Synthesis and Crystal Structure of Benzyl 4-(2-uoro-4-(triuoromethyl)phenyl)- 2,6,6-trimethyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate and Its DFT Analysis Combined with Bond Order Modeling in Terms of The Bond Critical Point Quantities

Inammation is the underlying cause of many diseases such as cardiovascular diseases, cancer and autoimmune diseases. Recently 1,4-dihydropyridine (1,4-DHP) compounds were found effective to reduce inammation which contributes to development of inammation associated diseases. Based on these data we synthesized to investigate this type of action of annulated 1,4-DHP molecule, benzyl 4-(2-uoro-4-(triuoromethyl)phenyl)-2,6,6-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate and proved the structure of this molecule by IR, 1 H-NMR, 13 C-NMR, HMRS and X-ray crystallography. X-ray analyses were conducted to nd out the exact 3D structure of the mentioned molecule. The molecular structure crystallizes in triclinic space group, P-1, with a = 7.0889(11) Å, b = 12.4861(18) Å, c = 14.338(2) Å, α = 66.899(4)°, β = 89.025(4)°, γ = 85.101(4)° and V = 1162.9(3) Å3. In the title molecule, C 27 H 25 F 4 NO 3 , the cyclohexene ring is in a sofa conformation and the 1,4-dihydropyridine ring is in a slight boat conformation. In the 2-uoro phenyl and benzyl rings form a dihedral angle of 13.6(1)°. In the crystal structure stabilized by the intra- and intermolecular N—H···O, C—H···O and C—H···F interactions. The molecules are linked together to form a dimer by N(1)—H(1N) ···O(1) i and C(2)—H(2A) ···O(1) i hydrogen bonds [symmetry code: (i) x+1,y,z ], producing two R 12 (6) rings. QTAIM, bond order, molecular planarity and molecular surface analyses have been performed on the optimized geometry by DFT. Considering the quantities obtained at the bond critical poins, the chemical bonds are discussed for classication. The correlation between bond critical point quantities and the bond orders based on different denitions have been explored considering different bond order models from the literature. The Laplacian Bond Order (LBO) gives best correlation for four of ve bond order models. All the bond order models with an exception of the model with parameter G have approximately same correlation degree for C-C bonds. For C-H bonds, only bond model with parameters of electron density and the principle curvatures for LBO gives relatively good correlation with R 2 value of 0.943. The two phenyl rings of the structure have aromaticity comparable to benzene as deduced from QTAIM quantities and molecular planarity metrics. As a result of molecular surface analysis, the mass density, molecular polarity index, v (the measure of charge balance), σ 2 tot .v (measure of intermolecular interactions) were calculated and compared with literature values.

Introduction 1,4-DHP scaffold which rstly discovered by Arthur Hantzsch has diverse biological activities depending on their effectiveness on different calcium channel types. Calcium channel modulators operate calcium entry through cells and by that cause cardiological bene ts. As a result of this pharmacological action, they are widely used for the treatment and prevention of cardiovascular diseases [1,2].
Chronic in ammation plays an important role of development of several in ammatory diseases.
In ammatory stimuli initiates signaling pathways those activate production of in ammatory mediators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Receptor activation by those mediators induce signaling pathways such as nuclear factor kappa-B (NF-κB), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT). Dysfunction of NF-κB, MAPK, or JAK-STAT activity is related with in ammatory, autoimmune and metabolic diseases, and cancer. Thus inhibiting those pathways are an promising approach to prevent previously mentioned in ammatory associated diseases [3,4].

Instrumentation
All chemicals used were purchased from Aldrich (USA) and were used without further puri cation. Purity of the synthesized compound was checked by thin layer chromatography (TLC) on Merck aluminum sheets (Germany), silica gel 60 F254, mobile phase ethyl acetate:n-hexane (1:1), and UV absorbing spots were detected under short-wavelength (254 nm) light (Camag UV Cabinet, Germany). Melting points were determined on a Thomas Hoover capillary melting point apparatus (USA) and uncorrected. Infrared spectra (IR) were recorded on Perkin Elmer Spectrum BX FT-IR (UK) equipped with MIRacle ATR accessory (PIKE Technologies, USA) and were reported in cm −1 . 1 H-NMR and 13 C-NMR spectra were taken in dimethyl sulfoxide (DMSO-d 6 ) solution on a Bruker Ultra Shield Spectrophotometer at 400 MHz using tetramethylsilane (TMS) as internal standard. Chemical shifts were reported in parts per million (ppm).
HRMS was realized by Agilent 6530 Accurate-Mass Q-TOF.
Computing details.
The geometry optimization of the molecule has been done the hybrid-GGA functional B3LYP [19] with standard basis set 6-31G(d,p). The an initial geometry input for the optimization is taken from the analysis of x-ray structure data. There was no restriction applied during the optimization. The vibrational spectrum of the optimized geometry has been calculated to make certain of minimum in potential energy surface by checking absence of imaginary frequency. The optimization and the calculations of vibrational spectrum together with some other properties of the optimized molecule were carried out by Gaussian 09 program package [20]. The properties such as bond orders, molecular surface quantities, partial charges, the parameters of the topology analysis have been calculated by Multiwfn 3.8 [21] using the checkpoint le obtained from the geometry optimization.

Results And Discussion
In this study, benzyl 4-(2-uoro-4-(tri uoromethyl)phenyl)-2,6,6-trimethyl-5-oxo-1,4,5,6,7,8hexahydroquinoline-3-carboxylate has been synthesized by Hantzsch reaction. According to this reaction mechanism aldehyde and β-ketoester formed an intermediate. This intermediate was converted to the target compound via ammonium acetate as nitrogen source. The structure of the compound was proved by using IR, 1 H-NMR, 13 C-NMR, HRMS and X-ray single-crystal diffraction. Detailed X-ray analyses were realized for further elucidation of the certain structure. The absorption bands of 3296 cm −1 , 1698 cm −1 and 1649 cm −1 veri ed nitrogen of the hexahydroquinoline ring, ester and ketone groups, respectively. In 1 H-NMR spectra of the compound, singlet signals belonging to -CH 3 group at 2nd position were seen at 2.27 ppm. Signals belonging to aromatic protons were seen at between 7.09-7.12 and 7.25-7.37 ppm.
Singlet signal belonging to N-H proton was seen at 9.27 ppm which indicates closure of the hexahyroquinoline ring. The peaks belonging to other protons were determined in expected values of 1 H NMR spectra. The peaks in the 13 C-NMR spectrum are consistent with the expected chemical shift values for the carbons in the molecule. In this context, C=O peaks in the 13 C-NMR spectrum have expected values for an ester and a ketone, 166.2 ppm, 199.2 ppm, respectively. The HRMS spectra of the compound were recorded using the electrospray ionisation-Q-TOF technique. Molecular ion peak (M+H) also base peak was seen in the spectra.
Selected bond lengths and angles are listed in Table 1. The molecular bond lengths and bond angles are in good agreement with related structures [22][23][24][25]. The (C1-C6) cyclohexene ring is in a sofa conformation with puckering parameters [26] (Table 3 and Fig. 2), producing two R (6) rings [27] into chains parallel to the b axis. In the crystal structure stabilized by the intra-and intermolecular N-H···O, C-H···O and C-H···F interactions.

Theoretical analysis
The optimized structure of the molecule is given in Figure 3. Some of the selected experimentally determined geometrical parameters are also given along with the calculated ones in Table 2. As seen from the gures ( Fig. 1 and 3) and the table, there is an general agreement between the calculated and experimentaly obtained geometrical parameters. There are some deviations on the dihedral angle values possibly due to geometry optimization in gaseous phase different from the experiment. In the discusion of theoretical analysis, the labels given in Fig. 3 are preferred to be used. Frontier orbitals are used to make predictions about the molecular properties. The HOMO and LUMO energy levels are calculated to be -5.817 and -1.635 eV, respectively. That is why the energy gap is equal to 4.183 eV, which can be used to compare its kinetic stability with similar molecules as done for other molecules [28]. HOMO and LUMO orbitals are shown in Figure 4. As seen from the gure, they are mostly located on and around the pyridine ring. The neighbouring phenyl ring connected to pyridine has a higher contribution in the case of LUMO. As seen from the gure, part of the orbitals located on the pyridine ring exhibits pi character.  The dipole moment of the molecule is calculated to be 6.854 Debye, as expected qualitatively from its asymmetric structure. This asymmetric charge distribution results in bonding regions and atoms. The partial charges accumulated in atoms can be calculated by using different charge models. The natural charges obtained as a result of NBO analysis [29] are given in Table 4. Partial atomic charges strongly depend on electronegativity and its environment. values ranging between -0.719 (C20) and 1.129 (C35). C20 is the methyl C which is bonded to C atom so it has strongly electropositive environment. On the other hand, C35 has a strong electroneative environment since it is C atom of -CF 3 group. There is one nitrogen bonded to two C atoms and having partial charge of -0.587 as expected. O atoms have very narrow partial charge range between -0.554 and -0.630, they are all connected to C atoms. There are four F atoms and orine is most electronegative known. However, the charges of orine atoms are between -0.318 and -0.368, which are less negative than some of the C atoms, all the O atoms and the N atom. Each F atom in -CF 3 has approximately the same partial charge value. However the F atom which is bonded to phenyl ring has relatively small negative charge value. Topological analysis of title molecule has been performed by means of QTAIM (quantum theory of atoms in molecules) [30]. QTAIM is one of the most powerfull tools to analyze chemical bonding [31]. Total 137 critical points have been obtained: There are 60 nuclear critical points associated with 60 nuclei, 68 bond critical points (63 of them is associated with chemical bonding and 5 of them correspond to nonbonding interactions) and nally 9 critical points associated with the rings formed with chemical bonding and nonbonding interactions. So, Poincare-Hopf relationship has been veri ed with the following equation: n NCP -n BCP + n RCP -n CCP =60 -68 + 9 -0 = 1 For that reason it may be assumed that all the critical points are found considering this veri cation [21b].
According to QTAIM, atomic nuclei behave as an attractor which electron density gets a maximum [32]. The electron density values corresponding to nuclear critical points were obtained within the range between 0.422 a.u. (H atom) and 419 a.u. (F atom). The electron density at nuclei of free atoms is approximately found to be proportional to Z 3 by Hartree-Fock approximation results [30]. This relationship is also seen approximately from these values. However, electron density value changes depending on molecular environment.
As stated before, 68 bond critical points have been obtained and they are given with some quantities calculated in Table 5 one is closer to 180 o , its distance is longer than that of the rst one. Therefore, the rst one is very high in strength with respect to second one. is less than 0.10 a.u. [32]. As given in Table 3, all the chemical bonds can be classifed as covalent due to their electron densities at the BCP. Its value vary between 0.236 a.u. ( C22-O7 ) and 0.405 a.u. ( C21-O6 ). O7 is bonded to two C atoms so that it is expected that it is approximately single bond since C21-O6 bond is expected to be double bond due to fact that it is only bonded to C21. Bader proposed bond order equation making a analogy with Pauling's relationship as follows: where a and b are the coe cients which can be determined considering single, double, triple C-C bond crtical point electron densities. By tting this equation, they are determined to be 6.862 and 0.243, respectively [30][31][32][33][34].The value of b is approximately equal to the single C-C bond critical point electron density. The ndings of this study for two C-O bonds given above are consistent with this eqaution. The lowest electron density value (0.237 a.u.) is obtained for C12-C19 which is clearly single bond because three hydrogen atoms bonded to C19. C9-C14 has the highest electron density value (0.333 a.u.), which seems to have the highest bond order. This is also reasonable considering the atoms bonded to atoms of this pair. The bond order of C-C bonds for the phenyl ring C29-C30-C31-C32-C33-C34 should be around 1.5. Their electron densities have a quite narrow range between 0.310-0.321 a.u.. This shows its aromaticity comparable to benzene.
The negative ratio of the kinetic to potential energy densities, -G(r)/V(r) can also be used to examine bonding character [35,36]. If this ratio is less than 0.5, then it is expected to covalent bonding. The value between 0.5 and 1.0 indicates partially ionic bond [35]. The values between 0.095 and 0.524 have been obtained for the bonds of the title molecule. C-O and C-F bonds have the values around 0.5 which is the border between covalent and partially ionic bond. This may be attributed to electronegativity differences between bonding atoms. H(r)/ρ(r)is de ned as Bond degree (BD) by Espinosa et al [37]. The more negative BD value indicates the stronger bond. In the table, the lowest BD value belongs to C21-O6 bond which has the highest electron density as well. Its value is -1.712 a.u. Both the electron density and bond degree indicate that this bond is the strongest bond. Depending on the sign of the Laplacian, ∇ 2 ρ(r), the type of bonding may be estimated also. The Electron Localization Function (ELF) [38] has a value between 0.0 and 1.0. The larger ELF value indicates more localized electrons which is a sign of a covalent bond, lone pair etc. As seen from The relationships between covalent bond order de ned by Cioslowski et al. [39] and the parameters derived from a topological analysis of the electron density have been explored by Howard and Lamarche [34]. They have studied the bonds between C and ve different elements (C, N, O, S, and P). The bond order values have been tted to equations including the electron density (ρ), the square of the electron density (ρ 2 ), the Laplacian of the electron density (∇ 2 ), the principle curvatures ( λ 12 (=λ 1 + λ 2 ) and λ 3 ), the kinetic energy density (G). As a result, the multiple linear description with ρ, λ 12 and λ 3 was concluded to recommend, since it worked well for all the bonds considered. In this study, the four bond models and Bader bond model considered in the study of Howard and Lamarche [34] have been used to study C-C and C-H bonds in the title molecule. Other bonds were not studied due to lack of su cient data for tting in the title molecule. Different from their study considering bond order de nition by Ciolowski et al., the four different bond order de nitions (MBO (Mayer Bond Order) [40], FBO (Fuzzy Bond Order) [41], LBO (Laplacian Bond Order) [42], IBSI (Intrinsic Bond Strength Index) [43]) have been considered to t the equations of four different bond order models. The tting was done using regression LibreO ce 7.1 Calc [44]. The coe cients obtained by tting are tabulated in Table 5.2. Among the bond order de nitions, LBO gives the best R-squared values (greater than 0.990) for the bond order models 1-3 and Bader bond order model. Only the bond order model 4 which is de ned by only the kinetic energy density (G) get the highest R-squared value for bond order de nition other than LBO. Average R 2 values for each bond order model is also given in the same table and they are approximately the same for the rst three models. The weakest correlation in average was obtained for Bader model. Although this model gives the weakest correlation in average, its R 2 value is good enough for LBO. In general, R-squared values of the LBO obtained in this study are better than that of the values obtained by Howard and Lamarche [34]. The best value among the all models and bond order de nitions was obtained for model 2 for LBO, although its R 2 value is little bit larger than Model 1 and 3 with a negligible extent. Model 2 is the recommended in the study by Howard and Lamarche [34]. They recommended this model due to their well working for all the cations, anions, neutral and charged radicals. Its elagent and simple physical interpretation play also important role for its recommendation, since ρ and λ 3 are the measure for the σ character and the curvatures perpendicular to the bond (λ 1 and λ 2 ) measure the degree of π character. As a result, it can be said that LBO has better correlations with the electron density (ρ), the square of the electron density (ρ 2 ), the Laplacian of the electron density (∇ 2 ), the principle curvatures ( λ 12 (=λ 1 + λ 2 ) and λ 3 ) with respect to other models. The model 4 which is exceptionally not best for LBO includes only the kinetic energy density (G) as a parameter and the best result is obtained for MBO with the R 2 value of 0.971. As stated before, there are 9 critical points associated with the rings formed with chemical bonding and nonbonding interactions. There are four rings formed as a result of chemical bonding completely. The quantities that belong to these rings are tabulated in Table 5 can be established [45]. QTAIM analysis of benzene optimized at the same theory level with title molecule has been done to get reference values for comparison of aromaticity. ρ(Γ BCP ) and ∇ 2 ρ(Γ BCP ) were calculated to be 0.020 and 0.163 a.u., respectively, for benzene. The curvature of electron density perpendicular to ring plane at its RCP was calculated to be -0.015 a.u. As the values for these quantities are the same with the values obtained for phenyl rings of the title molecule, these rings are conclude to have the same aromaticity leveel with the benzene. On the other hand, the other two rings have aromaticty lower than the phenyl rings, when the values are compared. In addition to QTAIM analysis, molecular planarity analysis based on two metrics proposed by Tian Lu [46] has been done for these rings. These two metrics, to characterize planarity of molecules quantitatively are molecular planarity parameter (MPP) and span of deviation from plane (SDP). These parameters are complementary to each other [46]. MPP and SDP values are given in the same table, both of them changes in parallel. As they get closer to zero, they tend to get more planarity. Two phenyl rings have the highest planarity. This is an expected result, since planarity is known to be important factor for aromaticity [47].  [48]. The van der Waals (vdW) surface of the molecule was used for performin the ESP analysis with the default value of 0.001 a.u. isosurface of electron density [49]. A general interaction properties function (GIPF) proposed by Murray et al. can be used to determine several properties by the quantites obtained from molecular surface analysis. Π (a measure of local polarity), σ 2 tot , (a measure of the variability of the potential on the surface), v (a measure of the balance between positive and negative regions) are used in their function [50]. The quantities obtained from this analysis are given Table 6.
Density values can be calculated by using expression M/V, where M is molecular mass and V is the van der Waals volume [51]. The density value calculated in this way is 1.49 g/cm 3 which is very close to experimental value 1.392 g/cm 3 given in Table 1. This calculation has been improved making electrostatic interaction correction by Politzer et al. [52]: Table 6 Quantities obtained from molecular surface analysis. where α = 0.9183, β = 0.0028 and =0.0443. In this equation, the second factor is the product of σ 2 tot and v is proposed to make correction for intermolecular interaction through surface electrostatic potentials in the solid phase. The values of σ 2 tot .v were obtained within a range of 0.5 and 62.5 kcal 2 /mol 2 for the molecules studied by Murray et al. [50]. The value for the title molecule is 43.4 kcal 2 /mol 2 which is higher than mean value considering this study. The parameters in the equation were obtained using the crystals containing C, H, N, O [50]. Although one of the elements of the title compound, F was not included and B3PW91 functional was used in this t, it may be meaningful to calculate the density of the title compound by using this equation to make comment on the effect of intermolecular interaction. By this equation, the density was found to be 1.53 g/cm 3 which has more deviation than previous result from experimental value. Of course, the increase does not come from the intermolecular interactions, since the coe cient of M/V is reduced to 0.9183. However, the equation shows that the density increase with an increase of interaction which results in the reduction of intermolecular space. A larger deviation probably comes from the extra atom which was not included and the different functional was used in the tting [52].
As stated before, v is the measure of charge balance between positive and negative regions. Its value get maximum value of 0.250 as in our results, if σ 2 tot for the positive and negative regions are equal. These values are aprroximately equal for the title molecule, so the molecular interaction with a similar extent through both its positive and negative regions may be concluded as stated in original study [50]. Π gives us a measure of local polarity (or charge separation) for a molecule. So, very large values may be obtained even for a molecule having a zero-dipole moment. It has values between 0 and approximately 20 kcal/mol for the molecules studied in the original reference [50]. It is obtained to be 11.2 kcal/mol for our molecule as given before. This value indicates that the title molecule has moderate value of local polarity considering the molecules in the original reference. For a similar purpose to quantify molecular polarity due to uneven ESP distribution, another index, MPI (the molecular polarity index) is de ned by Tian Lu and coworkers [53]. In their study, the calculated MPI values of the cyclo [18]carbon, ethane, ethene, and benzene are obtained to be 2.6, 2.6, 6.7, and 8.4 kcal/mol, respectively. The calculated value for MPI is 11.2 kcal/mol which is higher than benzene which has the highest value among the molecules given by Tian Lu and coworkers [53].
Natural charge, QTAIM, bond order, molecular planarity and molecular surface analyses have been performed on the geometry of title molecule optimized which has general agreement with experimental data. Two hydrogen bonds were identi ed and classifed as the weak and the weak to medium strength as a result of electron density at the BCP. The chemical bonds are classi ed based on the quantities obtained for BCPs. The correlation between BCP quantities and the bond orders based on different de nitions have been explored considering different bond order models from the literature. LBO gives the best correlation for four of ve bond order models. Considering slightly higher R 2 value and its practical usage, it can be said that the best bond order model includes the parameters ρ and λ 3 which are the measure for the σ character and the parameter λ 12 measuring the degree of π character for C-C bonds.
However all the bond order models with an exception of the model with parameter G have approximately same correlation degree. For C-H bonds, only bond model 2 for LBO gives relatively good correlation with R 2 value of 0.943. All the others have R 2 value lower than 0.900. Two phenyl rings of the structure have aromaticity comparable to benzene as deduced from QTAIM quantities and molecular planarity metrics.
The mass density, molecular polarity index, v (the measure of charge balance), σ 2 tot .v (measure of intermolecular interactions) were calculated based on molecular surface analysis. The molecular polarity index obtained is higher than benzen. The σ 2 tot .v value of the molecule was found to be higher than average value of the reference work [50]. The charge balance may be said to be nearly satis ed considering v value. Figure 1 Drawing of the C27H25F4NO3 molecule obtained with the Ortep III program. Displacement ellipsoids are drawn at the 50% probability level.

Figure 2
Page 29/30 The crystal packing of the title compound viewed along the b axis. Hydrogen bonds are shown as dashed lines-see Table 3 for details.

Figure 3
Optimized structure of title molecule by B3LYP.