, [D ]DMSO): δ = 0.71 (t, J = 7.3 Hz, 3H, CH ),

6

3

1

.63 (m, 8H, H-cyclopentyl and CH CH ), 1.96 (m, 2H, H-cyclopentyl), 4.22 (t, J = 7.3 Hz, 2H, N-

2 3

6

CH ), 4.55 (m, 1H, N -CH), 7.57 (m, 3H, H-Ph), 7.77 (m, 3H, H-Ph and NH), 8.24 (s, 1H, H-2);

2

Anal (C H N ) C, H, N.

1

9

23

5

General procedure for the synthesis of 9-alkyl-6-(tetrahydrofuran-3-ylamino)-8-phenyl-9H-

purine 20-22. 9-Alkyl-6-chloro-8-phenyl-9H-purines 9-11 (1 mmol) were, in turn, solubilized in 12

mL of dry THF, then tetrahydrofuran-3-ylamine toluene-4-sulfonic acid salt (4 mmol) and

triethylamine (12 mmol) were added to the solutions. The solutions were stirred at 80 °C for 40-60

h. After in vacuo evaporation of volatiles, the reaction mixtures were chromatographed on silica gel

columns eluting with suitable solvents.

9-Methyl-8-phenyl-6-(tetrahydrofuran-3-ylamino)-9H-purine (20). Compound 20 was obtained

from 9. The reaction mixture was chromatographed on silica gel flash chromatography eluting with

CHCl -cC H -CH OH (70:28:2). 20 was obtained as a white solid after crystallization from

3

6

12

3

1

methanol (169 mg, 57%); mp: 120–122 °C; H NMR (400 MHz, [D ]DMSO): δ = 2.00-2.30 (m,

6

2

9

H, THF), 3.61-4.00 (m, 7H, THF and N-CH ), 4.75 (m, 1H, N -CH), 7.59 (m, 3H, H-Ph), 7.89 (m,

3

H, H-Ph), 8.03 (br d, J = 6.6 Hz, 1H, NH), 8.29 (s, 1H, H-2); Anal (C H N O) C, H, N.

1

6

17

5

-Ethyl-8-phenyl-6-(tetrahydrofuran-3-ylamino)-9H-purine (21). Compound 21 was obtained

from 10. The reaction mixture was chromatographed on silica gel flash chromatography eluting

with CHCl -cC H -CH OH (70:28:2). Compound 21 was obtained as a white solid after

3

6

12

3

1

crystallization from EtOAc/petroleum ether (102 mg, 33%); mp: 105–107 °C; H NMR (400 MHz,

[

D ]DMSO): δ = 1.28 (t, J=7.1 Hz, 3H, CH ), 2.12 (m, 2H, THF), 3.96-3.79 (m, 4H, THF), 4.25 (q,

6

3

6

J = 7.0 Hz, 2H, N-CH ), 4.77 (m, 1H, N -CH), 7.57 (m, 3H, H-Ph), 7.78 (m, 2H, H-Ph), 7.98 (m,

2

1

H, NH), 8.27 (s, 1H, H-2); Anal (C H N O) C, H, N.

17 19 5

8-Phenyl-9-propyl-6-(tetrahydrofuran-3-ylamino)-9H-purine (22). Compound 22 was obtained

from 11. The reaction mixture was chromatographed on silica gel flash chromatography eluting

with CHCl -cC H -CH OH (70:29:1). 22 was obtained as a white solid after crystallization from

3

6

12

3

1

EtOAc/C H (136 mg, 42%); mp: 79–85 °C; H NMR (400 MHz, [D ]DMSO): δ = 0.71 (t, J=7.3

6

14

6

Hz, 3H, CH ), 1.65 (m, 2H, CH CH CH ), 2.00-2.30 (m, 2H, THF), 3.60-3.99 (m, 4H, THF), 4.22

3

2

3

6

(

t, J=7.4 Hz, 2H, N-CH ), 4.75 (m, 1H, N -CH), 7.58 (m, 3H, H-Ph), 7.78 (m, 2H, H-Ph), 8.00 (br

2

d, 1H, NH), 8.27 (s, 1H, H-2); Anal (C H N O) C, H, N.

1

8

21

5

General procedure for the synthesis of 9-alkyl-6-(cyclohexylamino)-8-phenyl-9H-purine 23-

5. 9-Alkyl-6-chloro-8-phenyl-9H-purines 9-11 (1 mmol) were, in turn, added to cyclohexylamine

13 mL). The reaction mixtures were stirred at room temperature for several hours. After that,

cyclohexylamine in excess was removed under vacuum and the pure products were obtained

2

(

ACCEPTED MANUSCRIPT

through silica gel flash chromatography of the reaction mixture residues by eluting with the suitable

solvent.

6-(Cyclohexylamino)-9-methyl-8-phenyl-9H-purine (23). Compound 23 was obtained from 9.

After 6 h the reaction was finished and the mixture was purified by flash chromatography eluting

with cC H -EtOAc (60:40). 23 was obtained as a white solid after further crystallization from

6

12

1

cyclohexane (218 mg, 71%); mp: 140–142 °C; H NMR (400 MHz, [D ]DMSO): δ = 1.07-1.98 (m,

6

1

2

6

0H, H-cyclohexyl), 3.80 (s, 3H, CH ), 4.14 (m, 1H, N -CH), 7.58 (m, 4H, H-Ph and NH), 7.87 (m,

3

H, H-Ph), 8.23 (s, 1H, H-2); Anal (C H N ) C, H, N.

1

8

21

5

-(Cyclohexylamino)-9-ethyl-8-phenyl-9H-purine (24). Compound 24 was obtained from 10.

After 20 h the reaction was finished and the mixture was purified by flash chromatography eluting

with cC H -EtOAc (60:40). 24 was obtained as a white solid after further crystallization from

6

12

1

cyclohexane (183 mg, 57%); mp: 117–119 °C; H NMR (400 MHz, [D ]DMSO): δ = 1.10-1.95 (m,

6

1

4

6

3H, CH and H-cyclohexyl), 4.12 (br m, 1H, N -CH), 4.25 (q, J = 7.4 Hz, 2H, N-CH ), 7.57 (m,

3

2

H, H-Ph and NH), 7.77 (m, 2H, H-Ph), 8.22 (s, 1H, H-2); Anal (C H N ) C, H, N.

1

9

23

5

-(Cyclohexylamino)-8-phenyl-9-propyl-9H-purine (25). Compound 25 was obtained from 11.

After 6 h the reaction was finished and the mixture was purified by flash chromatography eluting

with cC H -EtOAc (80:20). 25 was obtained as a white solid after further crystallization from n-

6

12

1

hexane (285 mg, 85%); mp: 128–130 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 0.70 (t, J=7.3 Hz,

6

3

H, CH ), 1.03-1.98 (m, 12H, H-cyclohexyl and CH CH ), 4.12 (m, 3H, N-CH and N -CH), 7.56

3

2

3

2

(

m, 4H, H-Ph and NH), 7.76 (m, 2H, H-Ph), 8.22 (s, 1H, H-2); Anal (C H N ) C, H, N.

20 25 5

General procedure for the synthesis of 8-aryl-6-chloro-9-methyl-9H-purine 26-36. A mixture

4

of the 4,5-diamino-6-chloro-N -methylpyrimidine (6, 1 mmol), the appropriate arylaldehyde (1.2

mmol), acetic acid (2.3 mmol) in methanol (4.3 mL) was stirred at room temperature for 16 h. The

reaction mixture was concentrated to dryness under reduced pressure. The residue was

azeotropically distilled with toluene (2 x 4 mL) and used directly in the next step. To the suspension

in ethanol (6 mL) of the crude imine, a solution of anhydrous FeCl (1 mmol) in ethanol (3 mL) was

3

added. The mixture was stirred at 80 °C for the time stated below for each product. The solvent was

removed and the 8-aryl-6-chloro-9-methyl-9H-purines 26-36 were obtained as pure compounds

after silica gel flash chromatography and crystallization.

6-Chloro-9-methyl-8-(pyridin-3-yl)-9H-purine (26). The intermediate imine was left to react 2 h

with FeCl . Flash column chromatography eluting with cC H -EtOAc (80:20) and recrystallization

3

6

12

from EtOAc afforded the desired product 26 as a white solid (34 mg, 14%); mp: 155–157 °C;

1

H NMR (400 MHz, [D ]DMSO): δ = 3.97 (s, 3H, CH ), 7.68 (m, 1H, H-Py), 8.39 (m, 1H, H-Py),

6

3

8

.84 (m, 2H, H-Py and H-2), 9.15 (m, 1H, H-Py); Anal (C H ClN ) C, H, N.

11 8 5

ACCEPTED MANUSCRIPT

6-Chloro-9-methyl-8-o-tolyl-9H-purine (27). The intermediate imine was left to react 2 h with

FeCl . Flash column chromatography eluting with cC H -EtOAc (80:20) and recrystallization from

3

6

12

methanol afforded the desired product 27 as a white solid (101 mg, 39%); mp: 169–172 °C;

1

H NMR (400 MHz, [D ]DMSO): δ = 2.56 (s, 3H, Ph-CH ), 3.65 (s, 3H, CH ), 7.49 (m, 4H, H-Ph),

6

3

8

.83 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

13

11

4

6-Chloro-9-methyl-8-m-tolyl-9H-purine (28). The intermediate imine was left to react 2 h with

FeCl . Flash column chromatography eluting with cC H -CHCl -CH OH (60:39:1) and

3

6

12

3

recrystallization from methanol afforded the desired product 28 as a white solid (70 mg, 27%); mp:

1

24–126 °C; H NMR (400 MHz, [D ]DMSO): δ = 2.44 (s, 3H, Ph-CH ), 3.93 (s, 3H, CH ), 7.50

6

3

(

m, 2H, H-Ph), 7.77 (m, 2H, H-Ph), 8.80 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

13 11 4

6-Chloro-9-methyl-8-p-tolyl-9H-purine (29). The intermediate imine was left to react 18 h with

FeCl . Flash column chromatography eluting with cC H -EtOAc (80:20) and recrystallization from

3

6

12

1

methanol afforded the desired product 29 as a white solid (98 mg, 38%); mp: 125–127 °C; H NMR

(

400 MHz, [D ]DMSO): δ = 2.44 (s, 3H, CH ), 3.93 (s, 3H, CH ), 7.45 (d, J = 8.0 Hz, 2H, H-Ph),

6 3 3

7

6

.88 (d, J = 8.0 Hz, 2H, H-Ph), 8.78 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

13 11 4

-Chloro-9-methyl-8-(2-chlorophenyl)-9H-purine (30). The intermediate imine was left to react 4

h with FeCl . Flash column chromatography eluting with cC H - EtOAc (90:10) and

3

6

12

recrystallization from EtOAc afforded the desired product 30 as a white solid (119 mg, 43%); mp:

1

8

6

54–157 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 3.67 (s, 3H, CH ), 7.58-7.77 (m, 4H, H-Ph),

3

.87 (s, 1H, H-2); Anal (C H Cl N ) C, H, N.

12

8

2

4

-Chloro-9-methyl-8-(3-chlorophenyl)-9H-purine (31). The intermediate imine was left to react 2

h with FeCl . Flash column chromatography eluting with cC H -EtOAc (90:10) and

3

6

12

recrystallization from EtOAc afforded the desired product 31 as a white solid (184 mg, 66%); mp:

1

49–151 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 3.94 (s, 3H, CH ), 7.70 (m, 2H, H-Ph), 7.95 (d,

3

J = 7.4 Hz, 2H, H-Ph), 8.02 (s, 1H, H-Ph), 8.84 (s, 1H, H-2); Anal (C H Cl N ) C, H, N.

1

2

8

2

4

6-Chloro-9-methyl-8-(4-chlorophenyl)-9H-purine (32). The intermediate imine was left to react 2

h with FeCl . Flash column chromatography eluting with cC H - EtOAc (80:20) and

3

6

12

recrystallization from an EtOAc/cC H mixture afforded the desired product 32 as a white solid

6

12

1

(

120 mg, 43%); mp: 146–148 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 3.93 (s, 3H, CH ), 7.71 (d,

3

J=8.8 Hz, 2H, H-Ph), 8.01 (d, J=8.8 Hz, 2H, H-Ph), 8.82 (s, 1H, H-2); Anal (C H Cl N ) C, H, N.

1

2

8

2

4

6-Chloro-9-methyl-8-(4-fluorophenyl)-9H-purine (33). The intermediate imine was left to react

1

8 h with FeCl . Flash column chromatography eluting with cC H -CHCl -CH OH (70:28:2) and

3

6

12

3

recrystallization from methanol afforded the desired product 33 as a white solid (134 mg, 51%);

ACCEPTED MANUSCRIPT

1

mp: 186–188 °C; H NMR (400 MHz, [D ]DMSO): δ=3.93 (s, 3H, CH ), 7.78 (m, 2H, H-Ph), 8.05

6

3

(

m, 2H, H-Ph), 8.81 (s, 1H, H-2); Anal (C H ClFN ) C, H, N.

12 8 4

6-Chloro-9-methyl-8-(4-methoxyphenyl)-9H-purine (34). The intermediate imine was left to

react 18 h with FeCl . Flash column chromatography eluting with cC H -CHCl -CH OH (80:18:2)

3

6

12

3

and recrystallization from isopropanol afforded the desired product 34 as a white solid (113 mg,

1

4

1%); mp: 134–137 °C; H NMR (400 MHz, [D

6

]DMSO): δ=3.88 (s, 3H, Ph-OCH ), 3.94 (s, 3H,

3

CH ), 7.18 (m, 2H, H-Ph), 7.95 (m, 2H, H-Ph), 8.77 (s, 1H, H-2); Anal (C H ClN O) C, H, N.

3

13 11

4

6-Chloro-9-methyl-8-(4-hydroxyphenyl)-9H-purine (35). The intermediate imine was left to

react 16 h with FeCl . Purification by preparative TLC eluting with CHCl -cC H -CH OH

3

6

12

3

(70:25:5) and recrystallization from ethanol afforded the desired product 35 as a white solid (162

1

mg, 62%); mp: 203–206 °C; H NMR (400 MHz, [D

6

]DMSO): δ=3.92 (s, 3H, CH ), 6.98 (d, J=8.4

3

Hz, 2H, H-Ph), 7.84 (d, J=8.4 Hz, 2H, H-Ph), 8.75 (s, 1H, H-2), 10.23 (s, 1H, Ph-OH); Anal

(

C H ClN O) C, H, N.

12 9 4

4-(6-Chloro-9-methyl-9H-purin-8-yl)benzoic acid (36). The intermediate imine was left to react 2

h with FeCl . Flash column chromatography eluting with CHCl -cC H -CH OH (70:25:5) and 2 gtt

3

6

12

3

of CH COOH and recrystallization from ethanol afforded the desired product 36 as a white solid

3

1

(

84 mg, 29%); mp: 246–248 °C; H NMR (400 MHz, [D

6

]DMSO): δ=3.96 (s, 3H, CH ), 8.14 (m,

3

4

H, H-Ph), 8.84 (s, 1H, H-2), 13.35 (s, 1H, COOH); Anal (C H ClN O ) C, H, N.

13 9 4 2

General procedure for the synthesis of 8-aryl-6-cyclopentylamino-9-methyl-9H-purine 37-47.

-aryl-6-chloro-9-methyl-9H-purines 26-36 (1 mmol) were, in turn, added to cyclopentylamine (10

8

mL). The reaction mixtures were stirred at room temperature for the time stated for each compound.

The solvent was evaporated and the pure products were obtained after silica gel flash

chromatography eluting with the suitable solvent.

6-(Cyclopentylamino)-9-methyl-8-(pyridin-3-yl)-9H-purine (37). Compound 37 was obtained

from 26. After 5 h of reaction time, the crude was chromatographed eluting with cC H -EtOAc

6

12

(70:30). Compound 37 was obtained as a white solid after crystallization from EtOAc (221 mg,

1

7

1

5%); mp: 145–147 °C; H NMR (400 MHz, [D

6

]DMSO): δ=1.47-1.75 (m, 6H, H-cyclopentyl),

.96 (m, 2H, H-cyclopentyl), 3.82 (s, 3H, CH ), 4.58 (br m, 1H, NCH), 7.61 (m, 1H, H-Py), 7.81 (d,

3

J=7.2 Hz, 1H, NH), 8.29 (m, 2H, H-Py and H-2), 8.74 (m, 1H, H-Py), 9.06 (m, 1H, H-Py); Anal

(

C H N ) C, H, N.

16 18 6

6-(Cyclopentylamino)-9-methyl-8-o-tolyl-9H-purine (38). Compound 38 was obtained from 27.

After 5 h of reaction time, the crude residue was chromatographed eluting with cC H - EtOAc

6

12

(70:30). Compound 38 was obtained as a white solid after crystallization from acetonitrile (252 mg,

1

8

2%); mp: 142–144 °C; H NMR (400 MHz, [D

6

]DMSO): δ=1.51-1.79 (m, 6H, H-cyclopentyl),

ACCEPTED MANUSCRIPT

1

.95 (m, 2H, H-cyclopentyl), 2.22 (s, 3H, Ph-CH ), 3.49 (s, 3H, CH ), 4.55 (br m, 1H, NCH), 7.41

3

(

m, 4H, H-Ph), 7.67 (d, J=6.6 Hz, 1H, NH), 8.24 (s, 1H, H-2); Anal (C H N ) C, H, N.

18 21 5

6-(Cyclopentylamino)-9-methyl-8-m-tolyl-9H-purine (39). Compound 39 was obtained from 28.

After 5 h of reaction time, the crude residue was chromatographed eluting with cC H - EtOAc

6

12

1

(

85:15). Compound 39 was obtained as a vitreous solid (212 mg, 69%); mp: n.d.; H NMR

400 MHz, [D ]DMSO): δ=1.48-1.75 (m, 6H, H-cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 2.41 (s,

H, Ph-CH ), 3.79 (s, 3H, CH ), 4.57 (br m, 1H, NCH), 7.42 (m, 2H, H-Ph), 7.65 (m, 3H, H-Ph and

(

6

3

1

3

NH), 8.23 (s, 1H, H-2).; C NMR (400 MHz, [D ]DMSO): δ = 154.6, 153.0, 151.6, 150.2, 138.7,

6

1

31.0, 130.4, 130.2, 129.2, 126.7, 52.2, 32.8, 31.0, 24.2, 21.7; Anal (C H N ) C, H, N.

18 21 5

6-(Cyclopentylamino)-9-methyl-8-p-tolyl-9H-purine (40). Compound 40 was obtained from 29.

After 16 h of reaction time, the crude residue was chromatographed eluting with cC H - EtOAc

6

12

(55:45). Compound 40 was obtained as a white solid after crystallization from acetonitrile (261 mg,

1

8

1

5%); mp: 157–158 °C; H NMR (400 MHz, [D

6

]DMSO): δ=1.44-1.75 (m, 6H, H-cyclopentyl),

.95 (m, 2H, H-cyclopentyl), 2.40 (s, 3H, Ph-CH ), 3.78 (s, 3H, CH ), 4.55 (br m, 1H, NCH), 7.37

3

(d, J=8.0 Hz, 2H, H-Ph), 7.67 (d, J=7.8 Hz, 1H, NH), 7.75 (d, J=8.4 Hz, 2H, H-Ph), 8.22 (s, 1H, H-

1

3

2

1

6

); C NMR (400 MHz, [D ]DMSO): δ = 154.4, 152.7, 151.3, 150.0, 139.9, 130.9, 129.7, 129.3,

6

27.5, 119.3, 52.1, 32.7, 30.7, 24.0, 21.4; Anal (C H N ) C, H, N.

1

8

21

5

-(Cyclopentylamino)-9-methyl-8-(2-chlorophenyl)-9H-purine (41). Compound 41 was obtained

from 30. After 2.5 h of reaction time, the crude residue was chromatographed eluting with cC H -

6

12

EtOAc (70:30). Compound 41 was obtained as a white solid after crystallization from EtOAc (272

1

mg, 83%); mp: 150–152 °C; H NMR (400 MHz, [D

6

]DMSO): δ=1.50-1.75 (m, 6H, H-

cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 3.52 (s, 3H, CH ), 4.55 (br m, 1H, NCH), 7.61 (m, 4H,

3

H-Ph), 7.80 (br d, 1H, NH), 8.26 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

1

7

18

5

6-(Cyclopentylamino)-9-methyl-8-(3-chlorophenyl)-9H-purine (42). Compound 42 was obtained

from 31. After 2.5 h of reaction time, the crude residue was chromatographed eluting with cC H -

6

12

1

EtOAc (70:30). Compound 42 was obtained as a vitreous solid (263 mg, 80%); mp: n.d.; H NMR

400 MHz, [D ]DMSO): δ=1.52-1.75 (m, 6H, H-cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 3.82 (s,

H, CH ), 4.55 (br m, 1H, NCH), 7.61 (m, 2H, H-Ph), 7.82 (m, 2H, H-Ph and NH), 7.95 (s, 1H, H-

(

6

3

Ph), 8.25 (s, 1H, H-2); Anal (C H ClN ) C, H, N.

1

7

18

5

6-(Cyclopentylamino)-9-methyl-8-(4-chlorophenyl)-9H-purine (43). Compound 43 was obtained

from 32. After 6 h of reaction time, the crude residue was chromatographed eluting with cC H -

6

12

EtOAc (70:30). Compound 43 was obtained as a white solid after crystallization from EtOAc (262

1

mg, 80%); mp: 173–176 °C; H NMR (400 MHz, [D

6]DMSO): δ = 1.47-1.75 (m, 6H, H-

cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 3.80 (s, 3H, CH ), 4.52 (br m, 1H, NCH), 7.64 (m, 2H,

3

ACCEPTED MANUSCRIPT

H-Ph), 7.78 (d, J = 7.4 Hz, 1H, NH), 7.91 (m, 2H, H-Ph), 8.24 (s, 1H, H-2); Anal (C H ClN ) C,

1

7

18

5

H, N.

-(Cyclopentylamino)-9-methyl-8-(4-fluorophenyl)-9H-purine (44). Compound 44 was obtained

from 33. After 6 h of reaction time, the crude residue was chromatographed eluting with cC H -

6

12

EtOAc (60:40). Compound 44 was obtained as a white solid after crystallization from cyclohexane

1

(

221 mg, 71%); mp: 95–98 °C; H NMR (400 MHz, [D

6]DMSO): δ = 1.50-1.72 (m, 6H, H-

cyclopentyl), 1.97 (m, 2H, H-cyclopentyl), 3.79 (s, 3H, CH ), 4.58 (br m, 1H, NCH), 7.42 (m, 2H,

3

1

3

H-Ph), 7.72 (d, J = 7.6 Hz, 1H, NH), 7.93 (m, 2H, H-Ph), 8.24 (s, 1H, H-2); C NMR (400 MHz,

D ]DMSO): δ = 164.8, 162.3, 153.1, 149.2, 132.1, 132.0, 127.1, 116.6, 116.4, 52.1, 32.8, 30.9,

[

6

2

4.2; Anal (C H FN ) C, H, N.

17 18 5

6-(Cyclopentylamino)-9-methyl-8-(4-methoxyphenyl)-9H-purine (45). Compound 45 was

obtained from 34. After 16 h of reaction time, the crude residue was chromatographed eluting with

cC H -EtOAc (60:40). Compound 45 was obtained as a white solid after crystallization from

6

12

1

EtOAc (233 mg, 72%); mp: 175–177 °C; H NMR (400 MHz, [D

6

]DMSO): δ = 1.50-1.75 (m, 6H,

H-cyclopentyl), 1.95 (m, 2H, H-cyclopentyl), 3.78 (s, 3H, CH ), 3.84 (s, 3H, Ph-OCH ), 4.55 (br m,

3

1H, NCH), 7.08 (m, 2H, H-Ph), 7.65 (d, J=7.6 Hz, 1H, NH), 7.81 (m, 2H, H-Ph), 8.21 (s, 1H, H-2);

Anal (C H N O) C, H, N.

1

8

21

5

6-(Cyclopentylamino)-9-methyl-8-(4-hydroxyphenyl)-9H-purine (46). Compound 46 was

obtained from 35. After 4 h of reaction time, the crude residue was chromatographed eluting with

cC H -CHCl -CH OH (70:25:5). Compound 46 was obtained as a white solid after crystallization

6

12

3

1

from isopropanol (164 mg, 53%); mp: 250 °C (dec); H NMR (400 MHz, [D ]DMSO): δ=1.45-1.75

6

(

m, 6H, H-cyclopentyl), 1.94 (m, 2H, H-cyclopentyl), 3.76 (s, 3H, CH ), 4.55 (br m, 1H, NCH),

3

6

1

4

.92 (d, J=8.6 Hz, 2H, H-Ph), 7.60 (d, J=7.6 Hz, 1H, NH), 7.69 (d, J=8.6 Hz, 2H, H-Ph), 8.20 (s,

1

3

H, H-2), 9.98 (s, 1H, Ph-OH); C NMR (400 MHz, [D ]DMSO): δ = 159.5, 152.3, 152.6, 150.5,

6

31.3, 131.2, 121.1, 116.1, 52.2, 32.9, 31.0, 26.2, 24.2; Anal (C H N O) C, H, N.

1

7

19

5

-(6-(Cyclopentylamino)-9-methyl-9H-purin-8-yl)benzoic acid (47). Compound 47 was obtained

from 36. After 5 h of reaction time, the crude residue was chromatographed eluting with CHCl₃-

cC H -CH OH (70:25:5). Compound 47 was obtained as a white solid after crystallization from

6

12

3

1

ethanol (213 mg, 63%); mp: 250 °C (dec); H NMR (400 MHz, [D ]DMSO): δ=1.45-1.72 (m, 6H,

6

H-cyclopentyl), 1.91 (m, 2H, H-cyclopentyl), 3.82 (s, 3H, CH ), 4.52 (br m, 1H, NCH), 7.81 (d,

3

J=7.2 Hz, 1H, NH), 7.99 (d, J=5.8 Hz, 2H, H-Ph), 8.22 (s, 1H, H-2), 13.22 (s, 1H, COOH); Anal

(

C H N O) C, H, N.

18 19 5

Biological assays

ACCEPTED MANUSCRIPT

Binding and adenylyl cyclase assays. All pharmacological methods followed the procedures as

described earlier [55]. In brief, membranes for radioligand binding were prepared from CHO cells

stably transfected with human adenosine receptor subtypes in a two-step procedure. In a first low-

speed step (1,000 x g) cell fragments and nuclei were removed. The crude membrane fraction was

sedimented from the supernatant at 100,000 x g. The membrane pellet was resuspended in the

buffer used for the respective binding experiments, frozen in liquid nitrogen and stored at -80°C.

For the measurement of adenylyl cyclase activity only one high speed centrifugation of the

homogenate was used. The resulting crude membrane pellet was resuspended in 50 mMTris/HCl,

pH 7.4 and immediately used for the cyclase assay.

3

For radioligand binding at A AR 1 nM [ H]CCPA was used, whereas 30 and 10 nM [ H]NECA

1

3

were used for A₂_Aand A ARs, respectively. Non-specific binding of [ H]CCPA was determined in

3

the presence of 1 mM theophylline, in the case of [ H]NECA 100 µM R-PIA was used. K -values

i

from competition experiments were calculated with the program SCTFIT [77].

Inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of

affinity of compounds. EC -values from these experiments were converted to K -values with the

50

i

Cheng and Prusoff equation [78].

Functional antagonism in GloSensor cAMP Assay. Chinese hamster ovary (CHO) cells stably

expressing human A AR were grown adherently and maintained in Dulbecco’s Modified Eagles

1

Medium with nutrient mixture F12 (DMEM/F12 with phenol red), supplemented with 10% Fetal

Bovine Serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5µg/ml amphotericin, 1mM

Sodium pyruvate and 0.1 mg/ml Geneticin (G418) at 37 °C and aerated with 5% CO : 95% O .

2

Cells were grown to ~70-80 % confluence and transient transfection with plasmid encoding the

biosensor were performed. After 48h, cells were harvested in CO independent media and counted

2

in a Neobauer chamber. The desiderate cell number was incubated in equilibration medium

containing a 3% v/v GloSensor cAMP reagent stock solution, 10% FBS and 87% of CO₂

independent media. After 2 hrs of incubation cells were dispensed in wells of 384 well plate and

when a steady-state basal signal was obtained CCPA at different concentrations was added. The

antagonist profile of the compounds was evaluated by assessing their ability to counteract an

agonist-induced decrease of cAMP accumulation [68]. The cells were incubated in the reaction

medium (10 min at room temperature) with different antagonist concentrations and then treated

with a fixed dose of CCPA (0.1 µM). Forskolin (FSK) 10 µM was added 10 min after the agonists

and various luminescence reads were performed at different incubation times.

Functional antagonism in mouse ileum. Animal testing was carried out according to the European

Communities Council Directive of 24 November 1986 (86/609/EEC). Experiments were performed

ACCEPTED MANUSCRIPT

on male mice (BALB/C; 25.5 ± 0.5 g body weight; 8 weeks old) according to a reported procedure

[

51]. Briefly ileum segments were connected to isometric force transducers (Ugo Basile, Biological

Research Apparatus, VA, Italy) and mechanical activity was visualized, recorded and analyzed on a

personal computer using the PowerLab/800 system (Ugo Basile, Italy). Longitudinal preparations

were subjected to an initial tension of 200 mg and were allowed to equilibrate for at least 30 min.

Rhythmic spontaneous contractions of varying amplitude developed in all preparations.

Concentration–response curve for CCPA was constructed by cumulative addition of the drug. The

concentration of agonist in the organ bath was increased approximately 3-fold at each step, with

each addition being made only after the response to the previous addition had attained a maximal

level and remained steady.

Antagonism study was carried out incubating tissues for 1 h with a dose of compound under study

before repeating the curve of CCPA. Time control experiments showed that a second curve to the

agonist was reproducible. Parallel experiments in which tissues did not receive any antagonist were

run in order to check any variation in sensitivity.

Statistical analysis. Values of concentration-response curves are given as mean ± SE of four or five

independent observations. Data are expressed as geometric means, with 95% confidence limits.

Student’s t-test was used to assess the statistical significance of the difference between two means.

Statistical significance was set at p < 0.05. Inhibitory effects induced by CCPA were estimated as

the decrease in the amplitude of the spontaneous contraction where 100% corresponding to

maximum amplitude. Curves were fitted by a non-linear regression using a Prism 4.0 program

(GraphPAD Software, San Diego, CA, USA). To quantify agonist (CCPA) potency, EC₅₀values

were calculated. EC₅₀is the concentration of agonist required to produce 50% of the maximum

effect. To quantify antagonist potency, K values were calculated from the equation K = (DR-1) -

b

[

B], where DR is the ratio of EC₅₀values of agonist after and before treatment with antagonist

concentration [B].

As far as the GloSensor cAMP Assay responses were expressed as percentage of the maximal

relative luminescence units (RLU) and concentration–response curves were fitted by a non-linear

regression using a Prism 4.0 programme. To quantify CCPA agonist potency, EC₅₀value was

calculated while to evaluate the antagonist profile of the understudy compounds, IC values were

5

0

determined. IC₅₀is the concentration of antagonists that produces 50% inhibition of the agonist

effect. Each concentration was tested three or four times in duplicate, and the values are given as

mean ± standard error (SE).

Molecular modelling

ACCEPTED MANUSCRIPT

Receptor refinement and energy minimization tasks were carried out using Molecular Operating

Environment (MOE, version 2014.09) suite [57]. Docking experiments were performed with CCDC

Gold [58] and Autodock (within PyRx interface) [59,60,79]. All ligand structures were optimized

using RHF/AM1 semiempirical calculations and the software package MOPAC [80] implemented

in MOE was utilized for these calculations.

A AR crystal structure refinement. The recently published crystal structure of the human A AR

1

was downloaded by the Protein Data Bank webpage (pdb code: 5UEN; 3.2-Å resolution [56]). The

A AR was then rebuilt by removing the covalently bound ligand DU172 and the engineered

1

segment bRIL and by restoring the IL3 missing loop and the wild type receptor sequence that was

changed with the N159A mutation inserted in the X-ray structure. The Homology Modeling tool of

MOE was used for these tasks. The protein coordinates were then minimized with MOE using the

-1

-

AMBER99 force field until the RMS gradient of the potential energy was less than 0.05 kJ mol Å

1

.

Molecular docking analysis. Docking analyses were performed by using CCDC Gold [58] and

Autodock [59,60]. Gold tool was used with default efficiency settings through MOE interface, by

selecting ChemScore as scoring function. Autodock 4.2.6 software was used with PyRx [79]

interface. Lamarckian genetic algorithm was employed for this analysis with the following settings:

50 runs for each ligand; 2,500,000 as maximum number of energy evaluations; 27,000 as maximum

number of generations; 0.02 as rate of gene mutation and 0.8 as rate of crossover. The grid box was

set with 50, 50, and 50 points in the x, y, and z directions, respectively, with the default grid spacing

of 0.375 Å.

A preliminary docking analysis was performed by considering as ligand the reference A AR

1

antagonist DPCPX. This compound presents very high similarity with the co-crystallized compound

DU172, as they share a 1,3-dipropylxanthine segment and a cycloalkyl group in the 8-position

(cyclopentyl and cyclohexyl for DPCPX and DU172, respectively). Docking analysis of DPCPX

within the A AR cavity gave as results ligand positions and conformations almost matching with to

1

the 8-cyclohexyl-1,3-dipropylxanthine fragment of DU172, by the two docking software employed.

Hence, both these programs were used also for the docking analysis of the compounds developed in

this study, with analogue settings.

ASSOCIATED CONTENT

Supporting Information

Elemental analysis data.

1

[

H]NMR spectra of compounds 12-25,37-47.

ACCEPTED MANUSCRIPT

13

[

C]NMR spectra of compounds 17, 39, 40, 44, 46.

ACKNOWLEDGMENT

This work was supported by the University of Camerino (Fondo di Ricerca di Ateneo and Progetto

FAR FPI000042) and by a grant from the Ministry of Research (PRIN N° 2015E8EMCM_008,

2015).

Notes

The authors declare no competing financial interest.

REFERENCES

[

1] B.B. Fredholm, A.P. IJzerman, K.A. Jacobson, K.-N. Klotz, J. Linden, International Union of

Pharmacology. XXV. Nomenclature and classification of adenosine receptors, Pharmacol. Rev., 53

(2001) 527-552.

[

2] B.B. Fredholm, A.P. IJzerman, K.A. Jacobson, J. Linden, C.E. Muller, International Union of

Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors-

-an update, Pharmacol. Rev., 63 (2011) 1-34.

[

3] M.V. Cohen, J.M. Downey, Adenosine: trigger and mediator of cardioprotection, Basic. Res.

Cardiol., 103 (2008) 203-215.

4] L.J. Knutsen, J. Lau, H. Petersen, C. Thomsen, J.U. Weis, M. Shalmi, M.E. Judge, A.J. Hansen,

[

M.J. Sheardown, N-substituted adenosines as novel neuroprotective A agonists with diminished

1

hypotensive effects, J. Med. Chem., 42 (1999) 3463-3477.

[

5] B. Johansson, L. Halldner, T.V. Dunwiddie, S.A. Masino, W. Poelchen, L. Gimenez-Llort, R.M.

Escorihuela, A. Fernandez-Teruel, Z. Wiesenfeld-Hallin, X.J. Xu, A. Hardemark, C. Betsholtz, E.

Herlenius, B.B. Fredholm, Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice

lacking the adenosine A receptor, Proc. Natl. Acad. Sci. U. S. A., 98 (2001) 9407-9412.

1

[

6] S.S. Gottlieb, S.L. Skettino, A. Wolff, E. Beckman, M.L. Fisher, R. Freudenberger, T. Gladwell,

J. Marshall, M. Cines, D. Bennett, E.B. Liittschwager, Effects of BG9719 (CVT-124), an A₁-

adenosine receptor antagonist, and furosemide on glomerular filtration rate and natriuresis in

patients with congestive heart failure, J. Am. Coll. Cardiol., 35 (2000) 56-59.

[

7] S.S. Gottlieb, Renal effects of adenosine A -receptor antagonists in congestive heart failure,

1

Drugs, 61 (2001) 1387-1393.

ACCEPTED MANUSCRIPT

[

8] S.S. Gottlieb, D.C. Brater, I. Thomas, E. Havranek, R. Bourge, S. Goldman, F. Dyer, M.

Gomez, D. Bennett, B. Ticho, E. Beckman, W.T. Abraham, BG9719 (CVT-124), an A adenosine

1

receptor antagonist, protects against the decline in renal function observed with diuretic therapy,

Circulation, 105 (2002) 1348-1353.

[

9] P.C. Obiefuna, V.K. Batra, A. Nadeem, P. Borron, C.N. Wilson, S.J. Mustafa, A novel A₁

adenosine receptor antagonist, L-97-1 [3-[2-(4-aminophenyl)-ethyl]-8-benzyl-7-{2-ethyl-(2-

hydroxy-ethyl)-amino]-ethyl}-1-propyl-3,7-dihydro-purine-2,6-dione], reduces allergic responses to

house dust mite in an allergic rabbit model of asthma, J. Pharmacol. Exp. Ther., 315 (2005) 329-

336.

[

10] A. Nadeem, P.C. Obiefuna, C.N. Wilson, S.J. Mustafa, Adenosine A receptor antagonist

1

versus montelukast on airway reactivity and inflammation, Eur. J. Pharmacol., 551 (2006) 116-124.

11] P. Kalk, B. Eggert, K. Relle, M. Godes, S. Heiden, Y. Sharkovska, Y. Fischer, D. Ziegler,

[

G.W. Bielenberg, B. Hocher, The adenosine A receptor antagonist SLV320 reduces myocardial

1

fibrosis in rats with 5/6 nephrectomy without affecting blood pressure, Br. J. Pharmacol., 151

(2007) 1025-1032.

[

12] B. Greenberg, I. Thomas, D. Banish, S. Goldman, E. Havranek, B.M. Massie, Y. Zhu, B.

Ticho, W.T. Abraham, Effects of multiple oral doses of an A adenosine antagonist, BG9928, in

1

patients with heart failure: results of a placebo-controlled, dose-escalation study, J. Am. Coll.

Cardiol., 50 (2007) 600-606.

[

13] S.S. Gottlieb, Adenosine A antagonists and the cardiorenal syndrome, Curr. Heart Fail. Rep.,

1

5

(2008) 105-109.

[

14] G. Cotter, H.C. Dittrich, B.D. Weatherley, D.M. Bloomfield, C.M. O'Connor, M. Metra, B.M.

Massie, The PROTECT pilot study: a randomized, placebo-controlled, dose-finding study of the

adenosine A receptor antagonist rolofylline in patients with acute heart failure and renal

1

impairment, J. Card. Fail., 14 (2008) 631-640.

[

15] W.F. Kiesman, E. Elzein, J. Zablocki, A adenosine receptor antagonists, agonists, and

1

allosteric enhancers, Handb. Exp. Pharmacol., (2009) 25-58.

16] S.S. Gottlieb, B. Ticho, A. Deykin, W.T. Abraham, D. Denofrio, S.D. Russell, D. Chapman,

[

W. Smith, S. Goldman, I. Thomas, Effects of BG9928, an adenosine A receptor antagonist, in

1

patients with congestive heart failure, J. Clin. Pharmacol., 51 (2011) 899-907.

[

17] P.S. Pang, M. Mehra, A.P. Maggioni, G. Filippatos, J. Middlebrooks, P. Turlapaty, D. Kazei,

M. Gheorghiade, Rationale, design, and results from RENO-DEFEND 1: a randomized, dose-

finding study of the selective A adenosine antagonist SLV320 in patients hospitalized with acute

1

heart failure, Am. Heart J., 161 (2011) 1012-1023 e1013.

ACCEPTED MANUSCRIPT

[

18] M. Suzuki, A. Tomaru, N. Kishibayashi, A. Karasawa, Effects of the adenosine A -receptor

1

antagonist on defecation, small intestinal propulsion and gastric emptying in rats, Jpn. J.

Pharmacol., 68 (1995) 119-123.

[

19] M. Kadowaki, Y. Nagakura, K. Tokita, K. Hanaoka, M. Tomoi, Adenosine A receptor

1

blockade reverses experimental postoperative ileus in rat colon, Eur. J. Pharmacol., 458 (2003) 197-

00.

20] L. Antonioli, M. Fornai, R. Colucci, N. Ghisu, M. Tuccori, M. Del Tacca, C. Blandizzi,

2

[

Regulation of enteric functions by adenosine: pathophysiological and pharmacological implications,

Pharmacol. Ther., 120 (2008) 233-253.

[

21] J.C. Bornstein, Purinergic mechanisms in the control of gastrointestinal motility, Purinergic

Signal., 4 (2008) 197-212.

22] K. Varani, F. Vincenzi, S. Merighi, S. Gessi, P.A. Borea, Biochemical and Pharmacological

[

Role of A Adenosine Receptors and Their Modulation as Novel Therapeutic Strategy, Adv. Exp.

1

Med. Biol., 1051 (2017) 193-232.

[

23] J.W. Daly, W. Padgett, M.T. Shamim, P. Butts-Lamb, J. Waters, 1,3-Dialkyl-8-(p-

sulfophenyl)xanthines: potent water-soluble antagonists for A - and A -adenosine receptors, J. Med.

1

2

Chem., 28 (1985) 487-492.

24] B. Hocher, Adenosine A receptor antagonists in clinical research and development, Kidney

[

1

international, 78 (2010) 438-445.

25] J.R. Pfister, L. Belardinelli, G. Lee, R.T. Lum, P. Milner, W.C. Stanley, J. Linden, S.P. Baker,

[

G. Schreiner, Synthesis and biological evaluation of the enantiomers of the potent and selective A₁-

adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbonyl)]-xanthine, J. Med. Chem., 40 (1997)

1

773-1778.

26] A.A. Wolff, S.L. Skettino, E. Beckman, L. Belardinelli, Renal effects of BG9719, a specific A₁

adenosine receptor antagonist, in congestive heart failure, Drug Dev. Res., 45 (1998) 166-171.

27] W.F. Kiesman, J. Zhao, P.R. Conlon, J.E. Dowling, R.C. Petter, F. Lutterodt, X. Jin, G. Smits,

[

M. Fure, A. Jayaraj, Potent and orally bioavailable 8-bicyclo [2.2.2] octylxanthines as adenosine A₁

receptor antagonists, J. Med. Chem., 49 (2006) 7119-7131.

[

28] C.R. Ensor, S.D. Russell, Tonapofylline: a selective adenosine-1 receptor antagonist for the

treatment of heart failure, Exp. Opin. Pharmacother., 11 (2010) 2405-2415.

29] H. Nonakay, M. Ichimura, M. Takeda, T. Kanda, J. Shimada, F. Suzuki, H. Kase, KW-3902, a

selective high affinity antagonist for adenosine A receptors, Br. J. Pharmacol., 117 (1996) 1645-

[

1

1652.

ACCEPTED MANUSCRIPT

[

30] B.M. Massie, C.M. O'Connor, M. Metra, P. Ponikowski, J.R. Teerlink, G. Cotter, B.D.

Weatherley, J.G. Cleland, M.M. Givertz, A. Voors, Rolofylline, an adenosine A receptor

1

antagonist, in acute heart failure, N. Engl. J. Med., 363 (2010) 1419-1428.

[

31] L.C. Chang, J. Brussee, A.P. IJzerman, Non-xanthine antagonists for the adenosine A₁

receptor, Chem. Biodivers., 1 (2004) 1591-1626.

32] R.D. Thompson, S. Secunda, J.W. Daly, R.A. Olsson, N ,9-disubstituted adenines: potent,

selective antagonists at the A adenosine receptor, J. Med. Chem., 34 (1991) 2877-2882.

6

[

1

[

33] F. Suzuki, J. Shimada, H. Nonaka, A. Ishii, S. Shiozaki, S. Ichikawa, E. Ono, 7,8-Dihydro-8-

ethyl-2-(3-noradamantyl)-4-propyl-1H-imidazo[2,1-i]purin-5(4 H)-one: a potent and water-soluble

adenosine A antagonist, J. Med. Chem., 35 (1992) 3578-3581.

1

[

34] S.A. Poulsen, R.J. Quinn, Synthesis and structure-activity relationship of pyrazolo[3,4-

d]pyrimidines: potent and selective adenosine A receptor antagonists, J. Med. Chem., 39 (1996)

1

4

156-4161.

35] A. Akahane, H. Katayama, T. Mitsunaga, T. Kato, T. Kinoshita, Y. Kita, T. Kusunoki, T.

Terai, K. Yoshida, Y. Shiokawa, Discovery of 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-

(6H)- pyridazinebutanoic acid (FK 838): a novel non-xanthine adenosine A receptor antagonist

[

1

with potent diuretic activity, J. Med. Chem., 42 (1999) 779-783.

36] P.L. Ferrarini, C. Mori, C. Manera, A. Martinelli, F. Mori, G. Saccomanni, P.L. Barili, L. Betti,

[

G. Giannaccini, L. Trincavelli, A. Lucacchini, A novel class of highly potent and selective A₁

adenosine antagonists: structure-affinity profile of a series of 1,8-naphthyridine derivatives, J. Med.

Chem., 43 (2000) 2814-2823.

[

37] S. Hess, C.E. Müller, W. Frobenius, U. Reith, K.-N. Klotz, K. Eger, 7-Deazaadenines bearing

polar substituents: structure-activity relationships of new A and A adenosine receptor antagonists,

1

3

J. Med. Chem., 43 (2000) 4636-4646.

38] E.W. van Tilburg, P.A. van der Klein, M. de Groote, M.W. Beukers, I.J. AP, Substituted 4-

[

phenyl-2-(phenylcarboxamido)-1,3-thiazole derivatives as antagonists for the adenosine A₁

receptor, Bioorg. Med. Chem. Lett., 11 (2001) 2017-2019.

[

39] L.C. Chang, R.F. Spanjersberg, J.K. von Frijtag Drabbe Kunzel, T. Mulder-Krieger, G. van den

Hout, M.W. Beukers, J. Brussee, A.P. IJzerman, 2,4,6-trisubstituted pyrimidines as a new class of

selective adenosine A receptor antagonists, J. Med. Chem., 47 (2004) 6529-6540.

1

[

40] N. Griebenow, L. Bärfacker, H. Meier, D. Schneider, N. Teusch, K. Lustig, R. Kast, P.

Kolkhof, Identification and optimization of substituted 5-aminopyrazoles as potent and selective

adenosine A receptor antagonists, Bioorg. Med. Chem. Lett., 20 (2010) 5891-5894.

1

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New potent and selective A1 adenosine receptor antagonists as potential tools for the treatment of gastrointestinal diseases

DOI: 10.1016/j.ejmech.2018.03.067

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