Table 1 show the calculated geometrical parameters for the two studied cyanopyridine derivatives in gas and aqueous solution phases by using the B3LYP/6-31G^* level of theory. The theoretical values were compared with the experimental ones determined by Mefetah et al. for 2-anilino-4,6-dimethylpyridine-3-carbonitrile by using X-ray diffraction method by means of the root mean square deviation (RMSD) [14] . The results show that for both molecules the calculated bond length and angles values are in agreement with the experimental ones with RSMD values for bond length between 0.004 and 0.005 Å while the difference for the bond angles are between 0.7˚ and 0.8˚. Comparing the calculated parameters for the two derivatives with those experimental obtained for 4-cyanopyridine [15] , we observed that the experimental CºN distance in it derivative is 1.137 Å while in the 4-cyanopyridinium dihydrogen phosphate-isonicotinonitrile-phosphoric acid derivative [16] is 1.142 Å and in 3-cyanopyridinium tetraehloroferrate(lll)-3-cyanopyridine is of 1.102 Å [17] .

The results clearly show that this bond is strongly dependent of the groups linked to the pyridine ring and of the position of the C-CºN group. On the other hand, the C-C bond linked to the CºN bond in 4-cyanopyridine is

^aThis work; ^bFrom Ref [38] .

1.439 Å [15] , in 4-cyanopyridinium dihydrogen phosphate-isonicotinonitrile-phosphoric acid [16] is 1.453 Å and in 3-cyanopyridinium tetraehloroferrate (III)-3-cyanopyridine [17] is between 1.452 and 1.480 Å, being the calculated values for (2-OH) and (2-Cl) between 1.428 and 1.422 Å. Thus, these values indicating that this bond is less depending of the position of the C-CºN group and of the groups linked to the pyridine ring. Also, in both derivatives we observed that the C1-N7 distances are slightly different from the C5-N7 distances indicating that both pyridinic bonds are dependent of the groups linked to the C1 or C5 atoms belonging to the C-N bonds, these are, in the (2-OH) derivative, C1-O18 and C5-CH₃ while in the (2-Cl) derivative are C1-Cl18 and C5-CH₃. Experimentally, the C-N distance in the symmetric molecule of 4-cyanopyridine [15] is 1.331 Å, in 4-cyanopyri- dinium dihydrogen phosphate-isonicotinonitrile-phosphoric acid [16] are 1.339 and 1.337 Å and, in 3-cyanopy- ridinium tetraehloroferrate(III)-3-cyanopyridine [17] those distances are between 1.301 and 1.351 Å. The differences observed in the latter molecule are justified because two molecules are linked in asymmetric form by the N atoms of both pyridine rings. Also, in the (2-OH) and (2-Cl) derivatives (Table 1) the two C-N distances are different because both molecules are asymmetrics. In aqueous solution, we observed that in both derivatives some distances slightly change with the hydration, being more evident in the C1-O18 and C1-Cl18 distances because both are sites of H bonds formation. In the (2-OH) derivative, the C-OH site is an electrophilic region while in the (2-Cl) derivative the C-Cl site is a nucleophilic region. On the other hand, Table S1 show a comparison of the total energies and the corresponding dipole moment values for both derivatives in the two media by using the B3LYP/6-31G^* method. Note that in both media the dipole moments for the (2-Cl) derivative are higher than the other ones, as expected because the Cl atom is a voluminous atom. For this reasons, the calculated molecular volumes for the (2-Cl) derivative in both media by using the Moldraw program [35] and the B3LYP/6-31G^* method are higher than the other ones, as observed in Table 2. In the (2-Cl) derivative, clearly there is a volume contraction with the hydration while in the (2-OH) derivative is observed a volume expansion, in agreement with the solvation energies values, as can be seen in Table 2.

The uncorrected solvation energies (DG_u), calculated as relative energies (DE) and defined as the difference between the total energies in aqueous solutions and the values in gas phase for the (2-OH) and (2-Cl) derivatives using the 6-31G^* basis set, are presented in Table 2. Here, the uncorrected (DG_u) and corrected (DG_c) solvation energies together with the total non electrostatic terms (DG_ne) due to the cavitation, dispersion and repulsion energies were calculated by using the PCM/SMD model [31] , in form similar as reported for other molecules in aqueous solution [32] -[34] . Note that for the (2-OH) derivative a higher DG_c value is obtained, probably due to its higher variation of volume in solution (Table 2), for this reason, a higher hydration is expected for this derivative in aqueous solution.

The molecular electrostatic potential values for both derivatives calculated in the two media by using the B3LYP/6-31G^* method are given in Table S2. In (2-OH), the most negatives molecular electrostatic potential values are observed on the N7, N17 and O18 atoms and the less negative on the H19 atom belonging to the OH group while in (2-Cl), the most negatives values are observed on the N7, N17 and Cl18 atoms and the less negative on the H atoms. Note that in both molecules the N17 atom have higher values than the N7 ones, for these rea- sons, strong red colorations are observed on the surfaces mapped of both derivatives, as observed in Figure S1, indicating in both derivatives those regions reacting with potential biological electrophiles. These colorations indicate probably capability of hydrogen bond formation with share of nitrogen atoms of the CºN or, in aqueous solution through hydrogen atom of -OH moiety. On the contrary, on the H19 atom a strong blue coloration is observed on the surfaces mapped of (2-OH) being this region reacting with potential biological nucleophiles. Another important result is the lower molecular electrostatic potential values observed on the H atoms of both

DG_c = DG_uncorrected^# − DG_{Totalnon electrostatic}; ^aThis work.

CH₃ groups in the chlorinated derivative in the two media in relation to the other one. These results justify the higher blue coloration on the surface mapped of those groups in the chlorinated derivative, as observed in Figure S1. In the (2-OH) derivative is expected a high reactivity due to the higher proximity between the N17 and O18 atoms (higher repulsion), whose distances in gas and aqueous solution phase are respectively of 3.559 and 3.496 Å while in the chlorinated derivative the N17-Cl18 distances in gas and aqueous solution phases are respectively of 3.743 and 3.698 Å. Note that in aqueous solution increase the reactivities of both derivatives due to the shortening in the distances between both involved atoms. The atomic charges derived from the ESPs (MK) [36] and the NPA charges by using the B3LYP/6-31G^* method were calculated for both derivatives in the two media, as can be seen in Table S3. The two charges show that despite the only difference between both derivatives are the presence of the OH group and the Cl atom the values are strongly dependent of the groups linked to the pyridine ring and of the position of the C-CºN group, as was also observed in the Section 3.1. These charges analysis also show that the (2-OH) derivative is less stable than the other one due to the higher charge values on the N17 and O18 atoms. Moreover, the different charge values observed on the N7, N17, O18 and Cl18 atoms suggest for the two derivatives a different hydration in solution. The bond orders expressed by Wiberg’s indexes for the two derivatives in both media are observed in Table S4. Note that the change of the OH group in (2-OH) by the Cl atom in (2-Cl) increase the bond order values for the N7, N17 and all the C atoms of the pyridine ring while decrease the bond order values of the H and C atoms belonging to the two CH₃ groups, thus, the pyridine ring has in gas phase a higher stability in the chlorinated derivative than the other one. Note that the bond order values in both derivatives practically not change in aqueous solution and only a little decreasing is observed in the bond order corresponding to the Cl atom with the hydration. This fact, in (2-Cl) is probably related with the contraction volume observed in aqueous solution.

The stability of both derivatives were studied by means of second order perturbation energies E⁽²⁾ (donor à acceptor) whose values are given in Table S5. Here, we considered only those contributions higher than 20 kJ/mol. These results for the (2-OH) and (2-Cl) derivatives show three different contributions to the total stabilization energies, which are, the DET_p®p*, DET_LP®s* and DET_s*®s* charges transfer, being the two latter interactions higher in the (2-OH) derivative than the other one while, the π-π interactions in the pyridine ring are higher in the chlorinated derivative. These results show that the calculated total stabilization energy favours to the (2-OH) derivative revealing thus a higher stability for this derivative in gas and aqueous solution phases. Here, it is very important to note that in the (2-OH) derivative the significant increase in the delocalization values in aqueous solution is related with the increase of the C-C and C-N double bonds of the pyridine ring as consequence of the hydration. This way, this analysis shows clearly that the DET_π-π interactions in the pyridine ring are higher in the chlorinated derivative and in both media while, in general, the DE_Total are higher in the (2-OH) derivative than the other one.

Both derivatives were also studied employing the AIM analysis [25] [26] . For the (2-OH) and (2-Cl) derivatives, the charge electron densities, (r) and the Laplacian of the electron densities, Ѳr(r) were calculated for the ring critical points (RCPs) belonging to the pyridine rings and the values can be seen in Table S6. The results show higher values in the topological properties for the chlorinated derivative and, also a slightly difference for this derivative with the hydration. Thus, the analysis clearly shows a higher stability for the pyridine ring of the chlorinated derivative in both media, contrarily to that result obtained by NBO analysis.

Many cyanopyridine derivatives have potentials antimicrobial and anticancer activities, for this reason, and to determine the exact nature of the interactions with electrophones and/or nucleophiles and, also to predict the behavior of both derivatives in gas and aqueous solution phases are very important the calculations of some descriptors. If both derivatives are used for the drugs design, the knowledge of these descriptors helps to understand the structural, dynamical, and functional properties of each derivative in both media. Thus, for both derivatives, the HOMO and LUMO orbitals, energy band gap, chemical potential (μ), electro negativity (χ), global hardness (η), global softness (S) and global electrophilicity index (ω) descriptors [18] [19] were calculated. These descriptors calculated in both phases at B3LYP/6-31G^* level of theory can be seen in Table S7. Comparing first the energy band gaps between both derivatives we observed that the chlorinated derivative has the highest values in both media indicating a higher stability for this molecule than the other one. Note that the OH group in the pyridine ring generate the reduction of the HOMO-LUMO gap indicating that in the (2-OH) derivative that group deactivating the ring and diminishing its potency when it is used as a drug. On the contrary, the presences of a Cl atom in the pyridine ring in (2-Cl) compared with (2-OH), active that ring and increase its potency. Comparing the calculated chemical hardness η, chemical potential m and global electrophilicity index ω values for (2-OH) with those obtained for (2-Cl) in both phases, we observed that (2-Cl) is more stable in both media (larger η) and has better capability to accept electrons (bigger electrophilicity index) than (2-OH) but, on the contrary, (2-OH) is better electrons donor than (2-Cl). Here, the lowest molecular electrostatic potentials observed on the H atoms of both CH₃ groups for the chlorinated derivative support the better capability to accept electrons while, the highest molecular electrostatic potentials observed on the N7 and N17 atoms of (2-OH) suggest that it derivative is better electrons donor than (2-Cl).

Experimental and calculated chemical shifts with the GIAO method using 6-311++G^** basis set for the ¹H and ¹³C nuclei of both derivatives are compared in Table S8 and Table S9 respectively. In general, the calculated shifts for the ¹³C nuclei are lower than the corresponding experimental values. Note that the calculated chemical shifts for the H nuclei of (2-OH) show a significant variation (0.81 ppm) than the (2-Cl) derivative (0.22 ppm), in relation to the corresponding experimental values [2] . Probably, the theoretical calculations do not correctly predict the hydrogen chemical shift of the H19 nucleus belonging to the OH group of (2-OH), as observed in Table S8, because that group is involved in intermolecular H-bonds. Thus, the higher hydration of this derivative is supported by the higher solvation energy in aqueous solution. Table S9 show that the calculated ¹³C chemical shifts for both derivatives are slightly different between them, as expected due to the different (OH and Cl groups) present in each structure.

OH modes. The broad and intense band in the IR spectrum of (2-OH) centred at 3350 cm⁻¹ and predicted in aqueous solution at 3523 cm⁻¹, is assigned to the O-H stretching as observed in compounds containing this group [34] [41] -[44] . The OH in plane deformation mode is assigned to the strong band at 1225 cm⁻¹ while the corresponding out-of-plane deformation mode is associated to the band at 495 cm⁻¹.

CH₃ modes. The IR bands between at 2980 and 2850 cm⁻¹ are assigned to the CH₃ antisymmetric and sym- metric stretching modes while the bands between 1465 and 1360 cm⁻¹ are clearly assigned to the ant symmetric and symmetric CH₃ deformation modes. The four expected rocking modes are assigned to the shoulder and

bands observed between 1095 and 1025 cm⁻¹. The twisting modes were not assigned because are predicted at 66 and 41 cm⁻¹.

C-CºN modes. Here, the IR band at 2220 cm⁻¹ is assigned to the CºN stretching mode while the strong band at 720 cm⁻¹ is assigned to the C2-C16 stretching mode, as observed in Table 3. The bending and out-of-plane deformation modes corresponding to the C2-C16 group are predicted at 236 and 138 cm⁻¹ and, for this reason, they were not assigned.

Skeletal modes. The C-N stretching modes corresponding to the pyridine ring are predicted by the calcula-

tions in different regions. Hence, the IR bands at 1480 and 1140 cm⁻¹ are associated respectively with those two C5-N7 and N7-C1 stretching modes while the strong band at 1375 cm⁻¹, is associated with the C-O stretching mode. Here, it is necessary to note that in aqueous solution there is a very important shifting in the wave numbers corresponding to those stretching modes, thus, the N7-C1 and C5-N7 stretching modes are predicted respectively at 1486 and 963 cm⁻¹, as observed in Table 3. This fact is related with the higher hydration of the (2-OH) derivative and with the increasing in the corresponding force constant values with the hydration as consequence of the increasing in the N7-C1 double bond character, as we will see later. Finally, the observed torsion and deformation modes of the pyridine ring are assigned as predicted by calculations and taking into account the assignments for similar molecules [45] -[49] . In this work, those modes are identified and assigned, as observed in Table 3.

CH₃ modes. As in the (2-OH) derivative, the CH₃ ant symmetric and symmetric stretching modes are assigned between 3010 and 2975 cm⁻¹ while the ant symmetric and symmetric CH₃ deformation modes bands are clearly assigned, as predicted by calculations, to the strong band at 1440 cm⁻¹. The four expected rocking modes are assigned to the bands between 1040 and 1000 cm⁻¹. In this derivative, the twisting modes were not assigned because both are predicted at 77 and 57 cm⁻¹. It is important to note that in this derivative the presence of the Cl atom in the structure shift the bands toward lower wave numbers, as observed in Table 3.

C-CºN modes. Here, the CºN stretching mode is assigned to the IR band at 2225 cm⁻¹ while the weak band at 700 cm⁻¹ is assigned to the C2-C16 stretching mode, as predicted by calculations.

In this derivative, with the hydration only is observed a shifting in the wave numbers related to the C5-N7 stretching mode, as indicated in Table 3. In (2-Cl), the C2-C16 bending mode is assigned to the weak band at 615 cm⁻¹ while the corresponding out-of-plane deformation mode is predicted at 241 cm⁻¹ and, for this, it mode is not assigned.

Skeletal modes. In this derivative, the C-N stretching modes corresponding to the pyridine ring are predicted by the calculations in the same regions, thus, both modes were assigned to the band and shoulder respectively at 1260 and 1250 cm⁻¹. The C-Cl stretching mode is predicted by calculations at 448 cm⁻¹ and assigned at 440 cm⁻¹. The bending and out-of-plane deformation modes corresponding to the C1-Cl18 group are predicted at 230 and 160 cm⁻¹, hence, these modes were not assigned. Finally, in accordance with similar molecules [41] -[49] the torsion and deformation modes corresponding to the pyridine ring are assigned as predicted by calculations, as observed in Table 3.

n, stretching; d, scissoring; wag and g, wagging or out of plane deformation; r, rocking; t, torsion, twist, twisting; a, antisymmetric; s, symmetric; R, ring; ^aThis work, ^bFrom Ref [38] , ^cFrom Ref [39] , ^dFrom scaled quantum mechanics force field B3LYP/6-31G^*, ^eFrom scaled quantum mechanics force field PCM/B3LYP/6-31G^*.