Synthesis of Heterocycles and Nucleosides Forming Higher—Order Structures

: Nucleic acid analogues play a multifaceted role in biology and materials science. Our efforts towards unveiling these roles led to xanthine derivatives that form higher–order structures with quadruplex-forming abilities. In this paper we present further modiﬁcations of the xanthine core resulting into 9-deaza and 8-aza-9-deaza heterocycles (pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines, respectively) that form tetrads and other higher–order structures. Addi-tionally, the ring contraction of 5-ﬂuoro-2 (cid:48) ,3 (cid:48) - O -isopropylideneuridine gave rise to the formation of an imidazolidine-4-carboxylic acid nucleoside derivative. Our computational predictions forecasted that the latter derivative will form stable triads.


Introduction
Beyond the well-known canonical adenine-thymine (A:T) and guanine-cytosine (G:C) base pairs in the double helical DNA, there are several other possibilities, resulting in a large number of polymorphic variants. Z-DNA, hairpin, triplex. i-motif and quadruplex nucleic acids (GQs) are probably the most studied non-canonical nucleic acid structures. A typical G quadruplex consists of at least two guanine G-tetrads (or tetramers), most frequently connected by loops of one or more nucleotides and feature various topologies [1,2]. The stability of the structure is ensured by stacking of the tetrads, cation coordination (K + , Na + , NH 4 + etc.), hydrogen bonding and hydrophobic effects [3][4][5]. The cations are located either in the central cavity of the G-tetrad or in the spaces between the stacking tetrads ( Figure 1) [6].
Earlier we have proved that the G base can be replaced by model 3-alkylxanthine derivatives (X) in tetrads/octads and the resulting cation-complexed tetrads and octads are comparable with the corresponding guanine derivatives in terms of interaction energies (Figure 2a) [7]. Furthermore, 3-alkylxanthines provide self-assembly structures at a solidliquid interface forming comb-like 2D structures [8] and melamine and 3-octadecylxanthine molecules form porous quasi-2D networks [9].
As an extension of these studies we have envisaged further modifications of the xanthine core resulting into 9-deaza and 8-aza-9-deaza heterocycles (pyrrolo [3,2-d]pyrimidines and pyrazolo [4,3-d]pyrimidines) that form tetrads and other higher-order structures (Figure 2b,c). These derivatives feature the same hydrogen bonding pattern as 7H-xanthines. Furthermore, we have anticipated that the ring contraction of 5-fluoro-2 ,3 -O-isopropyliden euridine, affording an imidazolidine-4-carboxylic acid nucleoside derivative, would result potentially in the formation of self-assembly structures.
isopropylideneuridine, affording an imidazolidine-4-carboxylic acid nucleoside derivative, would result potentially in the formation of self-assembly structures.

9-Deazaxanthines
The synthesis of 9-deazaxanthines (pyrrolo[3,2-d]pyrimidines) started from 6methyluracil (1) (Figure 3). Conventional nitration provided the 5-nitro derivative (2) [10]. The reaction of 2 with N,N-dimethylformamide dimethyl acetal gave the 6-dimethylaminomethylene derivative 3. Reduction of the nitro group and simultaneous ring closure provided the pyrrolo[3,2-d]-pyrimidine 4 in good yield. The purification of 4 has been hampered by the presence of zinc salts and its poor solubility. The best method was the recrystallisation of crude product from a large volume of ethanol. In the following step the glycosylation has been performed using the Vorbrüggen conditions employing 1-Oacetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (5) to furnish the glycoside 6 [11]. The subsequent debenzoylation took place smoothly with methanolic ammonia to afford nucleoside 7. isopropylideneuridine, affording an imidazolidine-4-carboxylic acid nucleoside derivative, would result potentially in the formation of self-assembly structures.

9-Deazaxanthines
The synthesis of 9-deazaxanthines (pyrrolo[3,2-d]pyrimidines) started from 6methyluracil (1) (Figure 3). Conventional nitration provided the 5-nitro derivative (2) [10]. The reaction of 2 with N,N-dimethylformamide dimethyl acetal gave the 6-dimethylaminomethylene derivative 3. Reduction of the nitro group and simultaneous ring closure provided the pyrrolo[3,2-d]-pyrimidine 4 in good yield. The purification of 4 has been hampered by the presence of zinc salts and its poor solubility. The best method was the recrystallisation of crude product from a large volume of ethanol. In the following step the glycosylation has been performed using the Vorbrüggen conditions employing 1-Oacetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (5) to furnish the glycoside 6 [11]. The subsequent debenzoylation took place smoothly with methanolic ammonia to afford nucleoside 7.

9-Deazaxanthines
The synthesis of 9-deazaxanthines (pyrrolo[3,2-d]pyrimidines) started from 6-methylur acil (1) (Figure 3). Conventional nitration provided the 5-nitro derivative (2) [10]. The reaction of 2 with N,N-dimethylformamide dimethyl acetal gave the 6-dimethylaminomethylene derivative 3. Reduction of the nitro group and simultaneous ring closure provided the pyrrolo[3,2-d]-pyrimidine 4 in good yield. The purification of 4 has been hampered by the presence of zinc salts and its poor solubility. The best method was the recrystallisation of crude product from a large volume of ethanol. In the following step the glycosylation has been performed using the Vorbrüggen conditions employing 1-O-acetyl-2,3,5-tri-O-benzoylβ-D-ribofuranose (5) to furnish the glycoside 6 [11]. The subsequent debenzoylation took place smoothly with methanolic ammonia to afford nucleoside 7.

8-Aza-9-Deazaxanthines
The 8-aza-9-deazaxanthines (pyrazolo[4,3-d]pyrimidines) have been obtained from the common nitro intermediate 2 ( Figure 4). Catalytic reduction of the nitro group followed by nitrosation gave the triazino intermediate (9) through the 5-amino derivative (8). Stannous chloride in hydrochloric acid gave rise to the formation of the pyrazolo[4,3d]pyrimidine derivative 10 [12,13]. Glycosylation as above [11] provided the nucleoside 11 which was in turn deprotected to furnish compound 12.  [14]. In particular, 5fluoro-2′,3′-O-isopropylideneuridine (14) undergoes ring contraction in basic medium to give an imidazolidinone carboxylic acid. We have anticipated that the amide and hydroxamic acid derivatives would be able to form higher-order structures (triads and/or tetrads). Thus, 5-fluorouridine (13) was isopropylidenated and compound 14 was treated with aqueous sodium hydroxide ( Figure 5). The sodium salt of carboxylate 15 was in situ methylated to circumvent the tedious isolation of the free carboxylic acid. The methyl ester 16 was then subjected to ammonolysis and allowed to react with free hydroxylamine, respectively, to provide the amide 17 and the hydroxamic acid derivative 18.

8-Aza-9-Deazaxanthines
The 8-aza-9-deazaxanthines (pyrazolo[4,3-d]pyrimidines) have been obtained from the common nitro intermediate 2 ( Figure 4). Catalytic reduction of the nitro group followed by nitrosation gave the triazino intermediate (9) through the 5-amino derivative (8). Stannous chloride in hydrochloric acid gave rise to the formation of the pyrazolo[4,3d]pyrimidine derivative 10 [12,13]. Glycosylation as above [11] provided the nucleoside 11 which was in turn deprotected to furnish compound 12.  [14]. In particular, 5fluoro-2′,3′-O-isopropylideneuridine (14) undergoes ring contraction in basic medium to give an imidazolidinone carboxylic acid. We have anticipated that the amide and hydroxamic acid derivatives would be able to form higher-order structures (triads and/or tetrads). Thus, 5-fluorouridine (13) was isopropylidenated and compound 14 was treated with aqueous sodium hydroxide ( Figure 5). The sodium salt of carboxylate 15 was in situ methylated to circumvent the tedious isolation of the free carboxylic acid. The methyl ester 16 was then subjected to ammonolysis and allowed to react with free hydroxylamine, respectively, to provide the amide 17 and the hydroxamic acid derivative 18.  [14]. In particular, 5fluoro-2 ,3 -O-isopropylideneuridine (14) undergoes ring contraction in basic medium to give an imidazolidinone carboxylic acid. We have anticipated that the amide and hydroxamic acid derivatives would be able to form higher-order structures (triads and/or tetrads). Thus, 5-fluorouridine (13) was isopropylidenated and compound 14 was treated with aqueous sodium hydroxide ( Figure 5). The sodium salt of carboxylate 15 was in situ methylated to circumvent the tedious isolation of the free carboxylic acid. The methyl ester 16 was then subjected to ammonolysis and allowed to react with free hydroxylamine, respectively, to provide the amide 17 and the hydroxamic acid derivative 18.

Computational Studies of Imidazolidinones
The complex forming capacity of imidazolidinone derivatives was investigated theoretically by high level quantum chemical methods. Density functional calculations with BLYP-D3/TZ2P level of theory was applied and two derivatives, namely the amide and the hydroxamic acid forms were calculated in higher-order structures. The optimized geometrics (top and side views) with important bond distances and average single hydrogen bond energies are presented in Figure 6. Comparing trimer structures, it is clear from the optimized geometries that the amide derivative is less flexible than the hydroxamic acid, thus it has less planar geometry, as well as larger bond distances were found. Consequently, the average hydrogen bond energy was also smaller in these cases. Thus, the rigidity causes weaker interaction, so the molecule with hydroxamic acid group looks more promising. Comparing the trimer complex with the tetrad, the trimer complexes were closer to a planar structure.

Computational Studies of Imidazolidinones
The complex forming capacity of imidazolidinone derivatives was investigated theoretically by high level quantum chemical methods. Density functional calculations with BLYP-D3/TZ2P level of theory was applied and two derivatives, namely the amide and the hydroxamic acid forms were calculated in higher-order structures. The optimized geometrics (top and side views) with important bond distances and average single hydrogen bond energies are presented in Figure 6.

Computational Studies of Imidazolidinones
The complex forming capacity of imidazolidinone derivatives was investigated theoretically by high level quantum chemical methods. Density functional calculations with BLYP-D3/TZ2P level of theory was applied and two derivatives, namely the amide and the hydroxamic acid forms were calculated in higher-order structures. The optimized geometrics (top and side views) with important bond distances and average single hydrogen bond energies are presented in Figure 6. Comparing trimer structures, it is clear from the optimized geometries that the amide derivative is less flexible than the hydroxamic acid, thus it has less planar geometry, as well as larger bond distances were found. Consequently, the average hydrogen bond energy was also smaller in these cases. Thus, the rigidity causes weaker interaction, so the molecule with hydroxamic acid group looks more promising. Comparing the trimer complex with the tetrad, the trimer complexes were closer to a planar structure. Comparing trimer structures, it is clear from the optimized geometries that the amide derivative is less flexible than the hydroxamic acid, thus it has less planar geometry, as well as larger bond distances were found. Consequently, the average hydrogen bond energy was also smaller in these cases. Thus, the rigidity causes weaker interaction, so the molecule with hydroxamic acid group looks more promising. Comparing the trimer complex with the tetrad, the trimer complexes were closer to a planar structure.

Results and Discussion
We have synthesized analogues of xanthine nucleosides by replacing noncritical nitrogen and carbon atoms with the purpose of obtaining derivatives that would participate in the formation of quadruplex structures. To this end, 9-deaza and 8-aza-9-deaza heterocycles (pyrrolo [3,2-d]pyrimidines and pyrazolo [4,3-d]pyrimidines, respectively) have been synthesized starting from 6-methyluracil. Additionally, the ring contraction of a 5-fluorouridine derivative allowed to obtain imidazolidinone carboxylic acid amide and hydroxamic acid. These latter derivatives have been subjected to density functional calculations and it turned out that they are potential candidates to form preferentially trimeric self-assemblies. The experimental study of the above derivatives in context of formation of higher-order structures is in progress.