Design, Synthesis and Assay of Novel Methylxanthine–Alkynylmethylamine Derivatives as Acetylcholinesterase Inhibitors

Abstract

Xanthine derivatives have been a great area of interest for the development of potent bioactive agents. Thirty-eight methylxanthine derivatives as acetylcholinesterase inhibitors (AChE) were designed and synthesized. Suzuki–Miyaura cross-coupling reactions of 8-chlorocaffeine with aryl(hetaryl)boronic acids, the CuAAC reaction of 8-ethynylcaffeine with several azides, and the copper(I) catalyzed one-pot three-component reaction (A³-coupling) of 8-ethynylcaffeine, 1-(prop-2-ynyl)-, or 7-(prop-2-ynyl)-dimethylxanthines with formaldehyde and secondary amines were the main approaches for the synthesis of substituted methylxanthine derivatives (yield 53–96%). The bioactivity of all new compounds was evaluated by Ellman’s method, and the results showed that most of the synthesized compounds displayed good and moderate acetylcholinesterase (AChE) inhibitory activities in vitro. The structure-activity relationships were also discussed. The data revealed that compounds 53, 59, 65, 66, and 69 exhibited the most potent inhibitory activity against AChE with IC₅₀ of 0.25, 0.552, 0.089, 0.746, and 0.121 μM, respectively. The binding conformation and simultaneous interaction modes were further clarified by molecular docking studies.

1. Introduction

The natural methylxanthines caffeine 1, theobromine 2, and theophylline 3 (Figure 1) are widely used in traditional medicines and also in the field of biological and medicinal chemistry [1]. Methylxanthines exert anti-inflammatory, antioxidative, and neuroprotective effects, so their consumption appears to be useful in the prevention of neurodegeneration [2]. Normally, four different mechanisms are proposed to mediate pharmacological methylxanthine activity at the cellular level: antagonism of adenosine receptors, modulation of GABA receptor action, acetylcholinesterase inhibition, and regulation of intracellular calcium levels [3,4,5,6,7]. In the past years, the impact of methylxanthines in neurodegenerative diseases has been extensively studied, and several new aspects have been elucidated [2,8,9]. Caffeine proved to be a relatively potent non-competitive inhibitor of acetylcholinesterase AChE [2,10,11,12,13,14]. Caffeine acts as a reversible inhibitor of acetylcholinesterase. It binds outside the catalytic site of cholinesterase but in its proximity to the so called anionic site and inhibition, it is of a non-competitive type in the acetylcholinesterase case while caffeine exerts minimal affinity toward the second type of cholinesterase, butyrylcholinesterase, found in the blood plasma and serum [14]. Further studies have confirmed that caffeine 1 is an agonist of nAChRs [15,16]. All the reviewed data on the activity of natural methylxanthines stimulated the development of methods for modifications of the substituent in positions N-1, N-7, and C-8 of the xanthine core to find new compounds with selective action and pharmacological interest.

Broad modifications of the substituent in position 8 of the xanthine core structure have resulted in high potency and selectivity for the A_2A [17] and A_2B [18] adenosine receptors. The synthesis and biological evaluation of xanthines with an N-benzyl-(piperidine or pyrrolidine) substituent and a methoxymethyl linker in N-7, C-8, or N-9 positions revealed dual inhibitors of acetylcholinesterase and butyrylcholinesterase [19,20]. Modification of caffeine at N-1 with a methylpentanoate moiety lead to pentoxyphylline, which exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC₅₀ > 50 μM) relative to the reference agent donepezil [21]. Connecting theophylline 3 with pyrrolidine substituent through a methylene chain of different lengths (three to seven carbon atoms) gave new 7-substituted theophylline derivatives that inhibited the AChE, of which the compound with the longest methylene chain showed the strongest effect. Electrophysiological studies showed that all compounds behave as agonists of the muscle and the neuronal α7 nAChR with greater potency than caffeine 1 [22].

Propargylamine as a versatile functional moiety has been reported to be useful for the design of CNS-targeting multi-target directed ligands for neurodegenerative disorders [23,24,25,26,27,28,29,30,31,32,33]. As represented by such modified preparation as ladostigil, donepezil-based hybrid ASS234, memantine-propargylamine, tacrine-selegiline, and chromene-propargylamine hybrids [34,35,36,37,38], the propargylamine fragment has been identified to be essential for the beneficial effects of these compounds.

In this work, we report the synthesis of new xanthine derivatives based on transformations of natural methylxanthines 1–3: 8-aryl(hetaryl) substituted, 8-(1,2,3-triazolyl)substituted, and substituted at the nitrogen in the side chain 8-propargylamino-based caffeine derivatives, 1-(4-aminobut-2-ynyl)theobromines, and 7-(4-aminobut-2-ynyl)theophyllines (Figure 2) intended as AChE inhibitors.

All derivatives were evaluated for AChE inhibition. To better understand the key structural requirements for effective receptor-ligand interactions in parallel with the biological evaluation, docking studies have also been performed.

2. Results and Discussion

2.1. Chemistry

8-Chlorocaffeine 4 and 8-bromocaffeine 5, the key compounds in the synthesis of all the desired 8-substituted xanthine derivatives, were prepared by reacting caffeine 1 with N-chlorosuccinimide or N-bromosuccinimide according to a general method [39] with minor modifications in the synthetic procedure [40]. In accordance with [41], the Suzuki cross-coupling reaction of 8-bromocaffeine 5 with benzeneboronic acid 6 in the presence of Pd(PPh₃)₄ (5 mol %) as the catalyst and K₃PO₄ (2 equiv.) as the base in DMF (110 °C, 6 days) led to 1,3,7-trimethyl-8-phenyl-3,7-dihydro-1H-purine-2,6-dione 7 isolated in 63% yield. Currently, as an efficient way to improve the rate of the cross-coupling reaction, the addition of tetraalkylammonium salts as additives, given their ability to generate a stable form of the catalyst [42], has been considered. Additionally, microwave-assisted organic synthesis (MAOS) has received increasing attention as a valuable alternative to conventional heating to speed chemical reactions (simplicity in operation, shortened reaction times, giving more pure products) [43]. We found that the reaction of 8-chlorocaffeine 4 with benzeneboronic acid 6 (1.2 equiv) in the presence of Pd(PPh₃)₄ (5 mol %) as the catalyst, K₂CO₃ (3 equiv.) as the base, and tetrabutylammonium bromide (1 equiv.) as the additive in toluene-water (1:1, v/v) under microwave irradiation at 130 °C for 1.5 h proceeds well and led to compound 7 isolated in 70% yield (Scheme 1). Carrying out the reaction by using an excess of triethylamine as a base was found to be ineffective. The coupling reaction of 8-chlorocaffeine 4 with arylboronic acids 8–13 under the mentioned conditions led to 8-arylcaffeine derivatives 14–19 in the yield of 47–87%. The yield of 8-arylcaffeines was dependent on the nature of arylboronic acid. The main decrease in the isolation yield to 27% was observed in the reaction of 4 with 2,5-dimetoxyphenylboronic acid 12. The cross-coupling reaction of compound 4 with furan-3-boronic acid 20 or indole-5-boronic acid 21 under the mentioned conditions afforded the desired 8-furyl- or 8-indolylsubstituted caffeine derivatives 22 and 23, respectively (Scheme 1).

Based on the Sonogashira cross-coupling reaction, a synthetic route was designed for the preparation of 8-ethynylcaffeine 24. We found that the reaction of 8-bromocaffeine 5 with (trimethylsilyl)acetylene 25 in the presence of Pd(PPh₃)₄ (5 mol %) and CuI (5 mol%) as catalysts and Et₃N (1.5 equiv.) as the base in toluene proceeds smoothly by heating to 95 °C for 6 h (TLC) with the formation of the 8-(3-trimethyl- silyl)ethynylcaffeine 26 (yield 93%). Compound 26 was further subjected to desilylation to furnish the terminal acetylene 24 (yield 88%). This approach to preparing the targeted xanthine derivative was found to be more economical and convenient compared to the previously described procedure [44]. With 8-ethynylcaffeine 24 in hand, we studied the CuAAC reaction with several organic azides 27a–f. Negligible activity was observed when 8-ethynylcaffeine 24 reacted with p-tolylazide 27a in the presence of sodium ascorbate (1.5 equiv.) and copper(II) sulfate (0.1 equiv.) in a mixture of water and chloroform (1v:5v). Low yield of the triazole 32 was obtained after heating to 50 °C for 24 h. Interestingly, we found that the use of CuI (0.1 equiv.) in conjunction with DIPEA (3.5 equiv.) in MeCN dramatically improved the catalytic efficiency, and the reactions were reasonably selective for 8-((1-p-tolyl)-1,2,3-triazol-4-yl)caffeine 28 (yield 84%). In this condition, the CuAAC reaction of alkyne 24 with azides 27b–e proceeded smoothly with the formation of triazole-substituted caffeine derivatives 28–32. The reaction of alkyne 24 with tertiary azide 27f required an additional selection of conditions for improving the conversion. The yield of the desired product 33 was increased to 69% by conducting the reaction at 90 °C in a sealed tube (Scheme 2).

Our main focus was the introduction of a variety of nitrogen-substituted aminopropargyl functional moieties in the C-8 position of methylxanthines (Figure 1). The literature pathway for the preparation of those compounds follows the Pd-Cu-catalyzed cross-coupling reaction of nitrogen-substituted propargylamines with 8-iodo- [45] or 8-bromoxanthines [46,47]. However, none of the above methods has found broad application for the preparation of 8-(aminopropargyl)xanthine derivatives, possibly due to moderate yields and also the low availability of nitrogen-functionalized propargylamines.

The copper(I) catalyzed one-pot three-component (A³-coupling) reaction among terminal alkyne, formaldehyde, and amine (the Mannich reaction) has become a popular approach to synthesizing propargylamines [48,49]. Therefore, the sequence of the Sonogashira cross-coupling reaction of 3-bromocaffeine 5 and the A³-coupling reaction of 8-ethynylcaffeine 24 has been exploited in our studies (Scheme 3). The reaction of compound 24, aq. formaldehyde (3 equiv.) and diethylamine 34 (3 equiv.) in THF in the presence of copper(I) iodide or copper(I) chloride (0.1 equiv.) at 75 °C (bath) proceeds smoothly, and after 3 h the alkyne was almost consumed and the desired 8-(3-(diethylamino)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione 35 was formed. By using copper (II) acetate monohydrate (0.1 equiv.) as the catalyst in the above conditions, compound 35 was isolated in a yield of 94% after column chromatography (Scheme 3). In this condition, 8-ethynylcaffeine 24 was reacted with secondary amines 36–38 to give the desired caffeine derivatives 39–41 in high isolated yields (57–96%). The reaction of pyrrolidine 42, azepane 43, azocane 44, homomorpholine 45, 4-methylpiperidine 46, morpholine 47, and N-substituted piperazines 48–50 with aq. formaldehyde and 8-ethynylcaffeine 24 in the presence of Cu(OAc)₂ × H₂O (conditions a) led to N-substituted 8-(1-(aminopropargyl))caffeine derivatives 51–59 in high yields (Scheme 3). All the caffeine derivatives were purified by column chromatography (chloroform:ethanol, solvent mixture). So we found that this A³-coupling approach allows for the direct installation of nitrogen-functionalized aminopropargyl substituent at the C-8 position, providing efficient access to different 8-(1-aminopropargyl)trimethylxanthines. It is well known that the reported reaction is thought to proceed through the alkyne activation forming a copper acetylide. After a nucleophilic addition on the intermediate formed by the reaction of formaldehyde and a secondary amine, the propargylamine derivative is obtained [48]. We performed also a one-pot deprotection–A³-coupling tandem procedure for obtaining compound 35. We found that the consecutive treatment of 8-(trimethylsilylethynyl)caffeine 26 in THF under argon flow with Bu₄NF (1.1 equiv.), amine 34, and aq. formaldehyde in the presence of Cu(OAc)₂ × H₂O as the catalyst (0.1 equiv.) gave the desired caffeine derivative 35 in the isolated yield 44%. No products of alkyne deprotection reaction (compound 24 or dimeric butadiyne) were observed in this condition. It should be noted that caffeine derivatives 51, 52, and 56 are known compounds, which were synthesized by the Sonogashira reaction of 8-bromocaffeine with subsequently substituted alkynes (yield 38–57%) [46,47]. Using an A³-coupling reaction between 8-ethynylcaffeine 24, formaldehyde, and secondary amines seems to be more effective; the reaction proceeds with higher isolated yield by using more available and stable secondary amines as the key reagents. The stage of synthesis of ethynylamines necessary for a cross-coupling reaction can be problematic due to side reactions, as well as to purify.

To further investigate the significance of the propynyl linker on enzyme inhibition assay, we synthesized caffeine derivatives 60, 61 with N-methylpiperidinyl- or N-Boc-piperazinyl- substituent at the C-8 position (Scheme 4). Compounds 60, 61 (yield 78–88%), we prepared by the reaction of 8-bromocaffeine 5 with an excess (3 equiv.) of 4-methylpiperidine 46 or 1-Boc-piperazine 49 in DMF.

Next, we concentrated our efforts on the design and synthesis of purine derivatives containing a nitrogen-substituted aminobut-2-ynyl residue in position N-1 or N-7 on the methylxanthine core. To our delight, we observed that the A³-coupling process was amenable to 1-(prop-2-yn-1-yl)-3,7-dimethylxanthine 62 obtained from the reaction of theobromine 2 with propargyl bromide 63 under basic conditions (Scheme 5). The three-component reaction of 63 with aq. formaldehyde and secondary amines, including diisopropylamine 36, azocane 44, or 1-(2-(pyrrolidin-1-yl)ethyl)piperazine 50 in THF in the presence of copper(II) acetate monohydrate was very efficient, and occurred with the formation of nitrogen substituted 1-(4-(amino)but-2-yn-1-yl)-3,7-dimethyl- 3,7-dihydro-1H-purine-2,6-diones 64–66 (54–90%) (Scheme 5).

A synthetic route was designed for the preparation of nitrogen-substituted 7-(aminobut-2-yn-1-yl)theophylline derivatives as depicted in Scheme 6. The reaction of theophylline 3 with propargyl bromide 63 led to the formation of 7-(prop-2-yn-1-yl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione 67. The three-component reaction of the alkyne 67 with an excess of formaldehyde and appropriate secondary amines 36, 44, or 50 in the presence of a catalytic amount of Cu(OAc)₂ × H₂O in THF afforded the target compounds 68–70 in an excellent yield (Scheme 6).

The composition and structure of all synthesized compounds were confirmed by IR, UV, ¹H and ¹³C NMR spectroscopy, and mass spectrometry (HRMS). The purity of all compounds was checked additionally by thin-layer chromatography and elemental analysis. The ¹H and ¹³C NMR spectra of all synthesized compounds agreed with their structures and contained one set of characteristic signals for the xanthine core and the corresponding substituent. The structures of 8-arylsubstituted and 8-triazolylsubstituted caffeine derivatives 18, and 32 were established by X-ray structure analysis. The refined molecules are shown in Figure 3. Aryl substituent on C8 in structure 18 has an interplane angle with caffeine core equaling 72.1º and the bond length of C8-C1′ 1.479(2) Å being longer than the same one in 32. The π…π–interactions of pyrimidine cycles form molecular dimers (Figure 3A, right), having the following geometric parameters: the intercentroid distances equaling 3.639(1) and distances from the centroid to plain 3.354(1) Å.

Notably, the triazole ring in structure 32 lays practically in the plain of caffeine (Figure 3B); the interplane angle between cycles is 1.3°, and the C8-C4 bond length equals 1.455 Å (the same as for a single bond in conjugated C=C-C=C [50]). Such an extended aromatic system and the presence of a hydroxy group lead to the formation of 1-D motifs along axes c (Figure 3B, right) due to interactions of the π-system, with the intercentroid distances equaling 3.497(2)–3.581(2), and distances from centroids to plain laying in the interval of 3.337(2)–3.366(2)^2x Å, together with hydrogen bond O3A-H…N2′ with parameters H…N 2.21, O…N 2.912(8) Å and O-H…N 144° [51].

2.2. Enzyme Inhibition

The synthesized 8-aryltrimethylxanthine derivatives 7, 14–19, 8-ethynylderivatives 24, 26, 8-triazolylcaffeines 28–33, substituted at nitrogen atom 8-(3-(R-amino)prop-1-yn-1-yl)caffeine derivatives 35, 39–41, 51–59, 8-(N-methylpiperidinyl)- 60, 8-(N-Boc-piperazinyl)caffeine 61, 1-(4-(amino)but-2-yn-1-yl)-3,7-dimethyl-3,7-dihydro- 64–66, 7-(4-(amino)but-2-yn-1-yl)- 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-diones 68–70, and the starting caffeine 1 were evaluated for their ability to inhibit AChE from electric eel (EeAChE) using Ellman’s method [52]. The enzymatic activity is quantified indirectly by spectrophotometric determination of the 5-thio-2-nitrobenzoate concentration formed in the reaction between the reagent 5,5′-dithio-bis(2-nitrobenzoic acid) and thiocholine, a product of acetylthiocholine hydrolysis catalyzed by cholinesterases. The efficacy of inhibitors is expressed as IC₅₀, i.e., the concentration required for 50% inhibition of the enzyme activity obtained from three independent experiments. Results are given in

Table 1. Structure of substituted 3,7-dihydro-1H-purine-2,6-diones and their inhibitory potencies for AChE.

Compound Type	Code	IC50, μM	Compound Type	Code	IC50, μM
8-Aryl(hetaryl)caffeines	7	510.4 ± 9.1	8-(3-R-amino)prop-1-yn-1-yl)-caffeines	35	4.7 ± 0.1
	14	201.4 ± 5.4		39	11.7 ± 0.1
	15	94.5 ± 18.9		40	50.1 ± 4.5
	16	105.0 ± 2.0		41	26.3 ± 0.5
	17	146.0 ± 1.0		51	43.7 ± 3.6
	18	142.0 ± 2.0		52	12.28 ± 0.31
	19	>1000		53	0.25 ± 0.001
R = Furan-3-yl	22	21.8 ± 0.2		54	124.0 ± 9.0
R = Indol-5-yl	23	>1000		55	2.5 ± 0.01
8-Triazolylcaffeines	28	531.0 ± 7.5		56	224.0 ± 5.0
	29	279.0 ± 6.0		57	60.0 ± 1.0
	30	242.0 ± 2.0		58	1.20 ± 0.01
	31	21.3 ± 0.4		59	0.552 ± 0.004
	32	35.3 ± 0.9		60	113.0 ± 2.0
	33	42.4 ± 1.0		61	42.0 ± 1.0
	24	101.0 ± 1.7		68	14.9 ± 0.4
	26	102.0 ± 4.3		69	0.121 ± 0.001
	64	2.4 ± 0.1		70	1.8 ± 0.1
	65	0.089 ± 0.001	Caffeine	1	30.0 ± 2.4
	66	0.746 ± 0.021	Galantamine		4.9 ± 0.2

. Galantamine, a clinically used drug acting as a balanced inhibitor of AChE, was used as a standard for IC₅₀ values comparison. The substituent at the C-8 position of caffeine has a great influence on AChE inhibition. IC₅₀ values for AChE for 8-aryl-, 8-triazolyl-, and 8-alkynyltrimethylxanthines ranged from 21.3 to 531.0 μM (31 and 28, respectively). When compared to caffeine, only two compounds 22 and 31 produced equal IC₅₀ values. Focused on 8-(3-aminoprop-1-yn-1-yl)caffeines, seven derivatives are more potent AChE inhibitors 35, 39, 52, 53, 55, 58, and 59, and two are comparable 41, 51 to caffeine (Table 1). Clearly, most of the derivatives 53, 55, 58, and 59 are stronger inhibitors of AChE than galantamine. Characteristically, these four compounds have a cyclic (azocanyl, 4-methylpiperidinyl, 4-Boc-piperazinyl or 4-((2-(pyrrolidin-1-yl)ethyl)piperazinyl) substituent in the propargylamine moiety.

Drawing a comparison among N-substituted 8-(1-aminopropargyl)caffeines 35, 39–41, 51–59, we can describe the following general SAR: For AChE inhibition, the presence of a cyclic substituent at the propargylamino moiety is essential. A remarkable increase in inhibition was observed for compound 53 with a bulk 8-(3-(azocan-1-yl)propargyl)substituent in the 8-th position of trimethylxanthine core (the best nanomolar AChE inhibitor, IC₅₀ = 0.255 ± 0.016 µM). Oxygen substituents in the cycle (morpholino and homomorpholino, compounds 54, 56) decreased the activity. 8-(1-Aminopropargyl)caffeines with piperazinyl substituents 58, 59 improved inhibition of AChE significantly (IC₅₀ = 0.552 and 1.20 µM) when compared to galantamine (IC₅₀ = 4.9 µM). Spacing the 1-Boc-piperazinyl substituent away from the xanthine core by a propargyl linker led to a great increase in the AChE inhibition (compared to compounds 58 (IC₅₀ = 1.20 µM) and 61 (IC₅₀ = 42.0 µM) (Table 1).

The introduction of 1-(4-(aminobut-2-yn-1-yl) substituent in the theobromine core (compounds 64–66) has improved the inhibition of AChE significantly. The 1-(4-(azocan-1-yl)but-2-yn-1-yl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (65) was the most active AChE inhibitor among all studied (IC₅₀ = 0.089 µM); characteristically, all three 1-(4-(aminobut-2-yn-1-yl))dimethylxanthines 64–66 were superior in inhibition of AChE compared to galantamine. The high inhibition of 64–66 of AChE is of interest because the starting compound 1,3,7-trisubstituted xanthine derivative (DMPX) 62 was the first A_2A adenosine receptor antagonist reported in the literature having a hA_2A Ki = 4.1 µM [53]. The introduction of the 7-(4-(amino)but-2-yn-1-yl) in the theophylline core (compounds 68–70) was also very useful for the construction of AChE inhibitors. Notably, in the series of these derivatives, compound 69 with the 7-(3-(azocan-1-yl)-but-2-yn-1-yl) substituent was more efficient than 68 and 70 derivatives in AChE inhibition; the bulk azocane substituent was helpful to the inhibitory activities (Table 1).

The obtained data indicated that the AChE inhibitory activity of 8-aminopropynyl-, 1-, and 7-aminobutynylmethylxanthines is sensitive to the nature of the nitrogen functionalities in the substituent. To the best of our knowledge, this structural type of AChE inhibitor is presented herein for the first time, and our results confirmed the potential value of this class of biologically active substances.

2.3. Molecular Docking Study

With the aim of obtaining some information about the possible interactions of the methylxanthine derivatives with the enzyme, which, in turn, could be useful in the design of more potent inhibitors, a molecular modeling study was performed comparing with donepezil, a nanomolar AChE inhibitor. The calculation of the interaction energy of donepezil in XRD model coordinates (score in place mode) gave an estimated binding energy of −14.806 kcal/mol. Molecular docking of the minimized donepezil molecule to the AChE active site showed a very close value of −14.817 kcal/mol (

Table 2. Results of molecular docking of methylxanthine derivatives to the AChE binding site.

Entry	Compound	Ligand Efficiency, kcal/mol	Binding Energy a, kcal/mol
1	64	−0.634	−15.207
2	Donepezil	−0.529	−14.817
3	65	−0.590	−14.742
4	68	−0.614	−14.733
5	66	−0.486	−14.590
6	69	−0.554	−13.840
7	59	−0.429	−12.860
8	55	−0.529	−12.693
9	58	−0.518	−12.432
10	39	−0.493	−11.833
11	53	−0.417	−10.691
12	70	−0.356	−10.542
12	41	−0.351	−9.562
13	52	−0.382	−9.561
14	22	−0.351	−7.931
15	32	−0.319	−7.712
16	31	−0.235	−7.053
17	24	−0.407	−6.512
18	1	−0.449	−6.284

^[a] Value is not genuine binding energy but estimated docking score.

). Compounds 64, 68, 65, 69, and 55 show a theoretical affinity for the AChE active site at the level of donepezil.

When comparing the conformations of donepezil in the XRD model and that obtained as a result of docking, the RMSD of the atomic coordinates was determined to be 0.363. Thus, the procedure of molecular docking under the conditions of the chosen AChE model makes it possible to quite accurately predict the position and conformation of the inhibitor. Molecular interactions of donepezil and active xanthine derivatives are shown in Figure 4.

The active site of AChE is saturated with aromatic amino acids, which provided the main type of non-covalent interactions with inhibitors-stacking and interactions of the π-systems of Trp286, Tyr341, Phe338, Tyr337, and Trp86 amino acids with π-systems and individual atoms of inhibitors. In this regard, the aromatic systems of the benzyl and dihydroindene rings of donepezil are actively involved in the interactions (Figure 4A). Apparently, the hydrogen bond between the oxygen of the keto group associated with the dihydroindene ring and the amino acid residue Phe295 is important for the formation of a stable interaction between the inhibitor and the AChE active site. The xanthine core of the new derivatives is actively stacked with the Trp286 and Tyr341 π-systems (except for compound 66). A common feature of all the most active compounds is the ability of the carbonyl groups of the purine cycle to form a hydrogen bond with Phe295. Due to the high saturation of xanthine with polar atoms, such a bond can arise at any orientation of the purine in the active site of AChE, depending on the site of the linker attachment.

Analysis of the SAR of compounds differing in the position of attachment of the same type of substituent to the xanthine backbone of caffeine shows that, apparently, in the series of compounds with the diisopropylamino function: 64, 68, and 39, the presence of a rotating bond in front of the propargylamine substituent has a significant effect on the theoretical affinity (Figure 4E). The most theoretically affine compounds in this series (64 and 68) are modified by adding an aminobut-2-inyl linker to diisopropylamino substituent at positions 1 or 7 of the xanthine core (Figure 4B,C), which allows the diisopropylaminobut-2-ynyl substituent to occupy a more advantageous position compared to compound 39 (Figure 4D), where the rigid aminopropargyl substituent is attached directly to the 8-th position of the core. The presence of a rigid acetylene bond in compound 39 causes the only possible position of the linear part, and (isopropylamino)propynyl substituent acquired the possibility of the formation of hydrophobic interactions of isopropyl moiety with Trp-86. When comparing compounds 65 and 69, with a bulky azocane moiety at the but-2-ynyl substituent, it is more advantageous for binding to attach a substituent at position 1 of the xanthine backbone compared to position 7 (Figure 4F–H). The differences in the estimated binding energies may be due to the fact that different carbonyl groups form a hydrogen bond with Phe295: in the case of derivative 65, this is the carbonyl oxygen in position 2 of the backbone, and for compound 69 – the carbonyl group in position C-6. The azocane substituent ensured the hydrophobic interaction with Trp86.

For compound 66 (pyrrolidinoethylpiperazinyl)butynyl substituent at position 1 of the backbone), the most energetically favorable conformation occurs at the “reverse” position of the methylxanthine core compared to the typical position for other active derivatives (Figure 4I). With this orientation, no stacking occurs with amino acids characteristic of AChE inhibitors, and no hydrogen bond is formed with Phe295; however, the carbonyl group at C-2 forms a hydrogen bond with Glu202. In this case, it is worth considering other, less energetically favorable conformations in comparison with other active compounds. Hydrophobic interaction of substituent: piperazine ring with Phe-338 and pyrrolidine ring with Tyr341 and Trp286 are maintained (Figure 4I). Compound 59 ((pyrrolidinoethylpiperazinyl)prop-2-ynyl substituent at position 8 of the backbone) does not form a hydrogen bond with Phe295 (Figure 4J). The binding mode for the hAChE pocket is characterized by π-π interactions of the xanthine core with Tyr341. The internal ethylpiperazinyl substituent at the alkyne in the C-8 position formed hydrophobic interaction with Phe 338. The pyrrolidinoethylpiperazinyl substituent of this derivative is U-folded. Compared to donepezil, its structure is less flexible due to the presence of a rigid propargylamine linker (Figure 4K). However, the pyrrolidine ring at the end of the substituent is able to establish the interaction with Trp86 (Figure 4I).

This molecular modeling helped to distinguish the key interaction of substituted methylxanthines and AChE. Molecular dynamics was performed to investigate the stability of the protein-ligand complexes in time (during 100 nanoseconds) Figure S2A–D, Supplementary Materials).

Donepezil. During the simulation, the protein-ligand complex was stable, but interestingly, based on the RMSD-time relation, two segments of the simulation could be distinguished, with the first being from 5 to 38 ns and the second from 54 to 100 ns. It should be noted that it corresponds to the conformational change “chair to chair” of the piperidine fragment of the donepezil ligand (Figure S2A, Supplementary Materials).

Compound 59. In general, the protein-59 complex was stable during the simulation; the insignificant deviations of ligand RMSD from 2 to 3 Å correspond to the conformational flexibility of the N-alkylpyrrolidine fragment of 59 (Figure S2B, Supplementary Materials).

Compound 64. Similar to 59, the protein-64 complex was stable during the simulation as well. As could be seen from the RMSD-time relation, a ligand reversible conformation change occurred. Analyzing the trajectory of the protein–ligand complex, we concluded that it was a result of conformational interconversion of the N-diisopropyl fragment of 64. In particular, a rotation took place across the -N-C- bond with a corresponding conformational change of the N-diisopropyl fragment from anti-gauche to gauche-anti (Figure S2C, Supplementary Materials).

Compound 69. As could be seen, the protein-69 complex was stable for the first 35 nanoseconds of the simulation, and after that compound 69 partially left the binding pocket. During the period from 35 to 55 ns, the ligand was bound with protein by an azocyl fragment. It seemed to be a formation of π-cation interaction between the protonated tertiary amino group and the side chain of Trp286. On the other hand, the formation of hydrogen bonds between the Tyr77 hydroxyl group and the C6-carbonyl group of the purine core is supposed to be possible. During the following simulation, after 55 ns, the dissociation of the protein–ligand complex occurred. Interestingly, after 90 ns, the ligand binds an opposite side of the protein in the region of 61–64 amino acids with the following dissociation after 98 nanoseconds (Figure S2D, Supplementary Materials).

3. Materials and Methods

3.1. Chemistry

General Information

¹H and ¹³C NMR spectra were acquired on ‘Bruker AV 300′ 300.13 (¹H) and 75.47 MHz (¹³C), respectively, (compounds 26, 30, 31, 32, 35, 40, 59), ‘Bruker AV 400′ 400.13 (¹H) and 100.61 MHz (¹³C) (15, 18, 19, 22, 24, 33, 39, 41, 52, 58), ‘Bruker DRX-500′ 500.13 (¹H) and 125.76 MHz (¹³C), (16, 23, 55, 60, 61) instruments. The ¹H spectra for compounds 28, 29, 53, 54, 56, and 57 were obtained on ‘Bruker AV 400′, and ¹³C spectra–on ‘Bruker DRX-500′. Deuterochloroform (CDCl₃) was used as a solvent, with residual CHCl₃ (δ_H = 7.24 ppm) or CDCl₃ (δ_C = 76.9 ppm) being employed as internal standards. NMR signal assignments were carried out with the aid of a combination of 1D and 2D NMR techniques that included ¹H, ¹³C, COSY, HSQC, and HMBC spectra. IR absorption spectra were recorded on a Vector 22 FT-IR spectrometer in KBr pellets. The UV spectra were obtained on an HP 8453 UV Vis spectrometer (Hewlett-Packard, Waldbronn, Germany). Melting points were determined using the Mettler Toledo FP900 (USA) thermosystem. HRMS spectra were recorded on a Thermo Scientific DFS mass spectrometer (evaporator temperature 190–250 °C, EI ionization at 70 eV). Elemental analysis was carried out on an 1106 Elemental analysis instrument (Carlo-Erba, Milan, Italy). The X-ray diffraction experiments for crystals 18 and 32 were carried out at ambient conditions on a Bruker KAPPA APEX II diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by the SADABS program [54]. The structures were solved by direct methods using SHELXS-97 [55] for 18 and the SHELXT 2014/5 [56] program for 32 and refined by anisotropic (isotropic for all H atoms and O in minor part of disordered OH group of 32) full-matrix least-squares method against F² of all reflections by SHELXL2018/3 [57]. The positions of the hydrogen were calculated geometrically and refined in a riding model. The OH group of compound 1 is disordered due to rotation, approximately as 9:1.

The reaction progress and the purity of the obtained compounds were monitored by TLC on Silufol UV-254 plates (CHCl₃-EtOH, 9:1; detection under UV light or by treatment with iodine vapor). Products were isolated by column chromatography on silica gel 60 (0.063–0.200 mm, Merck KGaA, Darmstadt, Germany) eluting with indicated solvent systems. The chemicals used: caffeine 1, theobromine 2, theophylline 3, arylboronic acids 6, 8–13, furan-3-boronic acid 20, indole-5-boronic acid 21, (trimethylsilyl)acetylene 25, azides 27a-f, formalin (30% formaldehyde in aq. solution), diethylamine 34, diisopropylamine 36, dibutylamine 37, dicyclohexylamine 38, pyrrolidine 42, azepane 43, azocane 44, homomorpholine 45, 4-methylpiperidine 46, morpholine 47, N-methylpiperazine 48, N-Boc-piperazine 49, 1-(2-(pyrrolidin-1-yl)ethyl)piperazine 50, propargyl bromide 63, CuI, CuCl, Cu(OAc)₂ × H₂O, tetra-n-butylammonium fluoride trihydrate (TBAF × 3H₂O), Bu₄NBr, Pd(PPh₃)₄, DIPEA, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), were purchased from Aldrich (St. Louis, MO, USA) or Alfa Aesar (GmbH, Karlsruhe, Germany). 5-Chlorocaffeine 4 and 5-bromocaffeine 5 were obtained according to published procedures [39,40]. Solvents (DMF, THF, toluene, MeCN, CHCl₃, CH₂Cl₂) and Et₃N were purified by standard methods. Copies of NMR spectra (¹H and ¹³C) are given in Supplementary Materials (S1–S70).

3.2. Synthesis and Spectral Data

3.2.1. General Method for the Preparation of 8-Aryl(hetaryl)-1,3,7-trimethyl- 3,7-dihydro-1H-purine-2,6-diones (7, 14–19, 22, 23)

8-Chlorocaffeine 4 (115 mg, 0.5 mmol) was dissolved in toluene (2 mL) in a microwave vial under argon. Boronic acid (1.2 equiv.) 6, 8–13, 20 or 21, (Pd(PPh₃)₄ (1–5 mol %), TBAB (161 mg, 0.5 mmol), and K₂CO₃ (0.21 g, 1.5 mmol) in H₂O (2 mL) were successively added. The mixture was stirred for 1.5 h in a microwave reactor at 130 °C. After cooling, the reaction mixture was diluted with NaCl (10 mL), the organic layer was separated, and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with H₂O (2 × 20 mL), dried over MgSO₄, and evaporated. The residue was purified by column chromatography (eluent petroleum ether-Et₂O, 2:1) to give compounds 7, 14–19, 22, 23.

1,3,7-Trimethyl-8-phenyl-3,7-dihydro-1H-purine-2,6-dione (7)

Yield 70%. White crystals, M.p. 180–181 °C (EtOH); M.p. 178 °C [41]. ¹H NMR, ¹³C NMR, and MS data are given in [41].

1,3,7-Trimethyl-8-o-tolyl-3,7-dihydro-1H-purine-2,6-dione (14)

Yield 87%. White solid, M.p. 194.5 °C (EtOH, decomp.); M.p. 193–194 °C [58]. ¹H NMR, ¹³C NMR, and MS data are given in [58].

8-(2-Aminophenyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (15)

Yield 53%. White solid. M.p. 195.6 °C (EtOH, decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.40 (3H, s, NCH₃), 3.58 (3H, s, NCH₃), 3.97 (3H, s, NCH₃), 4.48 (2H, s, NH₂), 6.84 (1H, d, J = 8.2 Hz, H-3′), 7.26–6.92 (2H, m, H-4′,5′), 7.45 (1H, d, J = 7.8 Hz, H-6′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.9 (NCH₃), 29.7 (NCH₃), 34.0 (N CH₃), 107.7 (C-5), 111.4 (C-1′), 116.8 (C-3′), 117.5 (C-5′), 129.8 (C-6′), 131.4 (C-4′), 146.7 (C-2′), 147.7 (C-4), 150.7 (C-8), 151.5 (C-2), 155.2 (C-6); IR (KBr, ν, cm⁻¹): 3410 (NH₂), 1655, 1693 (C=O), 1626, 1543, 1508, 1475, 746 (C=C,C=N); UV (MeCN) λ_max (lgε): 225 (4.49), 291 (4.04), 318 (3.91) nm. Anal. calcd for C₁₄H₁₅N₅O₂, %: C, 58.94; H, 5.30; N, 24.55; found, %: C, 58.57; H, 5.56; N, 24.22.

8-(3-Methoxyphenyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (16)

Yield 60%. White crystals. M.p. 171.0–171.6 °C (EtOH). ¹H NMR (500 MHz, CDCl₃, δ, ppm): 3.40 (3H, s, NCH₃), 3.60 (3H, s, NCH₃), 3.85 (3H, s, OCH₃), 4.03 (3H, s, NCH₃), 6.82 (1H, dd, J = 8.2, 1.8 Hz, H-4′), 7.28 (1H, dd, 8.2, 7.8 Hz, H-5′), 7.31 (1H, d, J = 1.8 Hz, H-2′), 7.60 (1H, d, J = 7.8 Hz, H-6′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.9 (NCH₃), 29.7 (NCH₃), 33.8 (NCH₃), 55.3 (OCH₃), 108.4 (C-5), 114.6 (C-2′), 116.0 (C-4′), 121.2 (C-6′), 129.3 (C-5′), 129.9 (C-1′), 148.0 (C-4), 151.6 (C-2), 151.8 (C-8), 155.4 (C-6), 159.7 (C-3′); IR (KBr, ν, cm⁻¹): 1659, 1691 (C=O), 1605, 1500, 1543, 1471, 1441, 747 (C=C, C=N); UV (MeCN) λ_max. (lgε): 217 (4.56), 298 (4.23) nm. Anal. calcd for C₁₅H₁₆N₄O₃, %: C, 59.99; H, 5.37; N, 18.66; found, %: C, 59.85; H, 5.84; N, 18.97.

8-(4-Methoxyphenyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (17)

Yield 53%. White crystals, M.p. 168.6 °C (EtOH, decomp.); M.p. 168–169 °C [58]. ¹H NMR, ¹³C NMR, and MS data are given in [58].

8-(2,3-Dimethoxyphenyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (18)

Yield 27%. White crystals. M.p. 200.7 °C (EtOH). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.41 (3H, s, NCH₃), 3.60 (3H, s, NCH₃), 3.65 (3H, s, OCH₃), 3.81 (3H, s, OCH₃), 3.90 (3H, s, NCH₃), 7.02 (1H, dd, J = 8.0, 1.8 Hz, H-4′), 7.06 (1H, dd, J = 7.6, 1.8 Hz, H-6′), 7.17 (1H, dd, J = 8.0, 7.6 Hz, H-5′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.9 (NCH₃), 29.7 (NCH₃), 33.3 (NCH₃), 55.9 (OCH₃), 61.3 (OCH₃), 108.2 (C-5), 114.5 (C-4′), 122.8 (C-1′), 122.9 (C-5′), 124.7 (C-6′), 147.4, 148.0 (C-2′,3′), 149.8 (C-4), 151.6 (C-2), 152.7 (C-8), 155.4 (C-6); IR (KBr, ν, cm⁻¹): 1657, 1707 (C=O), 1047 (C-O-C), 1543, 1481, 748 (C=C, C=N); UV (MeCN) λ_max (lgε): 217 (4.53), 286 (4.20) nm. Anal. calcd for C₁₆H₁₈N₄O₄, %: C, 58.17; H, 5.49; N, 16.96; found, %: C, 58.23; H, 5.99; N, 17.05. Crystallographic data for compound (18): C₁₆H₁₈N₄O₄, M 330.34, monoclinic, P2₁/n, a 8.0807(4), b 24.784(1), c 8.3115(3) Å, β 107.255(2), V 1589.6(1) ų, Z 4, D_calcd 1.380 g·cm^–3, μ(Mo-Kα) 0.102 mm^–1, F(000) 696, (θ 2.70–27.12°, completeness 99.6%), T 296(2) K, colorless, (0.96 × 0.50 × 0.30) mm³, transmission 0.6957–0.7455, 21885 measured reflections in index range −9 <= h <= 10, −31 <= k <= 31, −10 <= l <= 10, 3511 independent (R_int 0.0 305), 222 parameters, R₁ 0.0582 (for 2983 observed I > 2σ(I)), wR₂ = 0. 1628 (all data), GOOF 1.091, largest diff. peak and hole 0.419 and −0.276 e.A⁻³. Crystallographic data for compound (18) has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. 2208208. Copy of the data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44 122 3336033 or e-mail: [email protected]; internet: www.ccdc.cam.ac.uk).

8-(3,4,5-Trimethoxyphenyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (19)

Yield 47%. White crystals. M.p. 214.1^oC (EtOH, decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.40 (3H, s, NCH₃), 3.60 (3H, s, NCH₃), 4.03 (3H, s, NCH₃), 3.90 (6H, s, OCH₃), 3.88 (3H, s, OCH₃), 6.83 (2H, s, H-2′,6′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.9 (NCH₃), 29.7 (NCH₃), 33.8 (NCH₃), 56.2 (2 × OCH₃), 60.9 (OCH₃), 106.5 (C-2′,6′), 108.3 (C-5), 123.4 (C-1′), 140.0 (C-4′), 148.0 (C-4), 151.6 (C-2), 151.9 (C-3′,5′), 153.4 (C-6), 155.4 (C-8). IR (KBr, ν, cm⁻¹): 1664, 1707 (C=O), 1587, 1541, 1485, 1470, 1454, 695 (C=C, C=N); UV (MeCN) λ_max. (lgε): 222 (4.57), 251 (4.00), 303 (4.29) nm. Anal. calcd for C₁₇H₂₀N₄O₅, %: C, 56.66; H, 5.59; N, 15.55; found, %: C, 57.05; H, 5.37; N, 15.36.

8-(Furan-3-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (22)

Yield 33%. Yellowish solid. M.p. 205.7 °C (EtOH, decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.37 (3H, s, NCH₃), 3.56 (3H, s, NCH₃), 4.05 (3H, s, NCH₃), 6.85 (1H, s, H-4′), 7.54 (1H, s, H-5′), 7.92 (1H, s, H-2′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.8 (NCH₃), 29.6 (NCH₃), 33.0 (NCH₃), 107.9 (C-5), 109.7 (C-4′), 132.5 (C-3′), 143.9 (C-2′), 145.8 (C-5′), 148.1 (C-4), 151.5 (C-2), 154.3 (C-8), 155.2 (C-6); IR (KBr, ν, cm⁻¹): 1695, 1655 (C=O), 1567, 1539, 1464, 1429, 746 (C=C, C=N); UV (MeCN) λ_max. (lgε): 213 (4.41), 298 (4.16) nm. Anal. calcd for C₁₂H₁₂N₄O₃, %: C, 55.38; H, 4.65; N, 21.53; found, %: C, 55.48; H, 5.03; N, 21.24.

8-(1H-Indol-5-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (23)

Yield 52%. White solid. M.p. 292.7 °C (EtOH, decomp.). ¹H NMR (500 MHz, CDCl₃, δ, ppm): 3.33 (3H, s, NCH₃), 3.53 (3H, s, NCH₃), 3.95 (3H, s, NCH₃), 6.51 (1H, d, J = 3.0 Hz, H-3′), 7.22 (1H, d, J = 3.0 Hz, H-2′), 7.44 (1H, d, J = 8.5 Hz, H-7′), 7.63 (1H, s, H-4′), 7.81 (1H, d, J = 8.5 Hz, H-6′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.9 (NCH₃), 29.7 (NCH₃), 33.9 (NCH₃), 102.5 (C-3′), 108.2 (C-5), 111.7 (C-7′), 118.7 (C-6′), 122.0 (C-4′), 124.4 (C-2′), 126.1 (C-3a′), 127.7 (C-5′), 136.8 (C-7a′), 148.2 (C-4), 151.8 (C-2), 154.4 (C-8), 155.5 (C-6); IR (KBr, ν, cm⁻¹): 1448, 1543, 1620, 1647, 731 (C=N, C=C), 1689 (C=O), 3273 (NH); UV (MeCN) λ_max. (lgε): 240 (4.56), 278 (4.25), 306 (4.29) nm. Anal. calcd for C₁₆H₁₅N₅O₂, %: C, 62.13; H, 4.89; N, 22.64; found, %: C, 61.32; H, 4.81; N, 22.36.

3.2.2. 1,3,7-Trimethyl-8-((trimethylsilyl)ethynyl)-3,7-dihydro-1H-purine-2,6-dione {8-[(trimethylsilyl)ethynyl]-caffeine} (26)

A mixture of 8-bromocaffeine 5 (500 mg, 1.8 mmol), Pd(PPh₃)₄ (10 mg, 0.009 mmol), CuI (1.7 mg, 0.009 mmol), (trimethylsilyl)acetylene 25 (265 mg, 2.7 mmol), and Et₃N (0.27 g, 2.7 mmol) in toluene (15 mL) was stirred at 95 °C (bath) for 6 h under a stream of argon. After cooling, the solvent was removed under reduced pressure, and the residue was treated with water (10 mL) and extracted with CHCl₃ (3 × 20 mL). The combined organic solution was dried over MgSO₄, filtered, and evaporated in vacuo. The residue was purified by column chromatography (eluent CHCl₃-EtOH, 100:1→95:1) to afford compound 26 (495 mg, yield 93%). White powder. M.p. 166.1 °C (decomp.). ¹H NMR (300 MHz, CDCl₃, δ, ppm): 0.27 (9H, s, Si(CH₃)₃), 3.37 (3H, s, NCH₃), 3.53 (3H, s, NCH₃), 3.98 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃,δ, ppm): −0.82 (Si(CH₃)₃), 27.79 (NCH₃), 29.55 (NCH₃), 32.94 (NCH₃), 90.90 (C-11), 104.85 (C-10), 107.46 (C-5), 135.02 (C-8), 147.26 (C-4), 151.26 (C-2), 154.62 (C-6); IR (KBr, ν, cm^–1): 2164 (C≡C), 1711, 1664 (C=O), 1601, 1545, 1477, 760, 744 (C=C, C=N); UV (EtOH) λ_max (lgε): 234 (4.44), 307 (4.29) nm. HR-MS, m/z (I_rel., %): 290 (100), 276 (14), 275 (80), 143 (14), 142 (10), 82 (13), 67 (23), 58 (21), 57 (11), 56 (9); Calcd for C₁₃H₁₈N₄O₂Si: 290.1194; found [M]⁺ m/z: 290.1200. Anal. calcd for C₁₃H₁₈N₄O₂Si, %: C, 53.77; H, 6.25; N, 19.29; found, %: C, 53.87; H, 6.15; N, 19.40.

3.2.3. 8-Ethynyl-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione {8-Ethynylcaffeine} (24)

A stirred solution of compound 26 (3.00 g, 10.3 mmol) in dichloromethane (25 mL), was treated with a solution of tetra-n-butylammonium fluoride (12.4 mL of 0.1 M solution in THF, 12.3 mmol), and the mixture was stirred at 20 °C for 2 h. After completion based on TLC, the solvent was removed under reduced pressure, and the residue was treated with water (10 mL) and extracted with CHCl₃ (3 × 30 mL). The combined organic solution was dried over MgSO₄, filtered, and evaporated in vacuo. After column chromatography (eluent CHCl₃-EtOH, 100:0→50:1) compound 24 (1.99 g, yield 88%) was isolated as a cream powder. 212 °C (decomp.). M.p. 214–215 °C [44]. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.36 (s, 3H, NCH₃), 3.52 (1H, s, H-11), 3.56 (3H, s, NCH₃), 4.00 (3H, s, NCH₃). ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.89 (NCH₃), 29.62 (NCH₃), 33.03 (NCH₃), 71.37 (C-11), 85.32 (C-10), 107.80 (C-5), 134.27 (C-8), 147.31 (C-4), 151.33 (C-2), 154.70 (C-6); IR (KBr, ν, cm^–1): 3219, 2955, 2118 (C≡C), 1707, 1657 (C=O), 1599, 1547, 1473, 1419, 758, 744 (C=C, C=N); UV (EtOH), λ_max, (lgε): 226 (4.37), 299 (4.11) nm. HR-MS, m/z (I_rel, %): 218 (100), 181 (22), 133 (23), 131 (42), 100 (14), 82 (30), 69 (62), 67 (76), 56 (15), 52 (16); Calcd for C₁₀H₁₀N₄O₂: 218.0798; found [M]⁺ m/z: 218.0804. Anal. calcd for C₁₀H₁₀N₄O₂, %: C, 55.04; H, 4.62; N, 25.68; found, %: C, 54.74; H, 4.48; N, 25.36.

3.2.4. General Method for the Preparation of 8-(1,2,3-Triazol-4-yl)-3,7-дигидрo-1H-purine-2,6-diones (28–33)

A solution of 8-ethynylcaffeine 24 (300 mg, 1.4 mmol) in acetonitrile (10 mL) was treated with CuI (26 mg, 10 mol%), DIPEA (0.837 mL, 3.5 equiv.) and corresponding azide 27a–e (1 equiv.). The reaction mixture was stirred at 50 °C in argon for 24 h. After cooling the solvent was evaporated, and the residue was treated with brine (10 mL) and extracted with CHCl₃ (3 × 15 mL). The combined organic solution was dried over MgSO₄, filtered, and evaporated in vacuo. After column chromatography (eluent CHCl₃-EtOH, 100:0→20:1) compounds 28–32 were isolated. Compound 33 was obtained by heating the reaction mixture of 24, CuI, DIPEA, and azide 27f in a sealed tube to 90 °C for 24 h.

1,3,7-Trimethyl-8-(1-(p-tolyl)-1H-1,2,3-triazol-4-yl)-3,7-dihydro-1H-purine-2,6-dione (28)

Yield 84%. Yellow powder. M.p. 265.4 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 2.41 (3H, s, CH_3–4″), 3.38 (3H, s, NCH₃), 3.56 (3H, s, NCH₃), 4.45 (3H, s, NCH₃), 7.33 (2H, d, J = 8.4 Hz, H-3″,5″), 7.65 (2H, d, J = 8.4 Hz, H-2″,6″), 8.57 (1H, s, H-5′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 21.01 (CH₃-C4″), 27.86 (NCH₃), 29.57 (NCH₃), 34.30 (NCH₃), 108.30 (C-5), 120.29 (C-3″,5″), 122.79 (C-5′), 130.29 (C-2″,6″), 133.82 (C-1″), 139.60 (C-4″), 139.62 (C-8), 142.46 (C-4′), 148.00 (C-4), 151.47 (C-2), 155.13 (C-6); IR (KBr, ν, cm^–1): 3444, 3165, 3082, 2955, 1701 (C=O), 1655 (C=O), 1547, 1516, 1444, 822, 748 (C=C, C=N); UV (EtOH) λ_max, (lgε): 223 (4.45), 304 (4.38) nm. HR-MS, m/z (I_rel., %): 351 (71), 331 (20), 324 (22), 323 (100), 322 (44), 308 (32), 232 (20), 91 (17), 82 (29), 67 (39); Calcd for C₁₇H₁₇N₇O₂: 351.1438; found [M]⁺ m/z: 351.1441. Anal. calcd for C₁₇H₁₇N₇O₂, %: C, 58.11; H, 4.88; N, 27.90; found, %: C, 57.70; H, 4.76; N 27.69.

1,3,7-Trimethyl-8-(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)-3,7-dihydro-1H-purine- 2,6-dione (29)

Yield 70%. Orange powder. M.p. 290.1–291.4 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 3.42 (3H, s, NCH₃), 3.59 (3H, s, NCH₃), 4.50 (3H, s, NCH₃), 8.06 (2H, d, J = 9.0 Hz, H-3″,5″), 8.46 (2H, d, J = 9.0 Hz, H-2″,6″), 8.72 (1H, s, H-5′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.94 (NCH₃), 29.60 (NCH₃), 34.38 (NCH₃), 108.59 (C-5), 120.65 (C-2″,6″), 122.78 (C-5′), 125.60 (C-3″,5″), 140.27 (C-8), 140.55 (C-1″), 141.67 (C-4′), 147.52 (C-4), 147.98 (C-4″), 151.46 (C-2), 155.18 (C-6); IR (KBr, ν, cm^–11705, 1666 (C=O), 1595, 1344, 854 (NO₂), 1545, 1525, 1448, 748 (C=C, C=N); UV (EtOH) λ_max, (lgε): 222 (4.23), 305 (4.18) nm. HR-MS, m/z (I_rel., %): 382 (100), 354 (45), 308 (44), 307 (19), 85 (28), 83 (44), 82 (42), 67 (69), 47 (16), 42 (22); Calcd for C₁₆H₁₄N₈O₄, %: 382.1133; found [M]⁺ m/z: 382.1132. Anal. calcd for C₁₆H₁₄N₈O₄: C, 50.26; H, 3.69; N, 29.31; found, %: C, 50.60; H, 3.45; N, 29.01.

8-(1-Benzyl-1H-1,2,3-triazol-4-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (30)

Yield 65%. White powder. M.p. 243.1–248.2 °C. ¹H NMR (300 MHz, CDCl₃, δ, ppm): 3.37 (3H, s, NCH₃), 3.51 (3H, s, NCH₃), 4.41 (3H, s, NCH₃), 5.58 (2H, s, H-6′), 7.29–7.40 (5H, m, Ph), 8.06 (1H, s, H-5′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 27.87 (NCH₃), 29.56 (NCH₃), 34.28 (NCH₃), 54.43 (C-6′), 108.22 (C-5), 124.76 (C-5′), 129.07 (C-4″), 129.22 (C-2″,6″), 128.25 (C-3″,5″), 133.32 (C-1″), 139.34 (C-8), 142.70 (C-4′), 148.04 (C-4), 151.52 (C-2),155.20 (C-6); IR (KBr, ν, cm^–1): 1705, 1666 (C=O), 1545, 1441 (C=C, C=N), 744 (C=C); UV (EtOH) λ_max, (lgε): 231 (4.38), 300 (4.34) nm. HR-MS, m/z (I_rel., %): 351 (41), 322 (59), 91 (100), 69 (53), 67 (61), 57 (58), 55 (55), 43 (82), 41 (69), 18 (54); Calcd for C₁₇H₁₇N₇O₂: 351.1438; found [M]⁺ m/z: 351.1439. Anal. calcd for C₁₇H₁₇N₇O₂, %: C, 58.11; H 4.88; N 27.90; found, %: C, 57.95; H, 4.88; N, 27.75.

8-(1-Butyl-1H-1,2,3-triazol-4-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (31)

Yield 74%. White powder. M.p. 240.7–241.9 °C. ¹H NMR (300 MHz, CDCl₃, δ, ppm): 0.95 (3H, t, J = 7.2 Hz, H-9′), 1.37 (2H, sext, J = 7.2 Hz, H-8′), 1.93 (2H, sext, J = 7.2 Hz, H-7′), 3.38 (3H, s, NCH₃), 3.54 (3H, s, NCH₃), 4.41 (3H, s, NCH₃), 4.42 (2H, t, J = 7.1 Hz, H-6′), 8.14 (1H, s, H-5′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 13.26 (C-9′), 19.48 (C-8′), 27.81 (NCH₃), 29.53 (NCH₃), 31.93 (C-7′), 34.21 (NCH₃), 50.22 (C-6′), 108.11 (C-5), 124.67 (C-5′), 139.17 (C-8), 142.81 (C-4′), 147.98 (C-4), 151.46 (C-2), 155.12 (C-6). IR (KBr, ν, cm^–1): 3444, 3144, 2958, 2874, 1707 (C=O), 1666 (C=O), 1547, 1443, 1288, 1047, 995, 744. UV (EtOH) λ_max, (lgε): 230 (4.28), 300 (4.24) nm. HR-MS, m/z (I_rel., %): 317 (100), 252 (21), 247 (23), 246 (31), 112 (26), 69 (21), 67 (31), 55 (23), 43 (21), 41 (35); Calcd for C₁₄H₁₉N₇O₂: 317.1595; found [M]⁺ m/z: 317.1601. Anal. calcd for C₁₄H₁₉N₇O₂, %: C, 52.99; H, 6.03; N, 30.90; found, %: C, 52.68; H, 5.77; N, 30.59.

8-(1-(2-Hydroxyethyl)-1H-1,2,3-triazol-4-yl)-1,3,7-trimethyl-3,7-dihydro-1H-puri- ne-2,6-dione (32)

Yield 38%. White crystals. M.p. 232.0 °C (decomp.). ¹H NMR (300 MHz, CDCl₃, δ, ppm): 2.63 (1H, br.s, OH), 3.35 (3H, s, NCH₃), 3.52 (3H, s, NCH₃), 3.99 (2H, t, J = 5.0 Hz, C-7′), 4.36 (3H, s, NCH₃), 4.53 (2H, t, J = 5.0 Hz, C-6′), 8.34 (1H, s, H-5′); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 155.11 (C-6), 151.49 (C-2), 147.85 (C-4), 142.93 (C-4′), 138.50 (C-8), 126.25 (C-5′), 108.17 (C-5), 60.38 (C-7′), 52.81 (C-6′), 34.18 (NCH₃), 29.54 (NCH₃), 27.87 (NCH₃); IR (KBr, ν, cm^–1): 3433 (OH), 3142, 2955, 1705, 1668 (C=O), 1063, 1039 (C-O-C), 1545, 1443, 744 (C=N, C=C); UV (EtOH) λ_max, (lgε): 230 (4.29), 299 (4.23) nm. HR-MS, m/z (I_rel., %): 305 (100), 246 (27), 194 (52), 82 (22), 67 (57), 45 (39), 43 (22), 42 (24), 31 (43), 18 (25); Calcd for C₁₂H₁₅N₇O₃: 305.1231; found [M]⁺ m/z: 305.1234. Anal. calcd for C₁₂H₁₅N₇O₃, %: C, 47.21; H, 4.95; N, 32.12; found, %: C, 47.66; H, 5.21; N 32.54. Crystallographic data for compound (32): C₁₂H₁₅N₇O₃, M 305.31, orthorhombic, Pccn, a 14.420(2), b 27.125(4), c 6.9712(9) Å, V 2726.7(6) ų, Z 8, D_calcd 1.487 g·cm^–3, μ(Mo-Kα) 0.112 mm^–1, F(000) 1280, (θ 2.66–25.13°, completeness 98.9%), T 296(2) K, light-brown, (1.00 × 0.20 × 0.01) mm³, transmission 0.7507–0.9281, 31114 measured reflections in index range −17 <= h <= 17, −32 <= k <= 32, −5 <= l <= 8, 2416 independent (R_int 0.0432), 205 parameters, R₁ 0.0721 (for 1686 observed I > 2σ(I)), wR₂ = 0.211 (all data), GOOF 1.087, largest diff. peak and hole 0.507 and −0.482 e.A⁻³. Crystallographic data for compound 32 has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2179477. Copy of the data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44 122 3336033 or e-mail: [email protected]; internet: www.ccdc.cam.ac.uk).

8-(1-(tert-Butyl)-1H-1,2,3-triazol-4-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (33)

Yield 69%. White powder. M.p. 252.2 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.71 (9H, s, t-Bu), 4.41 (3H, s, NCH₃), 3.54 (3H, s, NCH₃), 3.36 (3H, s, NCH₃), 8.21 (1H, s, H-5′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.79 (NCH₃), 29.52 (NCH₃), 29.77 (3×CH₃, t-Bu), 34.20 (NCH₃), 60.15 (C-6′), 108.08 (C-5), 122.25 (C-5′), 138.58 (C-8), 143.09 (C-4′), 148.00 (C-4), 151.47 (C-2), 155.11 (C-6); IR (KBr, ν, cm^–1): 1705, 1670 (C=O), 1605, 1545, 1443, 744 (C=N, C=C); UV (EtOH) λ_max (lgε): 230 (4.33), 299 (4.28) nm. HR-MS, m/z (I_rel., %): 317 (100), 274 (14), 272 (15), 261 (28), 247 (15), 233 (25), 232 (17), 221 (13), 67 (13), 57 (12); Calcd for C₁₄H₁₉N₇O₂: 317.1595; found [M]⁺ m/z: 317.1598. Anal. calcd for C₁₄H₁₉N₇O₂, %: C, 52.99; H, 6.03; N 30.90; found, %: C, 52.72; H, 6.06; N, 28.47.

3.2.5. Preparation of 8-(Piperidinyl)- or 8-(piperazinyl)-1,3,7-trimethyl-3,7-dihydro-1H-purine- 2,6-diones (60,61)

To a vigorously stirred solution of 8-bromocaffeine (5) (100 mg, 0.37 mmol) in DMF (5 mL) was added an excess (3 equiv.) of 4-methylpiperidine 46 or N-Boc-piperazine 49. The reaction mixture was stirred at 120 °C for 3 h. After evaporation of the solvent, the residue was subjected to column chromatography (CHCl₃-EtOH, 100:0→99:1). Compounds 60 or 61 were isolated.

1,3,7-Trimethyl-8-(4-methylpiperidin-1-yl)-3,7-dihydro-1H-purine-2,6-dione (60)

Yield 78%. White powder. M.p. 131.6–131.7 °C. ¹H NMR (500 MHz, CDCl₃, δ, ppm): 0.97 (3H, d, J = 6.6 Hz, H-14), 1.32 (2H, quart.d, J = 12.4, 3.5 Hz, H_ax -12,12′), 1.49–1.61 (1H, m, H-13), 1.72 (2H, br.d, J = 12.4 Hz, H_eq-12,12′), 2.91 (2H, td, J = 12.4, 1.6 Hz, H_ax-11,11′), 3.34 (3H, s, NCH₃), 3.47 (2H, br.d, J = 12.4 Hz, H_eq -11,11′), 3.49 (3H, s, NCH₃), 3.68 (3H, s, N CH₃). ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 21.68 (C-14), 27.55 (NCH₃), 29.50 (NCH₃), 30.43 (C-13), 32.45 (NCH₃), 33.60 (C-12,12′), 50.06 (C-11,11′), 105.04 (C-5), 147.43 (C-4), 151.63 (C-2), 154.74 (C-8), 157.03 (C-6). IR (KBr, ν, cm^–1): 1703, 1659 (C=O), 1612, 1520, 1452, 804, 748 (C=C, C=N); UV (EtOH) λ_max. (lgε): 225 (4.28), 295 (4.21) nm. HR-MS, m/z (I_rel., %): 291 (100), 276 (27), 262 (10), 248 (15), 222 (24), 221 (25), 209 (12), 208 (12), 194 (16), 69 (15); calcd for C₁₄H₂₁N₅O₂: 291.1690; found [M]⁺ m/z: 291.1689. Anal. calcd for C₁₄H₂₁N₅O₂, %: C, 57.71; H, 7.27; N 24.04; found, %: C, 58.19; H, 7.61; N, 24.37.

tert-Butyl 4-(1,3,7-trimethyl-2,6-dioxo-3,7-dihydro-1H-purin-8-yl)piperazine-1- carboxylate (61)

Yield 88%. White powder. M.p. 163.9–166.9 °C (decomp.). ¹H NMR (500 MHz, CDCl₃, δ, ppm): 1.45 (9H, s, t-Bu), 3.18 (4H, t, J = 5.0 Hz, H-11,11′), 3.49 (3H, s, NCH₃), 3.35 (3H, s, NCH₃), 3.56 (4H, t, J = 5.0 Hz, H-12,12′), 3.73 (3H, s, NCH₃). ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.59 (NCH₃), 28.18 (3 × CH₃, t-Bu), 29.50 (NCH₃), 32.22 (NCH₃), 80.10 (C-12,12′), 49.43 (C-11,11′), 105.28 (C-5), 147.07 (C-4), 151.52 (C-2), 154.40 (C-8), 154.81 (C-6), 155.70 (C=O, t-Bu); IR (KBr, ν, cm^–1): 1697, 1657 (C=O), 1600, 1508, 1452, 1425, 741(C=C, C=N); UV (EtOH) λ_max. (lgε): 222 (4.29), 291 (4.20) nm. HR-MS, m/z (I_rel, %): 378 (69), 322 (62), 305 (19), 278 (16), 223 (19), 222 (100), 209 (56), 67 (12), 57 (31), 41 (14); calcd for C₁₇H₂₆N₆O₄: 378.2010; found [M]⁺ m/z: 378.2011. Anal. calcd for C₁₇H₂₆N₆O₄, %: C, 53.96; H, 6.93; N, 22.21; found, %: C, 53.82; H, 6.82; N, 22.42.

3.2.6. Synthesis and Spectral Data of 8-(3-Aminoprop-1-ynyl)-1,3,7-trimethyl- 3,7-dihydro-1H-purine-2,6-diones (35, 39–41, 51–59)

Conditions (a). A stirred mixture of 8-ethynylcaffeine (24) (218 mg, 1 mmol), 30% aq. formaldehyde (90 mg, 3 mmol), diethylamine (34) (220 mg, 3 mmol), Cu(OAc)₂ × H₂O (20 mg, 0.1 mmol) in THF (8 mL) was heated to 75 °C (bath) for 3 h under argon. After the consumption of the starting materials, the solvent was removed under reduced pressure, the crude material was diluted with water and the product was extracted with chloroform (3 × 15 mL). The combined organic solution was dried over MgSO₄, filtered and evaporated in vacuo. After column chromatography (eluent CHCl₃-EtOH, 100:0→6:1) compounds 35,39–41,51–59 were isolated.

Conditions (b). Synthesis of compound (35) from 8-(trimethylsilylethynyl)caffeine (26). A stirred solution of compound (26) (150 mg, 0.52 mmol) in THF (5 mL) was successive treated with aq. tetra-n-butyl ammonium fluoride (130 mg, 0.62 mmol) for 2 h, then formalin (0.155 mL, 3 equiv. of CH₂O), diethylamine 34 (114 mg, 1.56 mmol), and Cu(OAc)2 × H2O (10 mg, 0.05 mmol) in an argon flow. The reaction mixture was stirred 10 min at room temperature and then heated to 75 °C (bath) for 3 h and evaporated. The residue was diluted with water, and then extracted with chloroform (3 × 15 mL). The combined organic solution was dried over MgSO₄, filtered and evaporated in vacuo. After column chromatography (eluent CHCl₃-EtOH, 100:0→30:1) compounds 35 was isolated in the yield 44%.

8-(3-(Diethylamino)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (35)

Yield 94%^[a] or 44%^[b]. Brownish powder. M.p. 99.8–102.5 °C (decomp.). ¹H NMR (300 MHz, CDCl₃, δ, ppm): 1.09 (6H, t, J = 7.2 Hz, H-15,15′), 2.60 (4H, quart, J = 7.2 Hz, H-14,14′), 3.37 (3H, s, NCH₃), 3.53 (3H, s,NCH₃), 3.72 (2H, s, H-12), 3.97 (3H, s, NCH₃). ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 12.59 (C-15,15′), 27.87 (N CH₃), 29.64 (NCH₃), 32.98 (NCH₃), 41.21 (C-12), 47.42 (C-14,14′),72.98 (C-11), 93.73 (C-10), 107.44 (C-5), 135.37 (C-8), 147.45 (C-4), 151.38 (C-2), 154.71 (C-6). IR (KBr, ν, cm^–1): 2183 (C≡C), 1709, 1666 (C=O), 1601, 1547, 1485, 1429, 744 (C=C, C=N); UV (EtOH) λ_max. (lgε): 228 (4.40), 302 (4.23) nm. HR-MS, m/z (I_rel., %): 302 (2), 233 (21), 233 (9), 232 (75), 231 (100), 176 (10), 175 (21), 174 (56), 146 (23), 72 (9); calcd for C₁₅H₂₁N₅O₂: 303.1690; found [M-H]⁺ m/z: 302.1607. Anal. calcd for C₁₅H₂₁N₅O₂,%: C, 59.39; H, 6.98; N, 23.09; found, %: C, 58.98; H, 6.68; N, 22.70.

8-(3-(Diisopropylamino)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine- 2,6-dione (39)

Yield 96%. Orange powder. M.p. 113.8 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.11 (12H, d, J = 6.6 Hz, 4 × CH₃ (i-Pr)), 3.21 (2H, sept, J = 6.6 Hz, 2 × CH (i-Pr)), 3.37 (3H, s, NCH₃), 3.53 (3H, s, NCH₃), 3.70 (2H, s, H-12), 3.96 (3H, s, NCH₃); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 20.55 (4 × CH₃, i-Pr), 27.83 (NCH₃), 29.62 (NCH₃), 32.84 (NCH₃), 34.67 (C-12), 48.62 (2 × CH, i-Pr), 71.44 (C-11), 98.49 (C-10), 107.35 (C-5), 135.73 (C-8), 147.52 (C-4), 151.41 (C-2), 154.71 (C-6); IR (KBr, ν, cm^–1): 2970, 2931, 2875, 2226 (C≡C), 1701, 1668 (C=O), 1601, 1545, 1485, 1431, 746 (C=N, C=C); UV (EtOH) λ_max (lgε): 229 (4.42), 302 (4.24) nm. HR-MS, m/z (I_rel, %): 331 (4), 317 (76), 316 (37), 232 (48), 231 (100), 67(48), 57 (50), 43 (35), 42 (31), 41 (52); calcd for C₁₇H₂₅N₅O₂: 331.2003; found [M]⁺ m/z: 331.2001. Anal. calcd for C₁₇H₂₅N₅O₂,%: C, 61.61; H, 7.60; N, 21.13; found, %: C, 61.93; H, 7.35; N, 20.98.

8-(3-(Dibutylamino)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (40)

Yield 87%. Orange powder. M.p. 65.6–67.1 °C. ¹H NMR (300 MHz, CDCl₃, δ, ppm): 0.90 (6H, t, J = 7.2 Hz, H-17,17′), 1.27–1.37 (4H, m, H-16,16′), 1.39–1.50 (4H, m, H-15,15′), 2.50 (4H, t, J = 7.3 Hz, H-14,14′), 3.38 (3H, s, NCH₃), 3.54 (3H, s, NCH₃), 3.69 (2H, s, H-12), 3.98 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 13.92 (C-17,17′), 20.42 (C-16,16′), 27.90 (NCH₃), 29.60 (C-15,15′), 29.67 (NCH₃), 32.98 (NCH₃), 42.43 (C-12), 53.60 (C-14,14′), 72.95 (C-11), 94.29 (C-10), 107.46 (C-5), 135.48 (C-8), 147.48 (C-4), 151.42 (C-2), 154.74 (C-6); IR (KBr, ν, cm^–1): 2229 (C≡C), 1707, 1666 (C=O), 1601, 1547, 1485, 1429, 746 (C=C, C=N); UV (EtOH) λ_max. (lgε): 229 (4.44), 302 (4.27) nm. HR-MS, m/z (I_rel., %): 358 (1), 316 (5), 260 (2), 233 (4), 232 (25), 231(100), 217 (2), 174 (3), 146 (2), 128 (2); calcd for C₁₉H₂₉N₅O₂: 359.2316; found [M-H]⁺ m/z: 358.2219. Anal. calcd for C₁₉H₂₉N₅O₂,%: C, 63.48; H, 8.13; N, 19.48; found, %: C, 63.82; H, 8.01; N, 19.26.

8-(3-(Dicyclohexylamino)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine- 2,6-dione (41)

Yield 57%. Yellow powder. M.p. 147.4 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.14 (2H, t.t, J = 12.2, 3.2 Hz, 2 × H_ax-17), 1.20–1.40 (8H, m, 2 × H_ax-15,15′,16,16′), 1.60–1.67 (2H, m, 2 × H_eq-17), 1.74–1.83 (4H, m, 2 × H_eq -16,16′), 1.84–1.91 (4H, m, 2 × H_eq-15,15′), 2.81 (2H, tt, J = 10.8, 3.3 Hz, 2 × H-14), 3.37 (3H, s, NCH₃), 3.53 (3H, s, NCH₃), 3.75 (2H, s, H-12), 3.96 (3H, s, NCH₃); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 26.01 (2 × C-16,2 × C-16′), 26.14 (2 × C-17), 28.00 (NCH₃), 29.79 (NCH₃), 31.36 (2 × C-15,2 × C-15′), 32.96 (NCH₃), 35.70 (C-12), 57.53 (2 × C-14), 71.28 (C-11), 99.29 (C-10), 107.51 (C-5), 135.99 (C-8), 147.66 (C-4), 151.58 (C-2), 154.86 (C-6); IR (KBr, ν, cm^–1): 2227 (C≡C), 1713, 1662 (C=O), 1599, 1547, 1483, 1427, 744 (C=C, C=N); UV (EtOH) λ_max. (lgε): 229 (4.46), 302 (4.28) nm. HR-MS, m/z (I_rel., %): 411 (33), 356 (22), 355 (100), 328 (43), 273 (24), 272 (97), 232 (15), 231 (38), 55 (32), 41 (18); calcd for C₂₃H₃₃N₅O₂: 411.2629; found [M]⁺ m/z: 411.2634. Anal. calcd for C₂₃H₃₃N₅O₂,%: C, 67.13; H, 8.08; N, 17.02; found, %: C, 66.92; H, 7.90; N 16.80.

1,3,7-Trimethyl-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)-3,7-dihydro-1H-purine-2,6-dione (51)

Yield 62%. Orange powder. M.p. 154.1 °C (decomp.) (M.p. 149–150 °C [47]). IR (KBr, ν, cm^–1): 2243 (C≡C), 1705, 1664 (C=O), 1599, 1547, 1487, 1429, 744 (C=O); UV (EtOH) λ_max. (lgε): 228 (4.45), 302 (4.25) nm. ¹H NMR, ¹³C NMR, and MS data are given in [47].

8-(3-(Azepan-1-yl)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (52)

Yield 97%. Orange powder. M.p. 124.6 °C (decomp.). (M.p. 99–101 °C for hydrate of 52) [47]). IR (KBr, ν, cm^–1): 2239 (C≡C), 1705, 1666 (C=O), 1601, 1545, 1487, 1429, 744 (C=O); UV (EtOH) λ_max. (lgε): 229 (4.44), 302 (4.28) nm. ¹H NMR, ¹³C NMR, and MS data are given in [47].

8-(3-(Azocan-1-yl)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (53)

Yield 98%. Yellow powder. M.p. 166.1–167.6 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.51–1.62 (10H, m, H-15,15′,16,16′,17), 2.63–2.68 (4H, m, H-14,14′), 3.36 (3H, s, NCH₃), 3.52 (3H, s, NCH₃), 3.64 (2H, s, H-12), 3.97 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 25.77 (C-16,16′), 27.35 (C-17), 27.48 (C-15,15′), 27.85 (NCH₃), 29.62 (NCH₃), 32.97 (NCH₃), 48.18 (C-12), 53.11 (C-14,14′), 71.84 (C-11), 96.02 (C-10), 107.38 (C-5), 135.59 (C-8), 147.47 (C-4), 151.39 (C-2), 154.69 (C-6); IR (KBr, ν, cm⁻¹): 2226 (C≡C), 1699, 1670 (C=O), 1601, 1543, 1481, 1425, 750 (C=C, C=N); UV (EtOH) λ_max. (lgε): 229 (4.12), 302 (3.95) HM. HR-MS, m/z (I_rel., %): 343 (21), 286 (8), 272 (9), 259 (12), 233 (13), 232 (100), 231 (68), 174 (14), 112 (88), 42 (10); calcd for C₁₈H₂₅N₅O₂: 343.2003; found [M]⁺ m/z: 343.2006. Anal. calcd for C₁₈H₂₅N₅O₂,%: C, 62.95; H, 7.34; N, 20.39; found, %: C, 63.20; H, 7.27; N, 20.28.

8-(3-(1,4-Oxazepan-4-yl)prop-1-yn-1-yl)-1,3,7-trimethyl-3,7-dihydro-1H-purine- 2,6-dione (54)

Yield 90%. Orange powder. M.p. 118.7–121.7 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.91 (2H, pent, J = 6.0 Hz, H-18), 2.78–2.83 (4H, m, H-14,19), 3.33 (3H, s, NCH₃), 3.50 (3H, s, NCH₃), 3.68–3.73 (4H, m, H-12,17), 3.76 (2H, t, J = 6.2 Hz, H-15), 3.96 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.85 (NCH₃), 29.56 (C-18), 29.61 (NCH₃), 33.03 (NCH₃), 48.31 (C-12), 53.24 (C-14), 57.21 (C-19), 68.44 (C-15,17), 72.92 (C-11), 93.93 (C-10), 107.47 (C-5), 135.14 (C-8), 147.41 (C-4), 151.32 (C-2), 154.66 (C-6); IR (KBr, ν, cm^–1): 2241 (C≡C), 1705, 1662 (C=O), 1599, 1547, 1487, 1429, 742 (C=C, C=N); UV (EtOH, λ_max, nm) (lgε): 228 (4.44), 302 (4.26). HR-MS, m/z (I_rel., %): 331 (100), 272 (23), 233 (16), 232 (95), 231 (58), 194 (39), 100 (24), 82 (19), 67 (31), 42 (38); calcd for C₁₆H₂₁N₅O₃: 331.1639; found [M]⁺ m/z: 331.1642. Anal. calcd for C₁₆H₂₁N₅O₃, %: C, 57.99; H, 6.39; N, 21.13; found, %: C, 57.25; H, 6.35; N 20.67.

1,3,7-Trimethyl-8-(3-(4-methylpiperidin-1-yl)prop-1-yn-1-yl)-3,7-dihydro-1H- purine-2,6-dione (55)

Yield 91%. Yellow powder. M.p. 123.0–125.8 °C. ¹H NMR (500 MHz, CDCl₃, δ, ppm): 0.93 (3H, d, J = 5.9 Hz, CH₃-17), 1.26–1.39 (3H, m, H_ax -14,14′,16), 1.68 (2H, d, J = 11.6 Hz, H_eq-14,14′), 2.32 (2H, dd, J = 11.3, 12.8 Hz, H_ax-15,15′), 2.93 (2H, d, J = 11.3, H_eq -15,15′), 3.38 (3H, s, NCH₃), 3.54 (3H, s, NCH₃), 3.64 (2H, s, H-12), 4.00 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 21.65 (C-17), 27.90 (NCH₃), 29.66 (NCH₃), 29.98 (C-16), 33.07 (NCH₃), 34.01 (C-15,15′), 47.81 (C-12), 52.74 (C-14,14′), 73.12 (C-11), 94.02 (C-10), 107.49 (C-5), 135.31 (C-8), 147.46 (C-4), 151.39 (C-2), 154.73 (C-6); IR (KBr, ν, cm⁻¹): 2245 (C≡C), 1707, 1668 (C=O), 1601, 1545, 1487, 1429, 744 (C=C, C=N); UV (EtOH) λ_max. HM (lgε): 229 (4.45), 302 (4.27). HR-MS, m/z (I_rel., %): 328(16), 233 (17), 232 (100), 231 (43), 112 (20), 98 (56), 67 (21), 55 (16), 42 (21), 41 (18); calcd for C₁₇H₂₃N₅O₂: 329.1846; found [M-H]⁺ m/z: 328.1762. Anal. calcd for C₁₇H₂₃N₅O₂, %: C, 61.99; H, 7.04; N, 21.26; found, %: C, 61.52; H, 6.85; N, 20.92.

1,3,7-Trimethyl-8-(3-morpholinoprop-1-yn-1-yl)-3,7-dihydro-1H-purine-2,6-dione (56)

Yield 89%. White powder. M.p. 184.3–186.0 °C.). (M.p. 88–190 °C [47]). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 2.60 (4H, t, J = 4.5 Hz, H-14,14′), 3.35 (3H, s, NCH₃), 3.51 (3H, s, NCH₃), 3.60 (2H, s, H-12), 3.71 (4H, t, J = 4.5 Hz, H-15,15′), 3.97 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.86 (NCH₃), 29.60 (NCH₃), 33.05 (NCH₃), 47.66 (C-12), 52.07 (C-14,14′), 66.58 (C-15,15′), 73.53 (C-11), 92.96 (C-10), 107.51 (C-5), 135.00 (C-8), 147.42 (C-4), 151.32 (C-2), 154.68 (C-6); IR (KBr, ν, cm^–1): 2226 (C≡C), 1699, 1664 (C=O), 1545, 1487, 1429, 746 (C=C, C=N); UV (EtOH) λ_max. (lgε): 228 (4.43), 302 (4.26) nm. HR-MS, m/z (I_rel., %): 317 (20), 232 (100), 231 (31), 86 (21), 67 (40), 56 (46), 46 (23), 45 (48), 43 (26), 42 (38); calcd for C₁₅H₁₉N₅O₃: 317.1482; found [M]⁺ m/z: 317.1486. Anal. calcd for C₁₅H₁₉N₅O₃, %: C, 56.77; H, 6.03; N, 22.07; found, %: C, 56.42; H, 5.97; N, 22.00.

1,3,7-Trimethyl-8-(3-(4-methylpiperazin-1-yl)prop-1-yn-1-yl)-3,7-dihydro-1H- purine-2,6-dione (57)

Yield 53%. Orange powder. M.p. 144.1 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 2.25 (3H, s, CH₃-17), 2.38–2.54 (4H, m, H-15,15′), 2.60–2.69 (4H, m, H-14,14′), 3.34 (3H, s, NCH₃), 3.50 (3H, s, NCH₃), 3.60 (2H, s, H-12), 3.95 (3H, s, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 27.85 (NCH₃), 29.59 (NCH₃), 30.07 (NCH₃), 45.78 (C-17), 47.29 (C-12), 51.71 (C-14,14′), 54.69 (C-15,15′), 73.42 (C-11), 93.30 (C-10), 107.45 (C-5), 135.13 (C-8), 147.42 (C-4), 151.34 (C-2), 154.68 (C-6); IR (KBr, ν, cm^–1): 2243 (C≡C), 1705 1666 (C=O), 1599, 1547, 1487, 1427, 744 (C=C, C=N); UV (EtOH) λ_max. (lgε): 228 (4.44), 302 (4.26) nm. HR-MS, m/z (I_rel., %): 330 (57), 317 (50), 70 (49), 67 (57), 57 (100), 56 (94), 55 (49), 43 (76), 42 (92), 41 (81); calcd for C₁₆H₂₂N₆O₂: 330.1799; found [M]⁺ m/z: 330.1795. Anal. calcd for C₁₆H₂₂N₆O₂, %: C, 58.17; H, 6.71; N, 25.44; found, %: C, 57.87; H, 6.56; N, 25.81. 3.2.6.12. tert-Butyl 4-(3-(1,3,7-trimethyl-2,6-dioxo-3,7-dihydro-1H-purin-8-yl)prop- 2-yn-1-yl)piperazine-1-carboxylate (58). Yield 58%. Yellow powder. M.p. 151.0–152.0 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.42 (9H, s, t-Bu), 2.55 (4H, t, J = 4.8, H-14,14′), 3.36 (3H, s, NCH₃), 3.45 (4H, t, J = 4.8 Hz, H-15,15′), 3.52 (3H, s, NCH₃), 3.63 (2H, s, H-12), 3.97 (3H, s, NCH₃); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 27.87 (NCH₃), 28.24 (3 × CH₃, t-Bu), 29.62 (NCH₃), 33.05 (NCH₃), 47.51 (C-12), 51.61 (C-14,14′,15,15′), 73.59 (C-11), 79.76 (C, t-Bu), 92.93 (C-10), 107.56 (C-5), 135.02 (C-8), 147.46 (C-4), 151.36 (C-2), 154.47 (C=O, t-Bu), 154.71 (C-6); IR (KBr, ν, cm^–1): 2231 (C≡C), 1705, 1672 (C=O), 1545, 1487, 1423, 742(C=C, C=N); UV (EtOH) λ_max. (lgε): 229 (4.44), 302 (4.28) nm. HR-MS, m/z (I_rel., %): 416 (14), 343 (17), 316 (33), 315 (27), 232 (80), 231 (52), 85 (12), 84 (14), 57 (100), 41 (17); calcd for C₂₀H₂₈N₆O₄: 416.2167; found [M]⁺ m/z: 416.2172. Anal. calcd for C₂₀H₂₈N₆O₄, %: C, 57.68; H, 6.78; N, 20.18; found, %: C, 57.74; H, 6.82; N, 20.21.

1,3,7-Trimethyl-8-(3-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)prop-1-yn-1-yl) -3,7-dihydro-1H-purine-2,6-dione hydrate (59)

Yield 75%. Yellow powder. M.p. 208.4 °C (decomp.). ¹H NMR (300 MHz, CDCl₃, δ, ppm): 2.00–2.09 (4H, m, H-21,21′), 2.51–2.59 (4H, m, H-15,15′), 2.60–2.68 (4H, m, H-14,14′), 2.83 (2H, br.t, J = 6.3 Hz, H-17), 3.05 (2H, br.t, J = 6.3 Hz, H-18), 3.17–3.27 (4H, m, H-20,20′), 3.34 (3H, s, NCH₃), 3.50 (3H, s, NCH₃), 3.58 (2H, s, H-12), 3.96 (s, 3H, NCH₃); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 23.08 (C-21,21′), 27.90 (NCH₃), 29.64 (NCH₃), 33.14 (NCH₃), 47.25 (C-12), 51.76 (C-14,14′), 52.01 (C-18), 52.78 (C-15,15′), 53.29 (C-17), 54.15 (C-20,20′), 73.35 (C-11), 93.24 (C-10), 107.56 (C-5), 135.06 (C-8), 147.42 (C-4), 151.36 (C-2), 154.69 (C-6); IR (KBr, ν, cm^–1): 2226 (C≡C), 1701, 1672 (C=O), 1545,1421, 1340, 748 (C=C, C=N); UV (EtOH) λ_max. (lgε): 228 (4.41), 302 (4.24) nm. HR-MS, m/z (I_rel., %): 413 (2), 329 (26), 231 (11), 85 (12), 84 (100), 83 (18), 47 (9), 42 (13), 36 (8), 18 (13); calcd for C₂₁H₃₁N₇O₂: 413.2534; found [M]⁺ m/z: 413.2535. Anal. calcd for C₂₁H₃₁N₇O₂ × H₂O, %: C, 58.47; H, 7.65; N 22.73; found, %: C, 58.13; H, 7.48; N, 22.29.

3.2.7. Propargylation of Dimethylxanthines (2,3)

General Procedure

This procedure is a modification of the method used by Daly et al. [59]. To a stirred suspension of dialkylxanthine 2 or 3 (1000 mg, 5.6 mmol) and anhydrous K₂C0₃ (767 mg, 5.6 mmol) in DMF (30 mL) was added dropwise propargyl bromide 63 (660 mg, 5.6 mmol). The reaction mixture was stirred at rt for 24 h, and the volatile material was evaporated on a Petri dish at ambient temperature. The residue was treated with H₂O and extracted with several portions of CHCl₃. The combined extract was dried over MgSO₄, filtered, and the solvent was evaporated in vacuo. The obtained compounds 62 or 67 were used without further purification.

3.2.8. A³-Coupling of 1-Prop-2-ynyl-, or 7-Prop-2-ynyl- methylxanthines. Synthesis and Spectral Data of 1-(4-Aminobut-2-yn-1-yl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-diones (64–66) or 7-(4-Aminobut-2-yn-1-yl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-diones (68–70)

A stirred mixture of 1-prop-2-ynyl-, or 7-prop-2-ynylmethylxanthines (62, 67) (150 mg, 0.7 mmol), 30% aq. formaldehyde (63 mg, 2.1 mmol), secondary amine (2.1 mmol), Cu(OAc)₂ × H₂O (14 mg, 0.07 mmol) in THF (5 mL) was heated to 75 °C (bath) for 3 h under argon. After the consumption of the starting materials, the solvent was removed under reduced pressure, the crude material was diluted with water and the product was extracted with chloroform (3 × 15 mL). The combined organic solution was dried over MgSO₄, filtered, and evaporated in vacuo. After column chromatography (eluent CHCl₃-EtOH, 100:0→6:1), compounds 64–66 or 68–70 were isolated.

1-(4-(Diisopropylamino)but-2-yn-1-yl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (64)

Yield 90%. White powder. M.p. 46.5 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.00 (12H, d, J = 6.5 Hz, 4 × CH₃ (i-Pr)), 3.13 (2H, sept, J = 6.5 Hz, 2 × CH (i-Pr)), 3.34 (2H, t, J = 1.9 Hz, H-13), 3.49 (3H, s, NCH₃), 3.91 (s, 3H, NCH₃), 4.68 (2H, t, J = 1.9 Hz, H-10), 7.48 (1H, s, H-8); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 20.05 (4 × CH₃, i-Pr), 29.48 (NCH₃), 30.64 (C-10), 33.38 (NCH₃), 34.06 (C-13), 48.42 (2 × CH, i-Pr), 78.38 (C-12), 80.22 (C-11), 107.23 (C-5), 141.43 (C-8), 148.69 (C-4), 150.58 (C-2), 154.08 (C-6). HR-MS, m/z (I_rel., %): 331 (11), 317 (20), 316 (95), 274 (5), 232 (16), 231 (100), 229 (3), 181 (4), 136 (3), 94 (3); calcd for C₁₇H₂₅N₅O₂: 331.2003; found [M]⁺ m/z: 331.2006. Anal. calcd for C₁₇H₂₅N₅O₂, %: C, 61.61; H, 7.60; N, 21.13; found, %: C, 61.23; H, 7.37; N, 20.68.

1-(4-(Azocan-1-yl)but-2-yn-1-yl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (65)

Yield 83%. Yellow powder. M.p. 77.5°C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.40–1.53 (10H, m, H-16,16′,17,17′,18), 2.46–2.53 (4H, m, H-15,15′), 3.22 (2H, t, J = 1.8 Hz, H-13), 3.49 (3H, s, NCH₃),3.91 (3H, s, NCH₃), 4.70 (2H, t, J = 1.8 Hz, H-10), 7.47 (1H, s, H-8); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 25.76 (C-17,17′), 27.17 (C-18), 27.26 (C-16,16′), 29.48 (NCH₃), 30.53 (C-10), 33.35 (NCH₃), 47.53 (C-13), 52.78 (C-15,15′), 77.97 (C-12), 78.84 (C-11), 107.25 (C-5), 141.41 (C-8),148.68 (C-4), 150.58 (C-2), 154.09 (C-6). HR-MS, m/z (I_rel., %): 343 (26), 231 (80), 150 (40), 112 (100), 107 (21), 67 (28), 55 (32), 42 (48), 41 (40), 18 (32); calcd for C₁₈H₂₅N₅O₂: 343.2003; found [M]⁺ m/z: 343.2002. Anal. calcd for C₁₈H₂₅N₅O₂, %: C, 62.95; H, 7.34; N, 20.39; found, %: C, 62.61; H, 7.28; N, 19.86.

3,7-Dimethyl-1-(4-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)but-2-yn-1-yl)-3,7- dihydro-1H-purine-2,6-dione hydrate (66)

Yield 54%. Orange amorphous substance. M.p. not determ. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.47 (1H, s, H-8), 4.72 (2H, t, J = 1.8 Hz, H-10), 3.92 (3H, s,NCH₃), 3.51 (3H, s, NCH₃), 3.17 (2H, t, J = 1.8 Hz, H-13), 2.42–2.59 (16H, m, H-15,15′,16,16′,18,19,21,21′), 1.68–1.73 (4H, m,H-22, 22′); ¹³C NMR (101 MHz, CDCl₃, δ, ppm): 154.13 (C-6), 150.63 (C-2), 148.76 (C-4), 141.46 (C-8), 107.32 (C-5), 79.59 (C-11), 76.98 (C-12), 57.18 (C-18), 54.28 (C-21, C-21′), 53.36 (C-19), 53.21 (C-16, C-16′), 51.77 (C-15, C-15′), 46.93 (C-13), 33.41 (NCH₃), 30.52 (C-10), 29.55 (NCH₃), 23.14 (C-22,22′). HR-MS, m/z (I_rel, %): 413 (24), 330 (18), 329 (100), 317 (8), 316 (8), 231 (13), 180 (7), 149 (34), 84 (31); calcd for C₂₁H₃₁N₇O₂: 413.2534; found [M]⁺ m/z: 413.2532. Anal. calcd for C₂₁H₃₁N₇O₂ × H₂O, %: C, 58.47; H, 7.65; N 22.73; found, %: C, 58.13; H, 7.48; N, 22.29.

7-(4-(Diisopropylamino)but-2-yn-1-yl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (68)

Yield 98%. White powder. M.p. 63.7–65.3 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 1.00 (12H, d, J = 6.6 Hz, 4 × CH₃ (i-Pr)), 3.08 (2H, sept, J = 6.6 Hz, 2 × CH (i-Pr)), 3.31 (3H, s, NCH₃), 3.39 (2H, t, J = 1.9 Hz, H-13), 3.50 (3H, s, NCH₃), 5.07 (2H, t, J = 1.8 Hz, H-10), 7.77 (s, 1H, H-8); ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 20.27 (4 × CH₃, i-Pr), 27.69 (NCH₃), 29.52 (NCH₃), 34.04 (C-13), 36.91 (C-10), 48.25 (2 × CH, i-Pr), 74.08 (C-12), 87.56 (C-11), 106.44 (C-5), 140.15 (C-8), 148.55 (C-4),151.32 (C-2), 154.90 (C-6). HR-MS, m/z (I_rel, %): 331 (8), 317 (22), 316 (100), 232 (19), 231 (21), 152 (29), 138 (36), 94 (26), 43 (26), 41 (17); calcd for C₁₇H₂₅N₅O₂: 331.2000; found [M]⁺ m/z: 331.2003. Anal. calcd for C₁₇H₂₅N₅O₂, %: C, 61.61; H, 7.60; N, 21.13; found, %: C, 61.68; H, 7.77; N, 21.21.

7-(4-(Azocan-1-yl)but-2-yn-1-yl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (69)

Yield 84%. Yellow powder. M.p. 46.9 °C (decomp.). ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.85 (1H, s, H-8), 5.15 (2H, t, J = 1.7 Hz, H-10), 3.57 (3H, s, NCH₃), 3.40 (2H, t, J = 1.9 Hz, H-13), 3.38 (3H, s, NCH₃), 2.56–2.61 (4H, m, H-15,15′), 1.51–1.60 (10H, m, H-16,16′,17,17′,18). ¹³C NMR (126 MHz, CDCl₃, δ, ppm): 25.83 (C-17,17′), 27.27 (C-18), 27.42 (C-16,16′), 27.84 (NCH₃), 29.66 (NCH₃), 36.92 (C-10), 47.59 (C-13), 95.43 (C-11), 74.71 (C-12), 53.11 (C-15,15′), 106.63 (C-5), 140.30 (C-8), 148.76 (C-4), 151.50 (C-2), 155.10 (C-6). HR-MS, m/z (I_rel., %): 343 (1), 342 (18), 231 (24), 180 (37), 164 (63), 163 (37), 150 (100), 112 (56), 55 (28), 41 (33); calcd for C₁₈H₂₅N₅O₂: 343.2003; found [M-H]⁺m/z: 342.1922. Anal.calcd for C₁₈H₂₅N₅O₂,%: C, 62.95; H, 7.34; N, 20.39; found,%: C, 62.65; H, 7.27; N, 19.91.

1,3-Dimethyl-7-(4-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)but-2-yn-1-yl)-3,7- dihydro-1H-purine-2,6-dione (70)

Yield 74%. White amorphous powder; mp not determ. ¹H NMR (300 MHz, CDCl₃, δ, ppm): 1.76–1.84 (4H, m, H-22,22′), 2.49–2.71 (16H, m, H-15,15′,16,16′,18,19,21,21′), 3.32 (2H, t, J = 1.9 Hz, H-13), 3.37 (3H, s, NCH₃), 3.56 (3H, s, NCH₃), 5.15 (2H, t, J = 1.7 Hz, H-10), 7.80 (1H, s, H-8); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 23.22 (C-22,22′), 27.83 (NCH₃), 29.66 (NCH₃), 36.69 (C-10), 46.88 (C-13), 51.91 (C-15,15′), 53.17 (C-16,16′), 53.29 (C-19), 54.37 (C-21,21′), 56.72 (C-18), 76.59 (C-12), 83.11 (C-11), 106.59 (C-5), 140.36 (C-8), 148.72 (C-4), 151.49 (C-2), 155.08 (C-6). HR-MS, m/z (I_rel., %): 413 (1), 127 (4), 85 (5), 84 (100), 70 (4), 56 (7), 55 (5), 42 (12), 41 (4), 28 (3); calcd for C₂₁H₃₁N₇O₂: 413.2534; found [M]⁺ m/z: 413.2532. Anal. calcd for C₂₁H₃₁N₇O₂, %: C, 61.00; H, 7.56; N, 23.71; found, %: C, 60.61; H, 7.40; N, 23.31.

3.3. Biochemical Method

AChE activity inhibition was evaluated by the assay described by Ellman et al. [52]. 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), AChE (AChE, E.C.3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 units), and acetylthiocholine iodide were obtained from Sigma. A total of 100 μL of a solution containing 10 mM Tris-HCl pH7.5, 0.5 mM DTNB, and 1 μL of acetylcholinesterase at a concentration of 6.25 units × 10⁻³/mL was dropped into polystyrene wells in each. Then, 2 μL of solutions of inhibitors in DMSO were added. The control was supplemented with 2 µL of DMSO without inhibitors. Incubation was performed at room temperature for 15 min., then 5 min. at 4 °C on a planetary shaker. After that, 100 µL of a cooled solution containing 10 mM Tris-HCl pH7.5, 0.5 mM DTNB, and 1 mM acetylthiocholine iodide, prepared immediately before addition, was added to each well. The wells were placed in a reader (PerkinElmer 2103 Multilabel Reader), and the reaction was monitored at room temperature at a wavelength of 405 nm. Each inhibitor was applied at three concentrations in half-log increments (eg 10, 30, and 100) in triplicate. The measurements were carried out at a wavelength of 405 nm every 2 min. The 10th measurement was used for calculations (18 min. from the beginning of the reaction). For each well, the first measurement (background) was subtracted from the indicator of the last measurement, and the resulting numbers were used for further calculations.

Next, activity curves were built, where the point without inhibitor was taken as 100%. The points were used to build a trend line from which the inhibitor concentration where the activity was at 50% was calculated All constructions and determination of the mean and standard deviation were made in Excel 2007 and online services for solving equations. The curves for AChE inhibition by compounds 28, 64, 65, 66, and 70 are given in Supplementary Materials (Figure S1A–E).

3.4. Molecular Modelling and Molecular Dynamic Procedures

Molecular modeling was carried out in the Schrodinger Maestro visualization environment using applications from the Schrodinger Small Molecule Drug Discovery Suite 2016-1 package [60]. Three-dimensional structures of the derivatives were obtained empirically in the LigPrep application using the OPLS3 force field [61]. All possible tautomeric forms of compounds, as well as various states of polar protons of molecules in the pH range of 7.0 ± 2.0 were taken into account. For calculations, the XRD model of human AChE with PDB ID 6O4W (resolution 2.35 Å) was chosen [62]. To model a possible mechanism of inhibition of the selected target, molecular docking of new compounds was performed at the binding site of donepezil of AChE in the Glide application [63]. The search area for docking was selected automatically, based on the size and physico-chemical properties of the inhibitor. The extra precision (XP) algorithm of docking was applied. Docking was performed in comparison with donepezil. The molecular structure of the inhibitor was obtained in the PubChem database and prepared in the LigPrep application. Non-covalent interactions of compounds in the binding site were visualized using Biovia Discovery Studio Client [64].

The molecular dynamic simulation was performed by using NAMD v. 2.14 [65]. Topology files for the ligands were generated by using SwissParam [66], and simulations were performed by using the CHARMM force field [67]. The protein–ligand complexes were solvated using a cubic box with periodic boundary conditions, with the minimal distance from the protein to the boundary being 20 Å. Water molecules, located in the protein pocket and taken into the account during docking procedure, were added as well into the systems to be simulated. The systems were neutralized by the addition of sodium or chlorine ions. Simulations were performed in conditions of 0.15 M NaCl solution. The systems were minimized, annealed up to 310 K, and equilibrated during 1 nanosecond in an NPT ensemble with the movable of the backbone being constrained. The following simulations were performed during 100 nanoseconds in an NVT ensemble at 310 K. System generations and calculations of the ligand root-mean-square deviations (RMSD) were performed in VMD [68]. Plots of changes in the RMSD of atomic coordinates over simulation time, obtained as a result of molecular dynamics research, are given in Supplementary Materials, Figure S1A–D.

4. Conclusions

We have proposed A³-coupling approaches for combining methylxanthine scaffold with nitrogen-modified 8-(3-aminoprop-1-ynyl)-, 1-aminobut-2-ynyl-, and 7-aminobut-2-ynyl- substituent to prepare new candidate molecules as AChE inhibitors. Among these synthesized compounds, 8-(3-(azocan-1-yl)prop-1-ynyl)- 53, 8-(3-(4-(2-(pyrrolidin-1-yl)- ethyl)-piperazin-1-yl)prop-1-ynyl)caffeines 59, 1-(4-(azocan-1-yl)but-2-ynyl)-1-(4-(4- (2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)but-2-ynyl)theobromines 65, 66, and 7-(4- (azocan-1-yl)but-2-ynyl)theophylline 69 exhibited the AChE inhibitory potency in nanomolar concentrations. Molecular docking studies analyzed the binding mode of the most active molecules toward AChE. The majority of the active compounds interacted with the residues of Tyr341, Phe338, Trp86, and Phe295 amino acids, important residues of the AChE binding pocket, indicating that all ligands fitted well in the catalytic site, and might be an explanation for the relevant in vitro activities. In sum, some of the described methylxanthine derivatives showed promising properties for further study. In addition, due to the simplicity and effectiveness of the reported A³-coupling methods, it is likely that the new transformation of methylxanthine core will find use in the development of compounds that could be used as scaffolds toward accessing other libraries of bioactive compounds.