Synthesis, X-ray Diffraction and Computational Druglikeness Evaluation of New Pyrrolo[1,2-a][1,10]Phenanthrolines Bearing a 9-Cyano Group

: New 9-cyano-pyrrolo[1,2-a][1,10]phenanthrolines 5a – d , obtained by a 1,3-dipolar cycloaddition reaction between the corresponding N-ylides of 1,10-phenanthrolinium bromides 2a – d , generated in situ and acrylonitrile as a dipolarophile, were investigated by single-crystal X-ray diffraction and computational studies to assess their druglikeness and evaluate their structure-activity properties. The non-covalent interactions present within the supramolecular landscape of the new 9-cyano-pyrrolo[1,2-a][1,10]phenanthrolines were correlated with the SAR investigations with the aim of estimating the propensity for bioactivity in these compounds.


Introduction
The derivatives of fused polyazaheterocycles, known for their wide range of biological activities, are of considerable interest due to their extensive applications in medicinal chemistry [1].In order to increase the structural diversity of biologically active compounds, through structural combination strategies that aim to assemble different pharmacophores in the same molecular framework, several series of fused pyrrolophenanthrolines have been reported so far in the literature as possessing both biological impact and other interesting properties [2].
In silico investigations on physicochemical properties and pharmacokinetics are widely employed in medicinal chemistry to assess the potential of a small molecule to become an active therapeutic agent against specific targets, depending on their interaction strengths and binding affinity.Density Functional Theory (DFT) methods are used to predict properties [49] and key molecular descriptors and parameters related to pharmacokinetics and bioavailability [50,51] to rationally design new molecules with enhanced potential and specificity against various diseases.In drug development, the pharmacokinetics properties, such as absorption, distribution, metabolism and excretion (ADME profile), of drug candidates can be predicted using computational tools for specific molecular parameters which must comply with pre-established rules [52,53].
Herein we present some structural aspects of nitrile functionalized pyrrolo[1,2-a][1,10] phenanthroline, which can indicate their potential for in-depth pharmaceutical screening based on the nitrile pharmacophore group, their binding properties and non-covalent interactions of such compounds extracted from their interesting crystallographic structures.

X-ray Structural Analysis
Single-crystal X-ray diffraction data were collected by a XtaLAB Synergy, Dualflex, and HyPix diffractometer using Cu Kα radiation.The unit cell determination and data integration were carried out using the CrysAlisPro package from Oxford Diffraction [54].The multi-scan correction for absorption was applied.The structures were solved with the SHELXT program using the intrinsic phasing method and refined by the full-matrix least-squares method on F 2 with SHELXL [55,56].Olex2 was used as an interface to the SHELX programs [57].An anisotropic model was used for the refinement of non-hydrogen atoms.Hydrogen atoms were added in idealized positions and refined using a riding model.Compound 5b was crystallized as a chloroform semisolvate which was encountered before for similar structures [21].The crystal structures are reported for two triclinic solid-state phases of this compound, the room temperature phase 5bHT and low temperature phase 5bLT at 160 K. Upon cooling, the disordered solvent molecule became ordered and the unit cell volume doubled.Selected crystallographic data and structure refinement details are provided in Table 1 and the corresponding CIF files.The supplementary crystallographic data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.ca.ac.uk).

Computational Details
Computations regarding molecular and drug-like properties on the equilibrium geometries of the synthetized compounds were performed using Spartan v. 24 software from Wavefunction, Inc., Irvine, CA, USA [58], for the conformers showing the lowest energy of each structure, in vacuum conditions, at ground state using B3LYP [59] global hybrid density functional model with basis set 6-311 G (d, p) [60].Evaluation of druglikeness was conducted in accordance with Lipinski's rule of five [52] and Veber's guidelines [53] regarding specific properties for drug candidates.

Computational Details
Computations regarding molecular and drug-like properties on the equilibrium geometries of the synthetized compounds were performed using Spartan v. 24 software from Wavefunction, Inc., Irvine, CA, USA [58], for the conformers showing the lowest energy of each structure, in vacuum conditions, at ground state using B3LYP [59] global hybrid density functional model with basis set 6-311 G (d, p) [60].Evaluation of druglikeness was conducted in accordance with Lipinski s rule of five [52] and Veber s guidelines [53] regarding specific properties for drug candidates.
The structures of the new compounds were elucidated by NMR spectroscopy, which confirmed the substituted 9-cyano pyrrolophenanthroline framework.The H-10 hydrogen atom appears as a singlet in the range 7.36-7.41ppm.The most deshielded protons are H-2 and H-4 in the free pyridine ring of the phenanthroline system.The benzoyl moiety presents the signals of a para substitution pattern.The C atom in the CN group appears in the range 115-116 ppm.The C=O group appears in the region 183.8-184.4ppm as the most deshielded C-atom.

X-ray Crystallography
The results of X-ray structural analyses for 5a, 5bHT, 5bLT and 5c are illustrated in Figure 1, while the geometric parameters are summarized in Table S1.Compound 5b crystallized from chloroform solution as a semisolvate.This triclinic semisolvate on cooling undergoes a phase transition to another triclinic phase with a unit cell twice as large.Upon transition, ordering of the solvent molecules takes place.As was to be expected, by analogy with the structures of other 1-acyl-pyrrolo[1,2a][1,10]phenanthrolines [21,28,29], all studied molecules show severe overcrowding that results in their helical structure.The fused-ring system is twisted to avoid strong steric repulsions between the atoms of the terminal pyridine ring and the benzoyl substituent at the pyrrole fragment.The degree of helicity of the fused-ring system can be estimated by a dihedral angle between the planes of the terminal rings.These dihedral angles are similar and range from 19.54 • in 5bHT to 25.06 • in 5c.Despite the twisting of the fused-ring system, some intramolecular contacts remain quite short, as, for example, the non-bonding distance between the pyridine N1 and the carbonyl carbon C17 (2.513(2)-2.532(2)Å) that is ca.0.7 Å shorter than the sum of the van der Waals radii.A steric strain associated with this short distance leads to a significant displacement (0.485-0.560Å) of the carbonyl C17 atom from the plane of the virtually planar pyrrole ring and to a pyramidal arrangement of three bonds formed by C15.The C15 atom is displaced from the plane through N2, C14 and C17 by 0.468 Å in 5a to 0.560 Å in 5bHT.Due to a helical structure, the studied molecules are chiral and their two enantiomers can be assigned as P and M. Figure 1 shows P enantiomers, except 5bHT, where both enantiomers related by the pseudo-inversion center are shown.It should be pointed out that all three compounds crystallize in centrosymmetric space groups, and therefore their crystals have to contain both enantiomeric forms of molecules (Figure S1).
All crystal structures were analysed from the point of view of short intermolecular contacts.As there are no classical hydrogen-bond donors in the studied molecules, only

Hirshfeld Surface Analysis
Hirshfeld surfaces (HF) [62] generated for compounds 5a-c using CrystalExplorer [63] visually display the main interactions in the supramolecular structures.The red spots in dnorm mode present the locations of the non-covalent contacts with distances

Hirshfeld Surface Analysis
Hirshfeld surfaces (HF) [62] generated for compounds 5a-c using CrystalExplorer [63] visually display the main interactions in the supramolecular structures.The red spots in dnorm mode present the locations of the non-covalent contacts with distances under the van der Waals radii.The most relevant interactions represented by red spots

Hirshfeld Surface Analysis
Hirshfeld surfaces (HF) [62] generated for compounds 5a-c using CrystalExplorer [63] visually display the main interactions in the supramolecular structures.The red spots in d norm mode present the locations of the non-covalent contacts with distances under the van der Waals radii.The most relevant interactions represented by red spots (contact distances below the sum of the vdW radii) and are mainly visible (Figure 4) for the hydrogen bonds discussed in the X-ray chapter but also for short C For compounds 5a and 5bLT, the Shape index surfaces present the complementary spots specific for π•••π interactions involving the inner rings (II and III) of the pyrrolophenanthroline skeleton.This is enhanced by the curvedness surfaces, which confirm that the two rings are the most planar (Figure 4).The most numerous contacts, as extracted from the Hiershfild surface, are statistically H•••H contacts, which are similar (Figure 5) for 5a and 5bLT and present the same ratios for all the evaluated contacts.However, 5c, due to the introduction of the NO2 group, presents a steep decrease in H•••H contacts and an increase in O•••H contacts from around 7-8% for 5a,b to 25.5% for 5c.The statistical landscape of interactions in the Hirsfeld surface is a qualitative description and not a quantitative one.For example, N•••H contacts are the strongest in compound 5b, which presents the lowest percentage of interactions (10.6% compared to 13.3%-5a and 12.7%-5c).However, the HF surfaces and fingerprint plots are important descriptors of possible binding modes of such molecules in view of their further evaluation as potential bioactive molecules.There is no difference between the two helical enantiomers P and M in the HF percentages, with the exception of 5bLT, where one enantiomer shares more Cl•••H contacts with the chloroform solvent.The most numerous contacts, as extracted from the Hiershfild surface, are statistically H•••H contacts, which are similar (Figure 5) for 5a and 5bLT and present the same ratios for all the evaluated contacts.However, 5c, due to the introduction of the NO 2 group, presents a steep decrease in H•••H contacts and an increase in O•••H contacts from around 7-8% for 5a,b to 25.5% for 5c.The statistical landscape of interactions in the Hirsfeld surface is a qualitative description and not a quantitative one.For example, N•••H contacts are the strongest in compound 5b, which presents the lowest percentage of interactions (10.6% compared to 13.3%-5a and 12.7%-5c).However, the HF surfaces and fingerprint plots are important descriptors of possible binding modes of such molecules in view of their further evaluation as potential bioactive molecules.There is no difference between the two helical enantiomers P and M in the HF percentages, with the exception of 5bLT, where one enantiomer shares more Cl•••H contacts with the chloroform solvent.
An interesting aspect in the crystal landscape of such compounds is the presence of helical chirality devising the presence of the P and M helicoidal enantiomers (Figure 6).As in the case of many helicene-like molecules, this helical chirality is induced by the presence of four or more than four ortho-condensed rings [22,64].In structures with less condensed rings, such as pyrrolophenanthrolines, the helicoidal shape of the molecules can be induced by the presence of bulky substituents in the "Bay-region" of the molecule [21].
However, in the case of pyrrolophenanthrolines, intriguing intramolecular shortcontacts were observed [27,29] between the nitrogen in ring IV and the carbon atom in the C=O from the benzoyl moiety, raising the intuition that the pitch of the helix can be influenced by an interplay of this apparently attractive force and the repulsion forces generated by the molecular strain.In this case, this non-covalent interaction might be of type lp-π between the free electrons of the N atom and the empty π-orbital of the C=O group.Such kinds of bonds are typical, for example, in protein folding due to C=O•••C=O Symmetry 2024, 16, 911 9 of 15 interactions [65] (where one lp is donated by O instead of N).We measured the effect of this interaction using the value of the dihedral angle between the rings I and IV versus the contact distance between the N and C atom from C=O (Table 2).The results were not very conclusive, but it appears that such kinds of interactions can be further investigated as the measured contact lengths compared to sum of vdW radii could imply strong interactions.Another conceptual proof of this interaction might be given by the deformation of the carbonyl bond with the C atom going outside the C=O bond (pyramidalization).Also, a strong H-bond in the structure of CSD: ZIPQUM [38] leads to an increase in the molecular deformation.However, the negative correlation of the data in Table 2 shows that other effects are to be considered also.An interesting aspect in the crystal landscape of such compounds is the presence of helical chirality devising the presence of the P and M helicoidal enantiomers (Figure 6).As in the case of many helicene-like molecules, this helical chirality is induced by the presence of four or more than four ortho-condensed rings [22,64].In structures with less condensed rings, such as pyrrolophenanthrolines, the helicoidal shape of the molecules can be induced by the presence of bulky substituents in the "Bay-region" of the molecule [21].However, in the case of pyrrolophenanthrolines, intriguing intramolecular short-contacts were observed [27,29] between the nitrogen in ring IV and the carbon atom in the C=O from the benzoyl moiety, raising the intuition that the pitch of the helix can be influenced by an interplay of this apparently attractive force and the repulsion forces generated by the molecular strain.In this case, this non-covalent interaction might be of An interesting aspect in the crystal landscape of such compounds is the presence of helical chirality devising the presence of the P and M helicoidal enantiomers (Figure 6).As in the case of many helicene-like molecules, this helical chirality is induced by the presence of four or more than four ortho-condensed rings [22,64].In structures with less condensed rings, such as pyrrolophenanthrolines, the helicoidal shape of the molecules can be induced by the presence of bulky substituents in the "Bay-region" of the molecule [21].However, in the case of pyrrolophenanthrolines, intriguing intramolecular short-contacts were observed [27,29] between the nitrogen in ring IV and the carbon atom in the C=O from the benzoyl moiety, raising the intuition that the pitch of the helix can be influenced by an interplay of this apparently attractive force and the repulsion forces generated by the molecular strain.In this case, this non-covalent interaction might be of type lp-π between the free electrons of the N atom and the empty π-orbital of the C=O group.Such kinds of bonds are typical, for example, in protein folding due to C=O•••C=O interactions [65] (where one lp is donated by O instead of N).We measured the effect of this interaction using the value of the dihedral angle between the rings I and IV versus the contact distance between the N and C atom from C=O (Table 2).The results were not very conclusive, but it appears that such kinds of interactions can be further investigated as the measured contact lengths compared to sum of vdW radii could imply strong in- The helicoidal distortion of the molecules can influence the pitch of the helix and thus the structural similarity with bioactive molecules, such was the recent case of cholchicine mimick [5]-helistatin, the first known helicene showing real bioactive potential in relation with actual commercial drugs [66].The helicoidal distortion of the molecules can influence the pitch of the helix and thus the structural similarity with bioactive molecules, such was the recent case of cholchicine mimick [5]-helistatin, the first known helicene showing real bioactive potential in relation with actual commercial drugs [66].

Druglikeness Assessment
In Figure 7, the representation of the electrostatic potential maps is given, varying from blue (the most positive values) to red (the most negative values).The potential increases in the order: red < orange < yellow < green < blue.For structure 5a, values of the electrostatic potential range from 141.

Quantum Reactivity Parameters Calculated from FMO Predicted Energy Levels
Regarding the molecular properties assessment, in order to evaluate the potential suitability of compounds for further development, from the point of view of druggability and oral bioavailability, by applying property filters stated by Lipinski s rule of five (RO5), it can be observed that all structures, except 5c, exhibit one violation to RO5 in terms of hydrophilicity/lipophilicity balance, as shown from the values of logP listed in Table 3.Values of logP greater than five suggest highly lipophilic compounds.This fact

Quantum Reactivity Parameters Calculated from FMO Predicted Energy Levels
Regarding the molecular properties assessment, in order to evaluate the potential suitability of compounds for further development, from the point of view of druggability and oral bioavailability, by applying property filters stated by Lipinski's rule of five (RO5), it can be observed that all structures, except 5c, exhibit one violation to RO5 in terms of hydrophilicity/lipophilicity balance, as shown from the values of logP listed in Table 3.Values of logP greater than five suggest highly lipophilic compounds.This fact severely impacts on bioavailability and will require further special pharmaceutical formulations to increase the penetration in the physiological environment and to reduce the non-polar character of these compounds.Among the three structures, 5c is the most hydrophilic structure and 5b is the most hydrophobic structure, as expected, due to the phenyl substituent (R).Although other requirements of RO5 are met (mass, number of hydrogen bond donor's and acceptors) for 5a and 5b structures, and adding the conformity with the Veber rule concerning the total polar surface area and the number of rotatable bonds, keeping in mind the fact that it represents only one minor step in drug assessment, all compounds can be further considered as leads for development and investigation of their pharmaceutical potential.Concerning the 5c compound, the replacement of the R substituent on the skeleton structure with NO 2 will strongly increase the hydrophilic character and also will give the opportunity for more interactions within the active site of proteins targets due to the Lewis structure for NO 2 − and due to more hydrogen bond acceptors (HBA = 7).Additionally, predictive data for the global chemical reactivity of the investigated compounds are obtained from the frontier molecular orbitals's energy levels, as depicted in Figure 8, from their values given in Table 4.The quantum reactivity parameters are calculated according to Koopman's' theorem [67,68], which indicated relationships for the ionization potential (I) and the electron affinity (A) as the negative of HOMO and LUMO energies, respectively.Pearson's Hard and Soft Acids [69,70] is basis for calculation of the softness (σ) and hardness (η) parameters.The electronic reactivity (χ) is calculated according the Maximum Hardness Principle (MHP) [70].The electrophilicity index (ω), is obtained from an equation stated by Parr R.G and collaborators [71].The HOMO-LUMO energy gap is a measure of the kinetic stability and reactivity of molecules.According the calculated values, among the investigated compounds, the 5b derivative seems to be the more stable, revealing the larger energy gap (3.62 eV), and 5c is the most reactive, presenting the smaller energy gap (3.23 eV).The compounds are rather stable, and their non-covalent interactions in crystalline phase validated by the ESP provide insightful information regarding the affinity of the CN group as being pharmacophore or other possible interactions mainly driven by hydrogen bonds (given by the number of hydrogen bonds acceptors).The compounds 5a-c present large molecular masses and high logP values which might need further formulation studies in order to increase their bioavailability.Given the potential of such compounds confirmed by recent studies [8][9][10][11][12][13][14], and the potential use of the molecular distortion in mimicking com-mercial drugs [66], the new compounds are suitable for further expanding their structural diversity and investigating their potential anti-cancer or antibiotic properties.

Conclusions
In conclusion, we have synthesized and structurally characterized three new 9-cyanopyrrolo[1,2-a][1,10]phenanthrolines and investigated their supramolecular interactions, extracted from X-ray single crystal diffraction analysis, with the aim of analyzing their potential supra-molecular binding sites and binding patterns.The helicoidal structures could present interesting properties which might infer them with bioactive properties.
and C-H•••π interactions involving aromatic rings were expected among the specific intermolecular interactions influencing the crystal packing.In all cases, the carbonyl O1 atom takes part in a C-H•••O contact that is shorter or equal Symmetry 2024, 16, 911 6 of 15 2.60 Å.The shortest H•••O contact of 2.30 Å is found in 5bLT where the solvent CHCl 3 molecule acts as a donor in hydrogen bonding.In 5a and 5c, C-H•••O interactions involving the carbonyl group assemble the molecules into one-dimensional supramolecular arrays (Figures 2 and 3) that, in 5c, are further extended into double chains by C-H•••O interaction to the nitro group.In both crystalline phases of 5b, C-H•••O contacts involving the carbonyl group occur within centrosymmetric dimers formed via π•••π stacking interactions between the phenanthroline fragments of the fused-ring system.These dimers are further assembled into 1D-supramolecular arrays via π•••π stacking involving pyrrole and phenanthroline aromatic rings with centroid-to-centroid distances of 3.646 Å and 3.903 Å (Figure S2).Stacking interactions with the centroid-to-centroid distance of 3.636 Å between the inversion-center-related phenanthroline fragments of the neighboring one-dimensional supramolecular arrays are also found in 5a.In turn, in 5c, π•••π stacking interactions involving the benzene and pyrrole rings (centroid-to-centroid distance 3.635 Å) occur within the 1D supramolecular assembly formed via C-H•••O interactions (Figure 3), and C-H•••π interactions between the phenanthroline fragments extend these assemblies in a 2D array.Short C-H•••N contacts to the nitrile group (H•••N < 2.70 Å) are absent in 5a but are found in the remaining structures.In 5a and 5bHT, this interaction is generated between inversion-center-related molecules, resulting in dimers, whereas, in 5bLT, the molecules in dimer are related by the pseudoinversion center and the two H•••N distances of 2.44 and 2.64 Å differ significantly (Figure1c).

Symmetry 2024 ,
16,  x FOR PEER REVIEW 7 of 17 nitrile group (H•••N < 2.70 Å) are absent in 5a but are found in the remaining structures.In 5a and 5bHT, this interaction is generated between inversion-center-related molecules, resulting in dimers, whereas, in 5bLT, the molecules in dimer are related by the pseudoinversion center and the two H•••N distances of 2.44 and 2.64 Å differ significantly (Figure1c).

Symmetry 2024 , 17 Figure 5 .
Figure 5. Percentage interactions for compounds 5a-c extracted from their contribution to the HF surface.5bHT is exemplified for information; 5bLT presents both enantiomers, as, beside the helix directions, the two enantiomers present some other particularities due to the interaction with CHCl3 solvent.

Figure 6 .
Figure 6.Mirror image of the two helical enantiomers of compound 5a.

Figure 5 . 17 Figure 5 .
Figure 5.Percentage interactions for compounds 5a-c extracted from their contribution to the HF surface.5bHT is exemplified for information; 5bLT presents both enantiomers, as, beside the helix directions, the two enantiomers present some other particularities due to the interaction with CHCl 3 solvent.

Figure 6 .
Figure 6.Mirror image of the two helical enantiomers of compound 5a.

Figure 6 .
Figure 6.Mirror image of the two helical enantiomers of compound 5a.

Figure 8 .
Figure 8. FMO distribution and energy levels for compounds 5a-c.

Table 1 .
Crystal data and details of structure refinement for 5a

Table 2 .
Dihedral angles vs. distance between the N atom from pyridine ring and the C=O in the benzoyl moiety.

Table 3 .
Calculated druglike properties for the investigated structures.

Table 4 .
FMO distribution and energy levels for compounds 5a-c.