*3.2. Geometry*

Analysis of geometry will be focused on the lengths of CN bonds connecting the NO2 and NH2 substituents and the substituted system. As shown in Figure 5a, they vary depending on the substitution position and the tautomeric form. In the case of 5-NH2 derivatives, the shortest CN bond occurs in the **u2** tautomer and the longest in **u6**. The **u2** tautomer is also characterized by the highest electron-donating strength of the NH2 group among 5-NH2 derivatives (Table 2 and Figure 3). In the case of the **u6** tautomer in the gas phase, the NH2 group is rotated by 90 degrees in order to form a H2N···HO hydrogen bond with the OH group in the *ortho* position. This is accompanied by a significant extension of the CN bond, which reaches the length observed for the 5-NO2 group in u6. In 6-NH2 derivatives, CN bonds are shorter than in 5-NH2, which is connected with the strong electron-donating 6-NH2 group. A slightly longer bond relative to other tautomers occurs in **u1** and **u3**. This may be due to the presence of the NH group in the *ortho* position resulting in NH···HN steric interaction.

**Figure 5.** (**a**) The lengths of the CN bonds, connecting the substituent and substituted system in the gas phase and (**b**) differences between their lengths in formamide (the most polar solvent) and in the gas phase. Positive values of Δ*d*CN indicate longer bond in formamide than in the gas phase, while negative values indicate shorter.

In NO2 derivatives, shorter CN bonds are found in 5-NO2 than in 6-NO2 systems. This is in line with the electron-withdrawing strength of the 5-NO2 and 6-NO2 groups. In position 5, the shortest bond occurs in **u6**, where a strong NO···HO hydrogen bond is formed, while the second shortest is in **u3**, in which the NO2 group has the strongest electron-accepting properties among all systems. For 6-NO2 tautomers, clearly the shortest bonds occur in **u1** and **u3**, where the NH group is in the *ortho* position. This results from the attractive NO···HN interaction.

The rotation of the NO2 group in 5-NO2 derivatives causes the elongation of CN bonds, which is related to the disturbance of the resonance effect of the NO2 group. The largest elongation occurs in the u6 5-NO2 derivative. It is caused by breaking of the NO···HO hydrogen bond as a result of NO2 rotation. In the 6-NO2 systems, in four tautomers: **u2**, **u4**, **u5** and **u6** (*ortho* N), the NO2 rotation clearly shortens the CN bond. This is caused by the weakening of through-space repulsive interactions with the *ortho* endocyclic N atom. Thus, the main factor determining the CN bond lengths in the 5-NO2 derivatives is the resonance between the NO2 group and the substituted system, while in the 6-NO2 derivatives it is the *ortho* interaction.

The solvation effect is also reflected in the CN bond lengths. Figure 5b shows the difference in CN bond lengths between the values obtained in the aqueous solution and the gas phase. In NH2 derivatives, a stronger solvent effect occurs in 6-NH2 systems, while in the case of NO2 derivatives, in 5-NO2 systems. This is connected with the greater variability of the substituent's electronic properties in these systems (see, for example, Table 3). Thus, the bond shortening is related to an increase in the characteristic electronic properties of a given substituent, due to the increase in the solvent polarity.

## *3.3. Intramolecular Interactions between Non-Covalently Bonded Atoms*

An important aspect of the interaction between the substituent and the substituted system are through-space *ortho* interactions, which in some cases could already be seen by the cSAR(X) values and CN bond lengths. In order to identify these interactions, the lengths of two NH/NO bonds of the NH2/NO2 groups were plotted against each other (Figure 6). Deviations from the equal length of these two bonds may indicate the existence of an asymmetric through-space interaction. Such plots also provide information about the attractive/repulsive nature of these interactions, based on the location of a point above or below they=x line.

**Figure 6.** Plots between the lengths of the two (**a**) NH and (**b**) NO bonds of the NH2/NO2 groups. The dashed y = x line indicates a symmetry between the bonds. A system where asymmetry is present and the H-bond is detected (**u6** 5-NO2) has been marked appropriately. NH\_1 and NO\_1 are the bonds facing towards the lower atom numbers in the ring (for example, 4 in 5 substitution), while NH\_2 and NO\_2 towards higher numbers (see the atom numbering in Figure 1).

First of all, it should be noticed that the asymmetry in the bond lengths of the NO2 group is about four times greater than that of the NH2 group. Moreover, for the nitro group, the obtained results indicate greater variability of interactions, but as expected in systems with rotated groups, the lengths of both NO bonds are similar. Both repulsive and attractive interactions as well as hydrogen bonds are observed. In the latter case, the systems in which the interaction meets the Koch–Popelier criteria for hydrogen bonding [52] are depicted as H-bonds in Figure 6. Only one system (in the gas phase), visible in the plot, **u6** 5-NO2, fulfills the criteria. Additionally, an increase in the polarity of the solvent weakens the through-space interactions—an increase in the O···H distance and a decrease in O···HO angle, as shown in Figure 7. An interesting system in which, despite the symmetry between NH bonds, there is a strong H-bond is **u6** 5-NH2. In this case, the NH2 group rotates by 90◦, and forms a H2N···HO hydrogen bond. Moreover, the NH2 group in the formamide solution rotates slightly towards the coplanar conformation (76.7◦ dihedral angle) and the H-bond is weakened. This rotation is an interesting example of competition of attractive through-space interactions and the resonance between the group and the substituted system. In the gas phase, the H-bond has a greater influence on the structure, but in the polar solvent, due to the weakening of the H-bond, stabilization by resonance forces the group to be coplanar. The structures of **u6** 5-NO2 and **u6** 5-NH2 are shown in Figure 7.

**Figure 7.** Structures of several systems, in which an interesting intramolecular interaction is present, and geometric data for this interaction (distances in Å).

Based on the potential energy density at the critical point of each hydrogen bond, their energy was calculated from the Afonin equation (Equation (1)). For comparison, the hydrogen bond energy was also calculated using the rotational method [53], i.e., the difference between **u6** and **u5** rotamers. Both methods give similar results (Table 4), especially in the case of stronger hydrogen bonding in **u6** 5-NO2.


**Table 4.** Energies (in kcal/mol) of intramolecular hydrogen bonds in the gas phase calculated by means of rotational method (energy of **u6** minus **u5**) and from the Afonin equation (Equation (1)).

\* Calculated by rotating the OH group in the 4 position by 180◦ with NH2 group frozen in **u6** 5-NH2 conformation (perpendicular relative to the plane of the ring).

Figure 8 shows the energy scan along the dihedral angle between the amino group and the uracil ring plane. The global minimum corresponds to the conformer shown in Figure 7, the minimum near scan coordinate 300 corresponds to the form rotated by 180◦ from the global minimum, so that NH2···HO bifurcated contact is present. Two maxima correspond to forms with close NH···HO contacts (1.956 Å). Rotational barrier height is 5.08 kcal/mol, while the difference in energy between the two minima is 4.16 kcal/mol.

**Figure 8.** Energy scan for rotation of the NH2 group about the CN bond in the **u6** 5-NH2 system, shown in Figure 7.

The NCI analysis, shown in Figure 9, was performed to visualize all non-covalent interactions. In most cases, only weak interactions (green-shaded isosurfaces) are present. However, in systems where the asymmetry of two NH/NO bonds (Figure 6) was high, a blue color can be noticed on the isosurfaces between the interacting atoms. This indicates a stronger attractive character of these interaction. The **u1** 5-NH2 system, which has the highest bond length asymmetry (Figure 6) among the amino derivatives, has very slight blue features on the isosurface between NH and =O, which indicated stronger attractive interaction than in **u2**–**u5** 5-NH2 systems. The intramolecular H-bond in **u6** 5-NH2, discussed earlier, appears as a mostly blue isosurface between H2N and HO. The H-bond in the **u6** 5-NO2 system is so strong that the NCI analysis treats it as a partially covalent interaction, as the hole is pierced through the isosurface along the H···O line. In **u1** and **u3** 6-NO2 systems, some blue accents are noticeable on the isosurface corresponding to the NO···HN contact. Bond critical points of non-covalent interactions were found only in **u6** 5-NH2 and **u6** 5-NO2.

Interestingly, in several nitropurines, NO···HN interactions have a bond critical point [30]. It is possible that this interaction is on the edge of being classified as H-bonding. The reasons are probably low values of O···HN angles (105.6◦ in 1H 6-nitropurine vs. 101.4◦ in **u1** 6-NO2 uracil), which are close to the limit of 110◦ proposed by Desiraju [54], and rather high O···H distances (2.107 Å in 1H 6-nitropurine vs. 2.200 Å in **u1** 6-NO2 uracil).

**Figure 9.** NCI plots for all studied systems (gas phase geometry). Isosurfaces correspond to the value of reduced density gradient function of 0.5. Red shading indicates non-bonding (steric) contacts, green weakly attractive interactions (e.g., van der Waals) and blue strongly attractive interactions (e.g., hydrogen bonding).
