Synthesis and DFT analysis of non-covalent interactions in crystal structures of 6-R-2-alkoxy-, 2,3-di-, and 2,2,3-tri-tert-butylpyrrolo[1,2-b][1,2,4]triazines

Novel 7-amino-3-tert-butyl-2-OR1-6-R2-pyrrolo[1,2-b][1,2,4]triazine-8-carbonitriles (R1 = CH2CO2Et, CH2Boc, Me, n-Bu; R2 = CO2Et, CO2n-Bu, CO2t-Bu, C6H4CO2i-Pr) have been synthesized and investigated by X-ray diffraction. Nucleophilic replacement of an alkoxy group with t-BuLi afforded sterically hindered tert-butyl 7-amino-2,3-di-tert-butyl- and 2,2,3-tri-tert-butyl-8-cyanopyrrolo[1,2-b][1,2,4]triazine-6-carboxylates. The lengths and bond angles as well as packing modes of molecules in crystals have been considered. The non-covalent interactions such as the changes in the H-bonding and close contacts were analyzed by DFT and the Hirshfeld surfaces and compared for different substituents. Supplementary Information The online version contains supplementary material available at 10.1007/s11224-022-02006-x.


Introduction
Six-membered heterocycles containing one or two nitrogen atoms are ubiquitous in plants as alkaloids with a broad range of biological activities [1,2]. Triazines are rarely found in nature (e.g., fervenulin, toxoflavin [3,4], and fluviols [5]); nevertheless, they also exhibit antibacterial, antifungal, and anticancer properties [6], which make them an important target for research and various applications. Azolotriazines are particularly interesting in terms of their diverse chemical transformations and the bioisosteric nature [7]. The quantitative and qualitative structural analysis of known azolo [1,2,4]triazines, along with molecular modeling, has been successfully used to identify the most privileged scaffolds for further drug design [8][9][10]. These developments resulted in the production of the 4-aminopyrrolo[2,1-f] [1,2,4]triazine remdesivir, which is active against a number of viruses including Ebola virus and coronaviruses [11]. The interactions between an inhibitor and its molecular target are considered primarily non-covalent in nature and shape dependent. Therefore, the changes in the H-bonding and hydrophobic nature of the substituents can greatly affect the biological potency of the compounds [12][13][14]. It seems clear that investigation of such structural relationships, including novel hydrophilic and hydrophobic cases, can further shed the light on mechanism of influence of different substituent configurations on the binding affinity.

General experimental remarks
Melting points were determined on a STUART Melting point SMP30 apparatus. IR spectra were recorded in KBr pellets using Agilent Cary 660 FTIR infrared spectrophotometer. NMR spectra were recorded on Bruker AM-300, DRX-500, or AV-600 spectrometers operating at working frequencies of 300, 600 ( 1 H), 75, 126, or 151 MHz ( 13 C). Chemical shifts were related to that of the CHCl 3 ( 1 H), or CDCl 3 ( 13 C). High-resolution mass spectra were recorded on a Bruker MicroTOF II instrument in positive ion mode (capillary voltage 4500 V) using electrospray ionization (ESI) and methanol or acetonitrile as a solvent. Elemental analysis was performed on a PerkinElmer Series II 2400 Elemental Analyzer. All reagents were obtained from commercial sources and used without additional purification. All operations, except for chromatography, were carried out in argon atmosphere. Starting compound 1 was synthesized as described in literature [18].

General procedure for the synthesis of compounds 2a,b and 3 (Scheme 1)
Compound 1 (0.35 g, 1.61 mmol) was dissolved in 20 ml of dry DMF. To the resulting solution, powdered KOH (0.5 g, 8.91 mmol) was added in one portion. After stirring at r.t. for 15 min, the corresponding alkyl bromoacetate (5 mmol, for the synthesis of 2a,b) or isopropyl (p-bromomethyl)benzoate (2 mmol of BrCH 2 C 6 H 4 CO 2 i-Pr for 3) was added, and the reaction mixture was stirred at r.t. for 24 h (for the synthesis of 2a,b) or at 50 °C for 3 h (for the synthesis of 3). Next, an additional portion of alkyl bromoacetate (5 mmol) was added, and the stirring was continued at r.t. for 24 h. The reaction mixture was decanted and quenched with cooled H 2 O (200 ml) with vigorous stirring, followed by extraction with dichloromethane (3 × 50 ml). The combined organic phases were washed with H 2 O (3 × 100 ml), dried with crystalline K 2 CO 3 , and filtered. The solvents were removed in vacuo, and the residue was purified by flash column chromatography (eluted with EtOAc:heptane = 1:10-1:3) to give compounds 2a,b and 3. Spectral data for compound 2b, bright yellow powder, yield 0.63 g (1.41 mmol, 88%), mp. 185-186 °C, coincided with those described in literature [15].

General procedure for the synthesis of compounds 2c-e (Scheme 2)
Compound 2a or 2b (0.51 mmol) was dissolved in 10 ml of dry MeOH (for the synthesis of 2d), 5 ml of dry ethylene glycol (for 2e), or 5 ml of n-butanol (for 2c,f). Next, powdered K 2 CO 3 (0.1 g, 0.72 mmol) was added in one portion, and the reaction mixture was heated under reflux for 40 min (for 2d), 5 h (for 2c,f), or at 100 °C for 1 h (for 2e). After cooling to r.t., EtOAc (30 ml) was added with stirring. The resulting mixture was filtered, the solvents were removed in vacuo, and the residue was purified by flash column chromatography (eluted with EtOAc:heptane = 1:30-1:5) to give compounds 2c-f. Spectral and X-ray data for compound 2c, pale yellow crystals (CCDC 2,017,998), yield 0.17 g (0.44 mmol, 86%), mp. 159-160 °C, coincided with those described in literature [16].  After the addition was complete, the reaction mixture was further stirred at -100 for 20 min. Next, the cooling bath was removed, and 3 ml of a saturated KH 2 PO 4 /H 2 O solution was added dropwise over 1 min. The resulting mixture was stirred for 30 min (the inner temperature reached 0 °C), quenched with H 2 O (30 ml), EtOAc (30 ml), and heptane (20 ml). The organic phase was separated, washed with H 2 O (1 × 50 ml), dried with anhydrous MgSO 4 , and filtered. The solvents were removed in vacuo, and the residue was purified by column chromatography (eluted with EtOAc:heptane = 1:100-1:20) to give compound 4a, bright yellow crystals (CCDC 2,055,900), yield 20 mg (0.06 mmol, 13%), mp. 140-150 °C (decomp., spectral and X-ray data coincided with those described in literature [19]), and compound 4b as yellow crystals, yield 0.14 g (0.38 mmol, 75%), mp. 181-183 °C. IR (KBr)   was added dropwise over 5 min to a cooled (-110 ÷ -105 °C) solution of compound 4b (0.5 mmol) in 30 ml of dry THF, with vigorous stirring. After the addition was complete, the reaction mixture was further stirred at -85 ÷ -80 °C for 30 min. Next, the cooling bath was removed, and 3 ml of a saturated KH 2 PO 4 /H 2 O solution was added dropwise over 1 min. The resulting mixture was stirred for 30 min (the inner temperature reached 0 °C), and the product was isolated by chromatography, analogously as described above (for 4a,b). For X-ray single crystal studies, all compounds were recrystallized by slow solvent evaporation at r.t. from nearly saturated solutions in ethyl acetate/heptane mixture (2:1 v/v).

X-ray data collection and refinement
X-ray diffraction data were collected at 100 K (compounds 2a,e,f, 3, 4b, 5) or 250 K (compound 2d) on a Bruker Quest D8 diffractometer equipped with a Photon-III area-detector (graphite monochromator, shutterless φ-, and ω-scan technique), using Mo K α -radiation (0.71073 Å). The intensity data were integrated by the SAINT program [20] and were corrected for absorption and decay using SADABS [21]. The structures were solved by direct methods using SHELXT [22] and refined on F 2 using SHELXL-2018 [23]. All nonhydrogen atoms were refined with individual anisotropic displacement parameters. Locations of H-atoms of amino (H6A and H6B for 2a,f and 3, H6A, H6B, H6C, and H6D for 2d, H4A, H4B, H9A, and H9B for 2e, H2A and H2B for 4b, H1, H4A, and H4B for 5) and hydroxy (H2 and H6 for 2e) groups were found from the electron density-difference map; these atoms were refined with individual isotropic displacement parameters. Positions of atoms H6A and H6B in 3 were restrained at the distance of 0.85(3)Å from N6. All other hydrogen atoms were placed in ideal calculated positions and refined as riding atoms with relative isotropic displacement parameters. A rotating group model was applied for methyl groups in 5.
The SHELXTL program suite [20] was used for molecular graphics. Displacement ellipsoids are set to the 50% probability level on all figures below (see Electronic Supplementary Material (ESM) for more details on X-ray data collection and refinement).
Crystal data, data collection, and structure refinement details for 2a,d-f and 3, 4b, 5 are summarized in Table 1 and Table 2. Crystal data for compounds 2c (CCDC 2,017,998) and 4a (CCDC 2,055,900) have been previously described in literature [16,19]. Bond distances and angles, as well as additional ORTEP drawings, are presented in ESM for this paper. The structures 2a,d-f and 3, 4b, 5 have been deposited at the Cambridge Crystallographic Data Center with the reference CCDC numbers 2024439, 2,024,440, 2,077,346, 2,077,350-2,077,352, and 2,098,491; they also contain the supplementary crystallographic data. These data can be obtained free of charge from the CCDC via http:// www. ccdc. cam. ac. uk/ data_ reque st/ cif. A geometry optimization was calculated using GAUSSIAN 09 software [24] with the B3LYP/6-31G(d) basis set at the level of DFT theory. Hirshfeld surface analysis was calculated using CrystalExplorer 21.5 [25] and comprised 2D (two-dimensional) fingerprint plots and d norm surface plots [26]. The electrostatic potentials were mapped on the Hirshfeld surfaces using the B3LYP/6-31G(d) basis set using TONTO computational package integrated into CrystalExplorer software [27]. The crystallographic information files (CIF) of the compounds 2a, 2e, 3, 4a, 4b, and 5 were used as input for the analysis.
Nucleophilic heteroaromatic substitution [15,29] of the C2-OCH 2 CO 2 Et group in 2a with methanol or ethylene glycol proceeded on heating in the presence of a catalytic amount of potassium carbonate, to give compounds 2d and 2e, respectively (Scheme 2). The process probably involves coordination of a metal cation [30], as no reaction was observed when K 2 CO 3 was replaced with triethylamine. In the case of n-BuOH, a transesterification [31] of the C6-carboxyethyl group to give the corresponding n-butyl ester became the main competing process, which led to isolation of butyl 7-amino-2-butoxy-3-tert-butyl-8-cyanopyrrolo[1,2-b] [1,2,4]triazine-6-carboxylate 2f in good yield (Scheme 2).

Non-valence interactions
The crystals of the studied compounds are found to be rich in hydrogen bonding, which changes with the substitution pattern in heterocyclic nucleus. For instance, each molecule of compound 2a forms intramolecular H-bonds between one of the hydrogens of an amino group and the carbonyl oxygen in the C6 ring position, as well as the intermolecular N-H···O = C(OEt) bonds with the nearby molecule; the dimers form infinite nearly planar chains along the a axes via H-bonding between the C8-nitrile nitrogen and the second hydrogen atom of C7-NH 2 ( Fig. 1 and Table 5). The chains are held together by non-covalent π-π interactions, the intercentroid distances between stacking rings are in range 3.674 ÷ 3.787 Å. The p-phenylene linker in compound 3 eliminated any intramolecular H-bonds; however, the same tendency to form hydrogen bonds between amino-groups and the ester carbonyls of the nearby molecules as well as N-H···N≡C along the b axes is observed (Fig. 1). The presence of an additional hydroxyl moiety in the side chain of compound 2e allowed the construction of 3D H-bonded infinite framework (Fig. 3). Thus, two nearby molecules of 2e form the shortest hydrogen bonds within the series, with the distance O (6) (Table 5).
The nature and amount of the observed hydrogen bonds were also strongly dependent upon the size of the substituents. Thus, compound 2d form two types of H-bonds: C7-NH···O = C(OEt)-C6 and C7-NH···N≡C-C8 which parameters resemble that of 2a,e (Table 6). On switching to 2f, the bulkier C6-CO 2 n-Bu group considerably hindered the formation of NH···N≡C hydrogen bonds (∠ N(6)-H(6A)···N(7) = 136(2)°), while change to C6-Boc in 2c and 4a,b completely eliminated any intermolecular C7-NH···O non-valence interactions ( Fig. 4 and Table 6). However, this tendency is not observed in the case of compound 5, probably due to the re-distribution of the π-density in the pyrrole and non-conjugated triazine rings. The latter compound showed intermolecular C7-NH 2 ···O = C hydrogen bonds of the same type as in crystals of 2a,e,d, while the infinite chains along the a axes were formed via H-bonding between the C8-C≡N and N2-H (Fig. 4). Sterically accessible triazine ring in 4a was also involved in the intermolecular H-bonding of the type N2···NH 2 ···N≡C ( Fig. 4 and Table 6) and C2-H···N≡C (C2···N = 3.225(3) Å, ∠ C2-H···N = 145.5°).

Hirshfeld surface analysis
Hirshfeld surface analysis (HSA) has proved to be a useful tool for enhancing exploration of the intermolecular interactions in crystals of a wide range of organic molecules [27] including heterocycles [36,37]. To visualize the intermolecular contacts in the studied structures, HSA was performed as an additional method to X-ray diffraction analysis. In compounds 2a, 2e (hydrogen-bonded dimer), The d norm surfaces for 2a, 2e (hydrogen-bonded dimer), 3, 4a, 4b, and 5, H···N and H···O contacts (top) and their overall 2D fingerprint plots (bottom). Blue and red regions are weak and strong interactions, respectively. Isovalues range from − 0.39 to + 1.75 (2a), from − 0.48 to + 1.58 (2e), from − 0.25 to + 1.41 (3), from − 0.38 to + 1.60 (4a), from − 0.47 to + 1.62 (4b), and from − 0.38 to + 1.61 (5) 3, 4a, 4b, and 5, the main contribution to the energy of the non-valent interactions was made by the N···H bonds. For all the analyzed compounds, surface area also included the expected O···H reciprocal contacts (4.2% for 4b, 4.9% for 5, 6.1% for 4a, 11.1% for the dimer of 2e, 11.6% for 3, and 14.1% for 2a), which is consistent with the XRD data. These contacts are shown as colored sections on the graph of d norm surfaces where d norm = d i + d e and red (d norm < VdW radii), blue (d norm > VdW radii), white (d norm = VdW radii) (Fig. 6). The intermolecular energies in crystal packing and the fingerprint plots with d norm surfaces were calculated at B3LYP/6-31G(d) level of theory.
Analysis of the Hirshfeld surface for compound 3 also revealed notable H···H type of short contact between the isopropyl and phenyl groups which was not observed for other compounds. The calculated shortest interatomic distances H i-Pr ···H Ph lied in range 1.625-1.787 Å and were in satisfactory agreement with the experimental X-ray diffraction analysis (1.6898-1.8790 Å). The reciprocal contacts between nearby molecules and the resulting fingerprint plots are shown on Fig. 6.

Conclusions
To summarize, a series of novel 7-amino-3-tert-butyl-8cyanopyrrolo [1,2-b] [1,2,4]triazine-6-(p-phenylene)carboxylates bearing different substituents in the C2 ring position (OCH 2 CO 2 Et, OCH 2 Boc, OMe, OBu, OCH 2 CH 2 OH, H, t-Bu) have been synthesized by alkylation, 5-exo-dig cyclization, and nucleophilic substitution, and their structures were investigated by X-ray diffraction. The experimental results indicated the changes in the electron density distribution, as well as a marked increase in the bond lengths for the sterically hindered derivatives. The crystals of the studied compounds also showed diverse packing modes based on hydrogen bonding, which nature changed with the substitution pattern in heterocyclic nucleus and was successfully investigated by the DFT calculations and Hirshfeld surface analysis.