Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A1 and A2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K i = 0.28 μM) with fivefold A2A selectivity and the non-selective A1/A2A AR ligand pyrimidopurinedione 35 (K i A1 = 0.28 μM and K i A2A = 0.30 μM). The compounds were also evaluated for their affinity at human A1, A2A, A2B and A3 ARs. All of the obtained compounds were docked to the A2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds. Electronic supplementary material The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.

Adenosine, a major constituent of nucleic acids, which consists of the purine base adenine linked to the ribose moiety, has important and diverse effects on many biological processes. Some of the physiological actions of adenosine include effects on heart rate and atrial contractility, vascular smooth muscle tone, release of neurotransmitters, lipolysis, renal function and white blood cell functions [1,2].
Four adenosine receptor (AR) subtypes are known, A 1 , A 2A , A 2B and A 3 , all of which were cloned and pharmacologically characterized. The A 2A and A 2B receptors are positively coupled to adenylyl cyclase, while A 1 and A 3 adenosine receptors cause inhibition of cAMP formation. Adenosine acts via these different receptor subtypes, the affinity to which ranges from nanomolar ("high affinity" A 1 , 3-30 nM; A 2A 1-20 nM) to micromolar ("low affinity" A 2B , 5-20 μM; A 3 >1 μM) [3,4]. These receptors belong to the large superfamily of G protein-coupled receptors [2].
Adenosine A 1 receptors are ubiquitously expressed, e.g. in the central nervous system (CNS), especially in the brain, with high levels being expressed in many regions. The distribution of adenosine A 2A receptors is wide ranging but restricted, including lymphocytes, platelets, brain striatum, vascular smooth muscle and endothelium [2].
The prototypical antagonists of the A 1 adenosine receptor are the xanthines: theophylline and caffeine. Natural xanthines are non-specific adenosine antagonists. They are not selective for any of the adenosine receptor subtypes and have low affinity for the A 1 receptor. Due to their CNS-stimulating effects, A 1 adenosine receptor selective antagonists have been proposed as cognition enhancers for the treatment of dementias, such as Alzheimer's disease. These receptors have been shown to be involved in sedative, antiseizure and anxiolytic effects. New potential indications are being discovered and investigated: in heart (for the treatment of cardiac arrhythmias and oedemas and as positive inotropic and cardiac protectants), in kidney (for oedemas and nephritis treatment), in lung (for asthma, oedema and lung protection) and in CNS (for depression, stress and coma) diseases. A 1 AR antagonists are being investigated as antihypertensives and potassium-saving diuretics with kidney protective effects, for the treatment of depression and asthma and for the prevention of ischemiainduced injuries [5][6][7].
In the last years, numerous studies have confirmed the ability of A 2A adenosine receptor antagonists to prevent neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, ischemic brain damage and, recently, epilepsy and sensorimotor disorders (restless legs syndrome-RLS) [8][9][10][11][12][13][14]. Methylxanthines such as theophylline and caffeine have been known to enhance locomotor activity; however, these compounds are non-selective antagonists and have weak affinity for A 2A AR.
Our efforts were directed towards the development of new selective xanthine adenosine receptor ligands. Our main interest has focussed on the investigation of tricyclic xanthine derivatives [15][16][17][18][19]. The so far most active compounds are shown in Fig. 1. The most potent A 1 AR ligands were found among 1,3-dipropyl-substituted benzylpyrimidopurinediones (I, II) [17], while A 2A adenosine receptor ligands were 1,3- Table 1 Structures of the tested imidazo-, pyrimido-and diazepino[2,1-f]purinediones   dimethyl-substituted aryl-(III, IV) [16] cycloalkyl-(V) [19] and phenalkylpyrimidopurinediones (VI) [18] (Fig. 1). Several of the most active ligands at adenosine A 2A AR (e.g. III, IV and VI) [20] were demonstrated to exhibit antiparkinsonian effects. Among pyrimidopurinediones, compounds were found which displayed anticonvulsant properties-protective activity in subcutaneous pentylenetetrazole (ScMet) or in maximal electroshock (MES) and ScMet test; however, the mechanism of this action was not clear [16,19]. As a continuation of our search for potent adenosine A 1 and especially A 2A receptor ligands among cycloalkyl annelated xanthines, we have developed a new series of imidazo-, pyrimido-and diazepino[2,1-f]purinedione derivatives possessing aliphatic substituents (open congeners of cycloalkyl derivatives) in the annelated ring, e.g. alkyl, alkynyl and alkenyl chains. The compounds turned out to be selective A 1 or A 2A AR antagonists with moderate, submicromolar affinity as shown by radioligand binding studies at native rat receptors. Investigated compounds were examined also for A 2B and A 3 AR affinity at human recombinant AR. Additionally, the most active compounds were evaluated for affinity at human recombinant A 1 and A 2A AR affinity. Molecular modelling studies were performed to discuss affinity of the compounds through the docking onto the active sites of the A 2A adenosine receptor model. Investigated compounds were examined for their anticonvulsant activity as well.

Pharmacology
All compounds were tested in vitro in radioligand binding assays for affinity to A 1 and A 2A adenosine receptors in rat cortical membrane and rat striatal membrane preparations, respectively. The results are presented in Table 2. The nonselective AR ligand caffeine, selective A 2A AR antagonist Preladenant (SCH420814) [31] and selective A 1 AR antagonist PSB-36 [31,32] were included for comparison. Examined compounds were additionally tested for affinity at human recombinant A 1 , A 2A , A 2B and A 3 receptors stably expressed in Chinese hamster ovary (CHO) cells (Table 3). Tricyclic xanthine derivatives, previously obtained in our laboratory, showed anticonvulsant activity [15][16][17][18]. Therefore, compounds 5-35 were evaluated in vivo as potential anticonvulsants by the ADP (Antiepileptic Drug Development Program of the National Institute of Neurological Disorders and Stroke NINDS) according to the Antiepileptic Screening Project. Compounds were injected intraperitoneally as a suspension in 0.5 % methylcellulose into the mice and evaluated in the preliminary screenings with at least three dose levels (30, 100 and 300 mg/kg at 0.5-and 4-h time periods). Phase I of the evaluation included three tests: MES, ScMet seizure tests and the rotorod test for neurological toxicity (TOX). The tests were described in detail by Stables and Kupferberg [37][38][39].
The MES test is a model for generalized tonic-clonic seizures and identifies compounds which prevent seizure spread. The ScMet is a model to test compounds that raise seizure threshold. The minimal motor impairment was measured by the rotorod test. The results are given in Table 4.  Some compounds (9, 11, 13, 15, 16, 22 and 30) were also administered orally to rats and examined in the MES, ScMet screen and TOX test (Table 5). Compound 9 was tested also in the hippocampal binding model in rats to evaluate its ability to prevent or modify both the expression and acquisition of focal seizures [39] (Table 6). For two compounds (9 and 13), a quantitative test in mice (i.p.) was made (ED 50 and TD 50 determination). The results of these experiments compared with literature data for valproate [40] are collected in Table 7.

In vitro tests
Synthesized tricyclic xanthine derivatives displayed affinity towards both A 1 and A 2A ARs in radioligand binding studies performed at rat brain membranes ( Table 2). The most active but not selective A 1 AR ligand was 1,3-dipropylpyrimidopurinedione (35). In the group of diazepinopurinediones were found compounds which displayed submicromolar affinity towards adenosine A 2A receptors. Diazepinopurinedione 28 (K i = 0.28 μM) showed moderate A 2A AR selectivity (about sixfold). A 2A AR ligands with submicromolar affinity were also found among pyrimidopurinediones: compounds 11 and 13 displayed K i values of 0.82 and 0.87 μM, respectively. Nsubstituted derivatives displayed higher affinity than unsubstituted analogues (5, 25 and 33). Elongation of the straight N-alkyl substituents led to an increase in both A 1 and A 2A AR affinities in the group of diazepinopurinediones.
For pyrimidopurinediones, this tendency was not so obvious; it was only observed at A 1 AR. In the case of A 2A AR, the most active ones were compounds with 6 (comp. 11) and 3 (comp. 8) atom chains. Branched N-alkyl, especially αbranched substituents, generally were not favourable for AR affinity. In case of A 1 AR ligands, such modifications led to a loss of affinity (compounds 12, 14, 15, 16 and 18) or its decrease (compound 19). A 2A AR ligands were also sensitive to these modifications; however, compounds with branched N-alkyl substituents still showed micromolar affinity, and βbranched derivative 13 was the most active one with submicromolar affinity. Enlargement of the annelated ring was favourable for both A 1 and A 2A AR activities. Diazepinopurinediones with the same N-alkyl substituents as pyrimidopurinediones were up to sixfold more potent (compounds 9 and 28, and 8 and 27). An exception here was the Nisopropyl derivative 34 which possesses a five-membered annelated ring; however, there are only two members of this group for comparison. Introduction of N1,N3dipropyl substituents into the xanthine core definitely has favourable influence on A 1 AR affinity of Nalkylpyrimidopurinediones: compound 35 was 15-fold more potent than its N1,N3-dimethyl analogue 9. Ligands with unsaturated N-substituents (22-24) displayed only moderate A 1 and A 2A AR affinity.
Selected compounds are mainly those that were the most active ones at the A 2A AR (11, 13, 22, 27, 28 and 35) and were also tested for affinity to human A 1 and A 2A receptors    (Table 3). Generally, the affinity at human recombinant A 1 as well as A 2A receptors was worse than that for native rat receptors (in the range of 1.5-threefold lower affinity).
Only in the case of isobutylpyrimidopurinedione 13, the affinity for the human A 1 AR was better than that for the rat receptor and almost equal for A 2A AR. Affinity to the human A 2B and A 3 receptors of all of the investigated compounds was very weak; only the dipropyl derivative 35 showed micromolar affinity at A 2B AR and submicromolar affinity to the human A 3 AR. Only one compound with a double-branched, long chain (19) from the group of dimethyl derivatives showed affinity for A 3 AR in the micromolar range.
Two of the most potent rat and human A 2A receptor ligands (compounds 28 and 35) were investigated for their functional properties using cAMP accumulation assay. They were investigated for their potency to inhibit NECAinduced cAMP accumulation in CHO cells expressing the human A 2A receptor (Fig. 3). The compounds clearly behaved as competitive antagonists as the concentration-response curve of NECA was shifted to the right in a parallel fashion in their presence. K b values determined in living CHO cells expressing the human adenosine A 2A receptor were well in accordance with K i values determined in radioligand binding studies at membrane preparations of the same cell line. Owing to the structural similarity of all compounds in this series, we suppose that they are all antagonists.

Molecular modelling studies
In our previous molecular modelling studies on A 1 and A 2A adenosine receptors, the comparison of rhodopsin-and β 2adrenergic-based homology models through the docking studies was performed [41]. Since that time, a few X-ray structures of A 2A AR in complex with various ligands have been reported in the Protein Data Bank [42,43], among which  . Graphs from two independent experiments performed in duplicates with mean values ± SEM are shown. Both investigated compounds shifted the concentration-response curve for NECA in a parallel manner to the right, indicating competitive antagonism. Apparent K b values were as follows: 1,510±20 nM (28) and 1,210±130 nM (35) those co-crystallized with xanthines: XAC (PDB code: 3REY) and caffeine (PDB code: 3RFM) are of great importance for our research. Analysis of the ligand binding mode, observed in the crystals 3REY and 3RFM, compared to the binding mode of an inverse agonist ZM241385 bound to A 2A AR (PDB codes: 3PWH [42] and 3EML [43]) indicates flexibility of some amino acid side chains within the receptor binding cleft. In particular, the side chain of Asn253 (6.55), described as a crucial residue for ligand binding [44][45][46], in 3REY is rotated relative to other structures, e.g. 3RFM with caffeine as a ligand. Nevertheless, in both crystal structures of A 2A AR co-crystallized with xanthines, the terminal amino group of Asn253 (6.55) forms the hydrogen bond with the same carbonyl group present in purinedione core of ligands.
Due to the structure and the size of synthesized molecules, A 2A AR-XAC (3REY) crystal seems to be the best choice as a template for docking. To validate the utilized molecular docking methods, XAC ligand was redocked to its X-ray structure of A 2A receptor. In case of simulation without any constraints, the obtained highest ranked pose was distinct from the one in the crystal; superposition of both conformations gave a high RMSD value of 9.68 Å. In this pose, an imidazol nitrogen atom of the ligand plays a role of an H-bond acceptor involved in the contact with the distal amino group of Asn253 (6.55), while a phenyl ring is situated in the proximity of TM6 and ECL2. The docking was then repeated, setting the H-bond interaction between Asn253 (6.55) and one out of carbonyl groups of the ligand as a constraint. This time, the overlay with the experimental binding mode was better (Fig. 4, RMSD= 4.53 Å). The superposition of phenylpurinedione cores was almost perfect, giving an RMSD value of 0.42 Å, but calculated and experimental poses differ with orientation of a polar tail of XAC. However, in the crystal structure, the position of this flexible chain is not strictly fixed, as its electron density is not complete [42]. The protocol including constrained H-bond between a ligand and Asn253 (6.55) was chosen for further docking simulations.
As an additional validation to the tested series of xanthines, the structure of caffeine was added and docked together with the rest of the compounds. Docking simulations of the set of all 30 compounds to both templates gave results that can be grouped in three clusters according to the calculated docking score values.
In the two first, highest ranked clusters, the obtained poses adopt a reflected or rotated orientation of the heterocyclic core compared to the XAC conformation from the crystal. In this position, in both cases, the carbonyl group C2=O2 of the purinedione interacts with a side chain amino group of Asn253 (6.55), while C4=O4 either corresponds to the 2oxo fragment of XAC or points to the top of the receptor, between side chains of Glu169 and Leu267 (7.32).
However, it can be noticed that in both cases of published X-ray structures co-crystallized with xanthines, purinedione core interacts with Asn253 (6.55) in a similar way, with the same carbonyl oxygen atom (C4=O4) of the ligand, even if Asn253 in this crystal acid adopts two different conformations of the side chain. Taking into account positions of both XAC and caffeine in the binding site, we can presume that the synthesized tricyclic derivatives of xanthine would bind into the A 2A receptor in a similar mode (shown in Fig. 5a, b) despite lower docking scores. For this reason, the conformations belonging to the first two clusters were rejected.
The third cluster is created by poses predicted for most of the compounds including caffeine (Fig. 5a, b). Although docking scores are lower than those for the two first sets, this pose is in agreement with the position of the ligand XAC in the crystal structure. The carbonyl group C4=O4 interacts here with the terminal amino group of Asn253 (6.55), and the whole purinedione core of the modelled xanthines overlies the heterocyclic part of XAC with very good RMSD values (from 0.39 to 0.93 Å, for caffeine 0.58 Å), making π-π stacking with the aromatic part of Phe168. Two N-methyl (or N-propyl) substituents point towards the bottom of the binding site, likewise in case of XAC. The third annelated ring is situated between Phe168 and Glu169 of ECL2 from one side and Ile274 (7.39) and Met270 (7.35) from the other. The alkyl chain is buried in a similar pocket of the binding site to the phenoxy tail of XAC, in the long narrow cleft limited by residues from top fragments of TM2 (Ala63 (2.61), Ile66 (2.64) and Ser67 (2.65)) and TM7 (Leu267 (7.32) and Tyr271 (7.36)) as well as Tyr9 (1.35) (Fig. 5c).
The compounds 25 and 33, unsubstituted in the annelated ring, were not successfully docked in this position, while long alkyl chains in this position, branched or not, are easily adapted in the groove created by TM2 and TM7. Similarly, longer propyl fragments at 1-and 3-positions of 35 make hydrophobic contacts with residues Leu85 (3.33), Leu249 (6.51) and Met177 (5.38) from one side and Ile66 (2.64), Ala81 (3.29) and Val84 (3.32) from the other, increasing affinity to the receptor relative to short methyl substituents (35 vs. 9). Compounds 24 and 30 were not found among poses from this cluster either-the most probable explanation is that the linear structure of the triple bond in 24 or a doublebranched chain of 30 causes a steric clash with residues forming the cleft.
The performed docking experiments did not explain in detail the correlation between structure and A 2A AR affinity of the obtained xanthines. Nevertheless, it can be stated that both the third annelated ring and alkyl chains as Nsubstituents fit well to the A 2A binding pocket, forming additional interactions, and their presence has a big influence on the affinity to the adenosine receptors compared to caffeine-non-selective weak A 2A AR antagonist. The similar effect is observed for two N-propyl substituents and the phenyl ring of XAC, very potent adenosine receptor antagonist (35, Fig. 5b).

In vivo tests
Unsubstituted pyrimido-(5) and diazepinopurinedione (25) did not show protective activity in both electric and chemical seizures ( Table 4). Introduction of N-alkyl substituents resulted in anticonvulsant activity in the pyrimidopurinediones, whereas these modifications did not affect in the same way the group of diazepinopurinediones as only compounds 31 and 32 showed protective activity in a dose of 300 mg/kg, but after 4 h caused death of tested animals. Probably, enlargement of the annelated ring in the xanthine derivatives is not favourable for anticonvulsant activity. Unsaturated substituents introduced into the tricyclic xanthine derivatives (23 and 24) were also disadvantageous. Generally, N-alkyl derivatives showed good protective activity in mice (i.p.), especially in ScMet test in short time (0.5 h). The best compound was N-hexylpyrimidopurinedione 11, with 100 % protection at a dose of 100 mg/kg with no symptoms of neurotoxicity. The other two compounds with long lipophilic chains 18 and 19 also showed good protection: derivative 19 was active in both tests, and ligand 18 displayed protection in ScMet test. Both compounds did not show neurotoxicity. The N-butyl and N-pentyl derivatives 9 and 10 displayed protective properties in MES and ScMet tests but at the same time showed neurotoxicity. Compounds with short chains (6, 7, 8 and 14) caused death of test animals. The most toxic substance was N-propargylpyrimidopurinedione 24, causing death following clonic seizures and tonic extension.
The most active compounds in mice were also examined for activity in rats after oral administration, showing higher seizure protection ( Table 5). The best compound was the butyl derivative 9, showing 100 % protection in ScMet test in the dose of 50 mg/kg in short time (0.25 and 0.5 h) and 50 % protection in dose of 10 mg/kg after 1 2 and 4 h. Ligand 16 was active in MES test at 30 mg/kg and in ScMet test at 50 mg/kg. No symptoms of neurotoxicity were observed in both tested compounds. Ligand 9 was also tested in a hippocampal kindled seizure screen (Table 6). Results suggest the ability of 9 to prevent or modify fully kindled seizures. The N-butyl and N-isobutyl derivatives 9 and 13 were advanced for phase II of evaluation for quantification of activities (ED 50 and TD 50 ) against MESand ScMet-induced seizures in mice (i.p.) ( Table 7). These pharmacological parameters were compared with data for valproate [40,47]. Both tested substances were characterized by a PI ScMet higher than that of valproate, but a lower PI MES .
The anticonvulsant activity of the examined compounds was analysed for correlation with AR affinity. Some coincidence of adenosine A 2A affinity and anticonvulsant activity was observed. Compounds showing anticonvulsant activity in ScMet test (9, 11, 13, 14, 15, 16 and 18) were among the Comparing in vitro and in vivo activities of the studied tricyclic xanthine derivatives, there is no clear correlation between A 1 /A 2A AR affinity and anticonvulsant activity of the investigated ligands. So our previous observation that A 2A selectivity of AR antagonists may be important for anticonvulsant activity has not been confirmed in the present study [19].

Conclusions
Tricyclic pyrimido-and diazepinopurinediones are moderately potent A 1 and A 2A AR antagonists. Enlargement of the annelated ring caused an increase in affinity but not selectivity. Pyrimidopurinodiones showed anticonvulsant activity in mice (i.p.) and rats (p.o.), but lower homologs were toxic. Enlargement of the annelated ring was unprofitable for anticonvulsant activity and led practically to a lack of the activity. Although there is no apparent correlation between anticonvulsant activity and adenosine receptor affinity, it seems that a lipophilic substituent is necessary for both activities. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Experimental section
Chemistry Melting points were determined on a MEL-TEMP II apparatus, and they are uncorrected. IR spectra were taken as KBr discs on an FT Jasco IR 410 spectrometer. 1 H-NMR spectra (in CDCl 3 ) for compounds 10, 13, 14 and 22 were obtained at ambient temperature with a Bruker VM 250, for other compounds with a Varian Mercury 300-MHz spectrometer, using signal of undeuterated solvent as an internal standard. UV spectra were recorded on a Jasco UV/Vis V-530 apparatus in 10 −5 mole/L in methanol. Elemental analyses (CHN) were performed on an Elementar Vario-EL III apparatus and agreed with theoretical values within ±0.4 %. TLC data were obtained with Merck silica gel 60 F 254 aluminium sheets with benzene: acetone (7:3) as developing system. Spots were detected under UV light. Syntheses of compounds 5, 25 and 33 were described elsewhere [25,26].

Anticonvulsant screening
The anticonvulsant evaluation was carried but using reported procedures [39,40,47]. Male albino mice (Carworth Farms Nr 1, 18-25 g) and male albino rats (Spraque-Dawley, 100-150 g) were used as experimental animals. A group of three to five mice was used in MES and ScMet tests, and a group of four to eight animals was used in rotorod tests. For the evaluation of activity after oral administration, a group of four rats was used. The test compounds were suspended in 0.5 % methylcellulose water mixture.
In the preliminary screening, each compound was administered as an i.p. injection at three dose levels (30, 100 and 300 mg/kg), with anticonvulsant activity and neurotoxicity assessed at 30-min and 4-h intervals after administration. For some compounds, also intervals of 15 min and 1 h were used. Anticonvulsant efficacy was measured by MES, ScMet and neurological deficit in the rotorod tests, and the data are presented in Table 4. Some selected derivatives were examined for oral activity in the MES and ScMet and neurotoxicity screen (rotorod test) 30 and 50 mg/kg doses. The results are summarized in Table 5.
The pharmacological parameters estimated in the preliminary screening were quantified for compounds 9 and 13 in mice (i.p.) ( Table 7) and for comp. 9 also in rats (p.o.) ( Table 6).
Anticonvulsant activity was expressed in terms of the median effective dose (ED 50 ) MES and ScMet in mice and ED 50 ScMet in rats, and neurotoxicity was expressed as the median toxic dose (TD 50 ). For determination of ED 50 and TD 50 , groups of eight mice and rats were given a range of i.p. or p.o. doses of the test drug until at least three points were established in the range of 10-90 % seizure protection or minimal observed neurotoxicity. From the plot of these data, the respective ED 50 , TD 50 value (95 %) confidence intervals, slope of the regression line and the standard error of the slope were calculated by a computer program based on [38] methods described by Finney. For comp. 9, the hippocampal kindling model of focal seizures was applied ( Table 6). In this procedure, the bipolar electrodes were placed surgically in the ventral hippocampus of adult male Spraque-Dawley rats. Stage five behavioural seizures are produced by using a stimulus consisting of a 50-Hz, 10-s train of 1-ms biphasic 200-μA pulses delivered every 30 min for 6 h. A single dose of comp. 9 is then administered intraperitoneally, 15 min following the last stimulation. The anticonvulsant activity of 9 is assessed every 30 min for 2 h, starting 15 min after administering the test material. After each stimulation, seizure scores and after discharge durations were recorded (Table 6).

Molecular modelling
The 3D molecule models of 5-35 and caffeine were built using Schrödinger Maestro molecular modelling environment [56] basing on presented crystal structures of chosen pyrimido-and diazepinopurinediones (10 and 30, respectively). The geometry optimization was performed using the multiple minimization method as implemented in MacroModel with MMFF force field and Truncated Newton Conjugate Gradient options and terminated when the root means square (RMS) of conjugate gradient was below 0.05 kJmol −1 Å −1 . The minimization was carried out for water solvent, with distance-dependent dielectric constant as a way to treat electrostatic interactions.
The template 3REY was prepared to docking using the Protein Preparation Wizard option as a part of Schrodinger package. The ligand binding cavity was confined to a box with the crystal ligand in the center (size of the box set automatically), according to the receptor grid generation procedure. The studies of docking to the rigid receptor binding pocket were performed using Glide program, with standard precision (SP) mode.
For validation, redocking of XAC ligand was done. After initial docking, 10 poses for each ligand, with RMS deviation higher than 0.5 Å, were kept, and post-minimized, RMSD values between predicted and crystallographic positions of the ligand were calculated. Redocking of XAC was performed twice: (1) without any constraints and (2) with the amino hydrogen atom of the Asn253 (6.55) side chain used as a constrained H-bond donor for a carbonyl group of xanthine.

Molecular docking of A 2A AR antagonists
Docking simulations of low-energy conformations for all of the synthesized compounds and caffeine were performed with SP mode to both templates with limitation used during redocking: the constrained H-bond between the side chain amino group of Asn253 (6.55) and the ligand. Five poses obtained after docking for each ligand (RMS deviation higher than 0.5 Å) were post-minimized, and final poses were kept and analysed according to the obtained docking score values.
For the graphic presentation of the selected structures with the highest docking scores, representing individual clusters of poses, PyMOL [57] software was used.