*3.1. Electronic Properties of Substituents*

The raw data generated in this study and used in statistical analyses are available in the Supplementary Materials. Table 2 presents the cSAR values of the substituents in all studied systems. In the case of amino derivatives, the NH2 substituent in position 6 has more than twice, in the cSAR scale, stronger electron-donating properties than in position 5. In nitro derivatives, the NO2 group in position 5 is more electron-withdrawing than in position 6. Therefore, the substitution position, i.e., the position in relation to the nitrogen atoms in the ring, has a decisive influence on the properties of the substituent. In contrast, the effect of the tautomeric form of uracil is clearly less significant. It is also worth noting that in polar solvents, the characteristic properties of both NO2 and NH2 groups are enhanced, as shown by the difference between cSAR(X) values in the water and gas phase (Δ).

**Table 2.** Values of cSAR(X) (in elementary charge units, *e*) for X = NH2, NO2 groups in the gas phase. Δ indicates a difference between the cSAR(X) values in the aqueous solution (PCM) and the gas phase.


In 5-NH2 derivatives, electron-donating strength of the amino group decreases in the sequence: **u2** > **u5**~**u4**~**u1** > **u3** > **u6**. The clearly lower cSAR(X) for **u6** is a consequence of the rotation of the NH2 group by 90 degrees and the formation of the hydrogen bond, H2N···HO, which is discussed in more detail later in the paper. In this case, the large influence of the solvent on the cSAR(NH2) value is due to the rotation of the NH2 group to more planar conformation with respect to the ring in polar solvents. This strengthens the resonance effect.

In 6-NH2 derivatives, electron-donating strength of the amino group decreases in the sequence: **u3** > **u1**~**u5** > **u6**~**u4** > **u2**. Two systems containing the NH endocyclic group at the *ortho* position, **u3** and **u1**, have the greatest electron-donating properties. An interesting

difference is present between the **u5** form and its rotamers: **u4** and **u6**. Among them, the highest cSAR(NH2) value and the lowest Δ occur in **u5**, where the two OH groups are facing in the same direction. When they are in opposite directions, as in **u4** and **u6**, the value of cSAR(X) is lower, while Δ is higher. This may be due to the differences in the dipole moments in these two cases, as the conformation of the OH groups has a significant impact on the value and direction of molecular dipole moment (Table S1). By far the strongest solvent effect on cSAR(X) among the 6-NH2 derivatives occurs in **u1** and **u3** (highest Δ). These systems also have the highest values of the dipole moment (Table S1). All cSAR(NH2) values in 5-NH2 derivatives are lower than in aniline (0.094), while in 6-NH2 they are higher.

Generally, in all 5-NO2 tautomers, the NO2 group is withdrawing electrons more strongly than in nitrobenzene, where the cSAR(X) is higher, −0.140. Its rotation by 90 degree increases cSAR(NO2) by about 0.4 units. The only exception is **u6**, where a decrease in cSAR is observed; however, this is caused by the hydrogen bonding between the NO2 and *ortho* OH groups. In 5-NO2 systems, electron-withdrawing strength of the nitro group decreases in the sequence: **u3** > **u4**~**u5** > **u2** > **u1** > **u6**. The systems with the strongest electron-withdrawing NO2 groups (**u3**, **u4** and **u5**) have an electron-donating OH group in the *ortho* position, but its hydrogen atom is directed to the endocyclic N atom, so that NO···OH interaction can be expected. When NO···HO interaction is present (5-NO2 **u6**), the electron-withdrawing ability of the NO2 group is the weakest. Again, the greatest variability of cSAR(X) due to solvation occurs in the derivatives with the highest values of the dipole moments (**u1** and **u2**).

In the 6-NO2 derivatives, the cSAR(NO2) values are high, indicating weak electronwithdrawing properties. This is caused by the disturbance of the resonance interactions by ring nitrogen atoms in *ortho* and *para* positions. Weak resonance is also evidenced by a smaller increase in cSAR due to the rotation of NO2 by 90◦ as compared to the 5-NO2 derivatives. This increase is by about 0.2 units, with the exception of **u1** and **u3** where cSAR(NO2) is positive and its change due to rotation is smaller. Electron-withdrawing strength decreases in the sequence: **u2** > **u4** > **u6**~**u5** > **u1** > **u3**. The loss of electron-withdrawing properties (cSAR close to 0.0) of the 6-NO2 group occurs in the **u1** and **u3** derivatives, where the NH group is in the *ortho* position. Thus, apart from the relative position of the endo N atoms and the substituent, the NO···HN through-space interaction has an effect as well. The summary of the cSAR analysis in the form of a bar chart is shown in Figure 3.

**Figure 3.** Values of cSAR(X) (in *e*) for X = NH2, NO2 groups in the gas phase and in the aqueous solution (\_w). The 90◦ in parentheses indicates nitro derivatives where 90◦ rotation around the CN bond was forced.

In most cases, the dependences of cSAR(X) on 1/ε are well approximated by a linear function. The parameters of resulting cSAR (X) = *a*·(1/ε) + *b* functions are summarized in Table 3. The slope value, *a*, informs about the sensitivity of the electronic properties of the substituent in a given derivative to the solvent effect. In general, except for **u6** 5-NH2, large absolute values of the coefficient occur in systems with a large dipole moment, and small ones in systems with a small dipole moment (Table 3 and Table S1). In 6-substituted systems (6-NO2 and 6-NH2), the values of *a* in **u1** and **u3** (*ortho* NH) clearly differ from other tautomers (*ortho* N). This can be attributed to the influence of *ortho* interactions with endocylic N/NH groups. It can be concluded that the repulsive *ortho* interaction, NH···HN for 6-NH2 and NO···N for 6-NO2, causes high sensitivity of the substituent properties to the solvent effect, whereas the attractive interaction causes low sensitivity. A similar effect was observed in adenine and purine derivatives [29,30].

**Table 3.** Parameters of cSAR(X) = *a*·(1/ε) + *b* linear regressions: slopes, *a*, and determination coefficients, *R*2; unit cSAR(X) is *e*.


Properties of the =O/−OH groups of all studied forms of uracil, quantified by cSAR, are shown in Figure 4. Negative values correspond to the electron-withdrawing =O group, whereas positive values to the electron-donating −OH. Both the interactions with the substituent and the type of tautomer can affect the electron-donating (−OH) or -withdrawing (=O) properties of these groups. The electron-withdrawing properties of the =O groups are greater in the amino derivatives than in the nitro derivatives, which is shown by the more negative cSAR(=O) values in the 5-NH2 and 6-NH2 derivatives. In turn, the electron-donating properties of the −OH groups are greater in the nitro than in the amino derivatives. This is due to charge transfer between groups with opposite electronic properties. Global ranges of variation of cSAR are 0.143 for the =O group and 0.107 for the −OH group. The ranges for the =O group in C4 and C2 positions are 0.096 and 0.078, respectively, while the average values are −0.134 for C4 and −0.115 for C2. In the case of the −OH group, the ranges are 0.099 for C4 and 0.103 for C2 positions; the average values are 0.159 for C4 and 0.199 for C2. Thus, the characteristic electronic properties of the −OH group are on average stronger in the C2 position, while those of the =O group are stronger in the C4 position. Stronger electronic properties are accompanied by higher ranges of their variability.

The C2 position of the uracil ring is double *ortho* with respect to the two *endo* N/NH atoms/groups, while the C4 position is *ortho* and *para*. So, two electronegative atoms in the *ortho* position of the −OH group might enhance its electron-donating properties, while diminishing the electron-withdrawing by the =O group. A similar effect of *ortho* N atoms on the substituent properties was observed in our recent studies on nitro and amino derivatives of pyridine, pyrimidine, pyrazine and triazine [33].

**Figure 4.** Values of cSAR(=O) or cSAR(OH) (in *e*) for two groups in (**a**) C4 and (**b**) C2 position of uracil molecule. Data for NH2- and NO2-substituted uracil derivatives in the gas phase.
