Designing and Exploration of the Biological Potentials of Novel Centrosymmetric Heteroleptic Copper(II) Carboxylates

Copper(II) complexes with a general formula [Cu2(3,4-F2C6H3CH2COO)4(L)2], where L = 2-methylpyridine (1) and 3-methylpyridine (2), are reported here. The FTIR spectra of the complexes confirmed the bridging bidentate coordination mode of the carboxylate ligand. The low (475 and 449 cm−1) and strong (727 & 725 cm−1) intensity bands in the FTIR spectra, due to Cu-N stretches and pyridyl ring vibrations, confirmed coordination of the 2-/3-methyl pyridine co-ligands in complexes 1 and 2, respectively. A binuclear paddlewheel structural arrangement with a square pyramidal geometry was confirmed for copper atoms in the complexes via single-crystal X-ray analysis. The DPPH, •OH radical, and α-amylase enzyme inhibition assays showed higher activities for the complexes than for the free ligand acid. The binding constant (Kb = 1.32 × 105 for 1 and 5.33 × 105 for 2) calculated via UV-VIS absorption measurements and docking scores (−6.59 for 1 and −7.43 for 2) calculated via molecular docking showed higher SS-DNA binding potential for 2 compared to 1. Viscosity measurement also reflected higher DNA binding ability for 2 than 1. Both complexes 1 and 2 (docking scores of −7.43 and −6.95, respectively) were found to be more active inhibitors than the free ligand acid (docking score of −5.5159) against the target α-amylase protein. This in silico study has shown that the herein reported compounds follow the rules of drug-likeness and exhibit good potential for bioavailability.


Introduction
Heteroleptic paddlewheel binuclear copper(II) carboxylates containing monodentate N-donor co-ligands constitute an important class of compounds with potential applications in bioinorganic chemistry.This class of compounds can be represented by the general formula [Cu 2 (OOCR) 4 L 2 ], where four bridging carboxylate ligands form the base of the paddlewheel structure and the monodentate N-donor ligands occupy the apical positions.The two carboxylate-bridged copper(II) ions have square pyramidal geometry, and so are sterically less saturated, a structural feature of the complex that could be exploited for the interactions of the metal center with biomolecular receptors in biological activity [1].Apart from the possible interaction of metal center, the biological properties of these com-plexes could also be tuned up to varying extent by brining small structural changes in the attached carboxylate or N-donor ligands [2][3][4][5][6][7][8][9][10][11].
According to earlier reports, the attachment of N-donor ligands to Cu(II) ions enhances the planarity in the structure and reduces the maximum attainable geometry around the Cu(II) ions [12][13][14][15].In terms of biological activity, dinuclear complexes have shown greater activity than mononuclear complexes.Thus, the combination of carboxylates with less steric bulk and N-donor aromatic ligands provides an ideal platform for exploring their biological applications [16].
As a continuation of our previous work on copper(II) carboxylates [17], in this study, a simple synthetic route was adopted to introduce additional N-donor ligands to isolate complexes, consisting of 3,4-difluorophenylacetate and 2-/3-methylpyridine.The in vitro antioxidant and alpha-amylase inhibition potential of the synthesized complexes were evaluated.Since DNA is an important biological target of many anticancer and antiviral therapies, these compounds were screened for their DNA-binding ability using UV-visible spectroscopy and viscometry.A molecular docking study of the synthesized complexes was also performed to gain insight into the complex-DNA and complex-alpha-amylase interaction potential.
Pharmaceuticals 2023, 16,1462 2 of 21 so are sterically less saturated, a structural feature of the complex that could be exploited for the interactions of the metal center with biomolecular receptors in biological activity [1].Apart from the possible interaction of metal center, the biological properties of these com-plexes could also be tuned up to varying extent by brining small structural changes in the attached carboxylate or N-donor ligands [2][3][4][5][6][7][8][9][10][11].
According to earlier reports, the attachment of N-donor ligands to Cu(II) ions enhances the planarity in the structure and reduces the maximum attainable geometry around the Cu(II) ions [12][13][14][15].In terms of biological activity, dinuclear complexes have shown greater activity than mononuclear complexes.Thus, the combination of carboxylates with less steric bulk and N-donor aromatic ligands provides an ideal platform for exploring their biological applications [16].
As a continuation of our previous work on copper(II) carboxylates [17], in this study, a simple synthetic route was adopted to introduce additional N-donor ligands to isolate complexes, consisting of 3,4-difluorophenylacetate and 2-/3-methylpyridine.The in vitro antioxidant and alpha-amylase inhibition potential of the synthesized complexes were evaluated.Since DNA is an important biological target of many anticancer and antiviral therapies, these compounds were screened for their DNA-binding ability using UV-visible spectroscopy and viscometry.A molecular docking study of the synthesized complexes was also performed to gain insight into the complex-DNA and complex-alpha-amylase interaction potential.

Synthesis
The copper(II) complexes (1 and 2) were synthesized by reacting copper(II) chloride dihydrate with 3,4-difluorophenylacetic acid in the presence of either 2-methylpyridine (1) or 3-methylpyridine (2) as co-ligands in methanol under reflux condition.Sea green color complexes, soluble in methanol and DMSO, were obtained in moderate yields (Scheme 1).Scheme 1. Synthetic route of the syntheses of complexes 1 and 2.

FTIR Spectra
For the preliminary solid-state structural characterization, the FT-IR spectra of the synthesized complexes 1 and 2 were compared with that of the free ligand acid spectrum.Significant evidence confirming the complexes' synthesis was provided by this comparison.The most explicit evidence was the absence of a broad band at 3400-2400 cm −1 in the complexes' spectra.This broad band is typical for free carboxylic acid, showing OH bond vibrations [17].During complexation, deprotonation of the carboxylate ligand occurs, so this band disappears in the complexes.Also, low intensity bands, due to Cu-O bond vibrations, appeared at 418 and 432 cm −1 for complexes 1 and 2, respectively.The bands due to C=O and C-O bond vibrations at 1708 and 1279 cm −1 , respectively, of the free ligand Scheme 1. Synthetic route of the syntheses of complexes 1 and 2.

FTIR Spectra
For the preliminary solid-state structural characterization, the FT-IR spectra of the synthesized complexes 1 and 2 were compared with that of the free ligand acid spectrum.Significant evidence confirming the complexes' synthesis was provided by this comparison.The most explicit evidence was the absence of a broad band at 3400-2400 cm −1 in the complexes' spectra.This broad band is typical for free carboxylic acid, showing OH bond vibrations [17].During complexation, deprotonation of the carboxylate ligand occurs, so this band disappears in the complexes.Also, low intensity bands, due to Cu-O bond vibrations, appeared at 418 and 432 cm −1 for complexes 1 and 2, respectively.The bands due to C=O and C-O bond vibrations at 1708 and 1279 cm −1 , respectively, of the free ligand acid were absent in the spectra of the complexes.Instead, these bands were replaced by strong intensity bands at 1624, 1615 and 1397, 1396 cm −1 , due to COO asy and COO sym vibrations in complexes 1 and 2, respectively, indicating the bridging bidentate coordination of the carboxylate ligand to the copper center [17].The coordination of nitrogen-donor ligands was evidenced by low intensity bands at 475 and 449 cm −1 in the spectra of complexes 1 and 2, respectively [18].Strong bands due to pyridyl ring vibrations were observed at 727 and 725 cm −1 in complexes 1 and 2, respectively [19].Complexes 1 and 2 spectra also showed strong bands due to Ar-F bond vibrations at 1208 cm −1 .The structural findings of the IR spectra were also confirmed by single-crystal X-ray diffraction analysis of complexes 1 and 2.

Crystal Structure Description of the Complexes
The relevant crystallographic data for complexes 1 and 2 along with the details of structure refinement are listed in Table 1.Perspective views of complexes 1 and 2 are shown in Figures 1 and 2, respectively.The complexes are both C i -symmetrical complexes, as they lie across the inversion centers in the appropriate space groups.The asymmetric part of complex 2's structure actually contains two symmetry-independent halves across two different inversion centers, at 1,1/2,0 and 0,0,1.The tendency towards such symmetry is common for similar compounds.In the CSD, there are 712 fragments with bis(µ 2 -benzoato-O, O')copper(II) complexes with N atoms at the fifth coordinating position, and among these, 612 fragments contain symmetrical molecules.shown in Figures 1 and 2, respectively.The complexes are both Ci-symmetrical complexes, as they lie across the inversion centers in the appropriate space groups.The asymmetric part of complex 2's structure actually contains two symmetry-independent halves across two different inversion centers, at 1,1/2,0 and 0,0,1.The tendency towards such symmetry is common for similar compounds.In the CSD, there are 712 fragments with bis(µ2-benzoato-O, O`)copper(II) complexes with N atoms at the fifth coordinating position, and among these, 612 fragments contain symmetrical molecules.In both the complexes, Cu(II) ions are five coordinated (O 4 N), exhibit square pyramidal geometry, with four equatorial oxygen atoms belonging to the carboxylate group of four bridging 3,4-difluorobenzoate anions (Cu1-O eq distances in the range of 1.958(3)-1.971(3)Å in 1 and 1.9595(17)-1.9828(17)Å in 2 (Table 2)) and the pyridine nitrogen atom of the methylpyridine.The methyl groups of the two pyridine ligands are pointing opposite to each other in square planar geometry, to minimize any steric strain.The elongated Cu1-N1 bond distance of 2.210(3) Å in 1 compared to 2.139 Å (average) in 2 could be attributed to the steric crowding of 2-methylpyridine in close proximity to the Cu center.The same could be argued for the longer Cu•••Cu separation [2.6775(9) Å] in 1 than [2.6333(6)Å (average)] in 2, but well within the range known for such compounds [17].Table 2 lists all the relevant geometrical parameters.In general, this geometry is typical for similar compounds.Hirshfeld surface analyses were performed to quantify and visualize the closed intermolecular atomic contacts in the crystal structures of 1 and 2, and visualization of the Hirshfeld three-dimensional d norm surfaces of the title compounds are shown in Figures S1 and S2, respectively.The intensive red spots on the surfaces, colored according to d norm , are related to interactions involving fluorine and oxygen atoms corresponding to the H-bonds.Figures S3 and S4 show the fingerprint plots of complexes 1 and 2, respectively, indicating significant contributions due to different intermolecular interactions.For both compounds, the highest contributions of total Hirshfeld surfaces are attributed to

Antioxidant Activities
DPPH and hydroxyl (•OH) radical scavenging assays based on spectroscopic measurements are simple in vitro methods used to evaluate the antioxidant potential of test compounds.The promising results of these assays could be exploited as preliminary tests in search of drugs with potential anticancer, anti-aging, and anti-inflammatory activities [20].Within this context, the abilities of the free ligand acid (HL) and synthesized complexes (1 and 2) to scavenge DPPH and hydroxyl radicals were determined in order to evaluate their potential antioxidant ability compared to that of vitamin C as a reference compound.
The dose-dependent (15-240 µM) responses and IC 50 values of the compounds compared to those of standard vitamin C are shown in Tables 3 and 4 for DPPH and hydroxyl (•OH) radical scavenging assays, respectively.The studied radicals with a single unpaired electron each are capable of accepting an electron, and thus not only lead to a decrease in the UV absorption at 517 nm, but also the color changes from violet to yellow.A decrease in absorption generally occurs when it accepts a hydrogen atom or electron from an antioxidant, to form a stable molecule.With an increase in the sample's concentration, the radicle scavenging rates of the test compounds increase subsequently.The complexes showed significantly higher free radical scavenging ability compared to that of the free ligand acid in both the assays.This reflects the importance of the redox active copper center as a free radical scavenger.The presence of an unpaired electron in copper(II) ion could be a possible reason for the enhanced ability of the complexes to arrest the free radicals.The two complexes, despite their very close structural/composition resemblance, showed significantly different activities.Complex 1 showed higher activity compared to that of complex 2. The difference in the antioxidant activity of the two complexes reflects the importance of the functional group position on the nitrogen-donor ligand.A slight change in the position of the functional group has a pronounced impact on the free radical scavenging ability of the complex.In complex 1, the 2-methyl pyridine ligand has better electron donating ability than the 3-methyl pyridine of complex 2, thus making the copper an electron-rich center for interactions with the free radicals.Complex 1 was more effective against DPPH compared to •OH radical, while complex 2 showed better efficiency against •OH than DPPH.This radical scavenging selectivity of the complexes is not unexpected, as many complexes are reported in the literature with a somewhat similar behavior against different free radicals [21].

Alpha-Amylase Inhibition Assay
The α-amylase catalyzed hydrolysis of carbohydrate results in elevated blood sugar levels, especially after meals, and can lead to hyperglycemia [22].Therefore, inhibition of this enzyme is considered to be an effective therapeutic approach to control hyperglycemia [23].In the current study, the inhibition effects of the free ligand acid and synthesized complexes on the α-amylase enzyme were investigated and compared to acrabose as a standard (Table 5).A dose-dependent enzyme inhibition response was noticed for the test compounds with the following activity order: acrabose > 1 > 2 > HL.In this study, the free ligand acid was the weakest inhibitor of α-amylase (IC 50 value of 728.17 µg/mL) compared to acrabose (IC 50 value of 207.72 µg/mL).Coordination of the deprotonated carboxylate ligand to the copper center sufficiently improved the α-amylase inhibition potential of the synthesized complexes (IC 50 values, 437.73 µg/mL (1) and 455.32 (2) µg/mL).However, no significant activity differences were observed in the case of synthesized complexes.The enhanced activity of the metal complexes compared to the free ligand acid could be a consequence of the copper(II) ion's ability to interact with the donor atoms present in the enzyme [24].UV-visible absorption measurements can be used to monitor changes occurring in λ max values (red or blue shifts) and absorption intensity (hyper-or hypochromism) of either a test compound or DNA as a result of any possible interaction.These changes can be used to find some details about the mode of interaction of metal complexes with DNA [25].In the present study, absorption titrations were made with a fixed concentration (200 µM) of complexes against varying concentrations (5-30 µM) of SS-DNA.
The synthesized complexes showed pronounced hypochromism with a very small red shift of about 1 nm upon successive addition of SS-DNA, as shown in Figures 3A and 4A for complexes 1 and 2, respectively.This behavior suggests a mixed binding mode of the complexes with DNA, consisting of partial intercalation of the complexes into the base pairs of the DNA, along with groove binding [19,25].
2.6.DNA Binding Study 2.6.1.DNA Binding Study by Absorption Spectroscopy UV-visible absorption measurements can be used to monitor changes occurri λmax values (red or blue shifts) and absorption intensity (hyper-or hypochromism) ther a test compound or DNA as a result of any possible interaction.These change be used to find some details about the mode of interaction of metal complexes with [25].In the present study, absorption titrations were made with a fixed concentration µM) of complexes against varying concentrations (5-30 µM) of SS-DNA.
The synthesized complexes showed pronounced hypochromism with a very red shift of about 1 nm upon successive addition of SS-DNA, as shown in Figures 3A 4A for complexes 1 and 2, respectively.This behavior suggests a mixed binding mo the complexes with DNA, consisting of partial intercalation of the complexes into the pairs of the DNA, along with groove binding [19,25].The extent of complex-DNA binding was assessed by calculating intrinsic bin constant (Kb) values using the well-known Benesi-Hildebrand equation [26].
where Kb is the binding constant, A is the absorbance of complex-DNA adduct, Ao absorbance of pure complex solution, [DNA] is the concentration of SS-DNA in mol/ G is the molar absorption co-efficient of each complex-DNA adduct, and εG is the m absorption co-efficient of pure complex.The Kb value was calculated from the interce slope ratio of the plot of 1/[DNA] along the abscissa vs. Ao/A-Ao along the ordina shown in Figures 3B and 4B for complexes 1 and 2, respectively.The calculated Kb values were found to be 1.32 × 10 5 and 5.33 × 10 5 for comple and 2, respectively.The Kb values show significant interaction of the synthesized plexes with the SS-DNA compared to the previously reported structurally related pa wheel copper(II) complexes [17,27].The difference in Kb values of the complexes re the importance of the functional group position on the same ligand.The position functional group on a coordinated ligand can significantly affect the relative orient of a complex during its interaction with a biomolecule that can affect the bioactivity The extent of complex-DNA binding was assessed by calculating intrinsic binding constant (K b ) values using the well-known Benesi-Hildebrand equation [26].
where K b is the binding constant, A is the absorbance of complex-DNA adduct, A o is the absorbance of pure complex solution, [DNA] is the concentration of SS-DNA in mol/L, ε H-G is the molar absorption co-efficient of each complex-DNA adduct, and ε G is the molar absorption co-efficient of pure complex.The K b value was calculated from the intercept to slope ratio of the plot of 1/[DNA] along the abscissa vs.A o /A−A o along the ordinate, as shown in Figures 3B and 4B for complexes 1 and 2, respectively.
The calculated K b values were found to be 1.32 × 10 5 and 5.33 × 10 5 for complexes 1 and 2, respectively.The K b values show significant interaction of the synthesized complexes with the SS-DNA compared to the previously reported structurally related paddlewheel copper(II) complexes [17,27].The difference in K b values of the complexes reflects the importance of the functional group position on the same ligand.The position of a functional group on a coordinated ligand can significantly affect the relative orientation of a complex during its interaction with a biomolecule that can affect the bioactivity of the metal complexes.The calculated ∆G values of −29.20 and −32.67 KJ/mol for the complexes 1 and 2, respectively, reflect the spontaneity of the complex-DNA interaction.

DNA-Binding Study by Viscometry
The interaction mode of the compounds with DNA was further investigated via viscometry, and the results are shown in Figure 5.The free ligand acid has no effect on the relative viscosity of the DNA.However, the synthesized complexes significantly increased the relative viscosity of the DNA.Complex 2 increased the viscosity of DNA more than complex 1.The significant increase in the relative viscosity of DNA in the presence of complexes compared to that in the free ligand acid reflects the importance of metal in DNA interactions.Changes in viscosity are sensitive towards length changes in DNA molecules.In the case of the intercalation mode of action, a DNA molecule lengthens to adjust the binding complex and as a result the viscosity of the complex-DNA adduct increases [28].In groove binding, a steady increase in the relative viscosity of DNA occurs with an increasing concentration of complex [28].A pronounced increase in the relative viscosity of DNA upon addition of complexes 1 and 2 followed by a slight increase in complex-DNA adduct viscosity show intercalation and groove binding as the possible mode of complex-DNA interactions.

Docking of Complexes with Alpha-Amylase
The molecular docking studies of the synthesized complexes showed a wide range of potential interactions with the substrate-binding site (Figure 6A) of alpha-amylase.In order to assess the accuracy of the MOE docking, the co-crystallized ligand (PDB ID 3BAJ) was extracted out of the active site and docked once again into the binding site of alphaamylase.Our procedure was quite accurate, as shown by the RMSD value of 1.0214 between the top-ranked docked conformation and the co-crystallized ligand [29] (Figure S8).The binding site residues involved in the interactions with the complexes and control compound included Thr 163, His 201, Glu 233, Asp 300, Gly 304, and His 305.The interaction details and docking scores are listed in Table 6.The control, i.e., acarbose, (docking score of −8.73) established significant interactions with the catalytic site residues of amylase.Thr 163, Glu 233, and Asp 300 developed hydrogen bond donor interactions, while His 201 showed one hydrogen bond acceptor interaction with acarbose, as shown in

Molecular Docking Studies 2.7.1. Docking of Complexes with Alpha-Amylase
The molecular docking studies of the synthesized complexes showed a wide range of potential interactions with the substrate-binding site (Figure 6A) of alpha-amylase.In order to assess the accuracy of the MOE docking, the co-crystallized ligand (PDB ID 3BAJ) was extracted out of the active site and docked once again into the binding site of alpha-amylase.Our procedure was quite accurate, as shown by the RMSD value of 1.0214 between the top-ranked docked conformation and the co-crystallized ligand [29] (Figure S8).The binding site residues involved in the interactions with the complexes and control compound included Thr 163, His 201, Glu 233, Asp 300, Gly 304, and His 305.The interaction details and docking scores are listed in Table 6.The control, i.e., acarbose, (docking score of −8.73) established significant interactions with the catalytic site residues of amylase.Thr 163, Glu 233, and Asp 300 developed hydrogen bond donor interactions, while His 201 showed one hydrogen bond acceptor interaction with acarbose, as shown in Figure 6E.Complex 1 (docking score of −7.43) formed two hydrogen bond donor interactions and one C-H-pi interaction with Gly 304 and His 305 amino acids residues of the target protein, as shown in Figure 6B.Complex 2 (docking score of −6.95) formed two hydrogen bond donor interactions with His 305 and Asp 356.His 305 was also found in the C-H-pi interaction with complex 2, as shown in Figure 6C.The free ligand acid was found to be the least active compound with a docking score of −5.5159 and having developed two H bond donor interactions with Glu 233 active residues of the target protein, as shown in Figure 6D.The interaction of the synthesized compounds with SS-DNA was further confirmed theoretically by a molecular docking analysis in order to gain insight about the mechanism of interactions.The MOE's rigid molecular docking approach was utilized to evaluate the binding modes of the synthetic complexes with SS-DNA duplex.The whole DNA duplex was considered for docking to determine the preferred binding sites, binding strength, and the orientation of complexes 1 and 2 within the DNA groove and base pairs.The interaction details of both the complexes (1 and 2) and the free ligand acid are presented in Table 7.The docking analysis reveals that the complexes electrostatically interact with the oxygen atom on the phosphate backbone of DNA outer edge stacking.The synthesized complexes form hydrogen bond donors and C-H-pi interactions in contact with the DNA  The interaction of the synthesized compounds with SS-DNA was further confirmed theoretically by a molecular docking analysis in order to gain insight about the mechanism of interactions.The MOE's rigid molecular docking approach was utilized to evaluate the binding modes of the synthetic complexes with SS-DNA duplex.The whole DNA duplex was considered for docking to determine the preferred binding sites, binding strength, and the orientation of complexes 1 and 2 within the DNA groove and base pairs.The interaction details of both the complexes (1 and 2) and the free ligand acid are presented in Table 7.The docking analysis reveals that the complexes electrostatically interact with the oxygen atom on the phosphate backbone of DNA outer edge stacking.The synthesized complexes form hydrogen bond donors and C-H-pi interactions in contact with the DNA functional groups that define the minor groove because they fit snugly into the curved contour of the target DNA in the groove.The theoretical interaction analysis of the complexes strongly validated the experimental finding of the spectroscopic analysis of the complexes.Complex 2, with a docking score of −7.43, forms two hydrogen bond donor interactions with DA17 and one C-H-pi interaction with DA18, as shown in Figure 7B.The C-H-pi interaction is a type of interaction that involves the π system.This type of bond is formed between a hydrogen atom and the π cloud.This type of interaction exists where aliphatic or aromatic CH groups are involved.Complex 1 (docking score of −6.59) shares one hydrogen bond donor and one C-H-pi interaction with DT 7 and DC 21, respectively, as shown in Figure 7A.The free ligand acid, with a docking score of −5.11, forms one H-donor interaction with DC 9, as shown in Figure 7C.The C-H-pi interaction is a type of interaction that involves the π system.This type of bond is formed between a hydrogen atom and the π cloud.This type of interaction exists where aliphatic or aromatic CH groups are involved.Complex 1 (docking score of −6.59) shares one hydrogen bond donor and one C-H-pi interaction with DT 7 and DC 21, respectively, as shown in Figure 7A.The free ligand acid, with a docking score of −5.11, forms one H-donor interaction with DC 9, as shown in Figure 7C.

In Silico Studies
Table 8 presents the physicochemical parameters including solubility, lipophilicity, toxicity risks, drug-likeness, pharmacokinetics and medicinal chemistry of the synthesized compounds, evaluated according to the following filters: Lipinski [30], Ghose [31], Veber [32], Egan [33] and Muegge [34] using the SwissADME server [35].An orally-active compound should follow these filters and should not have more than one violation.Since both complexes 1 and 2 have the same molecular weight and formula, their SwissADME data is the same.Drug likeness of a compound indicates the resemblance between the compound and effective drugs [36,37], and it is determined from its Fsp 3 value which corresponds to the percentage of sp 3 -carbon atoms in the molecule.About 84% of commercially available drugs have Fsp 3 ≥ 0.42 [38].For the synthesized compounds, the Fsp 3 values are 0.12 (HL) and 0.14 (1, 2).The molar refractivity values for the screened compounds are 37.90 (HL) and 102.50 (1, 2).The predicted octanol/water partition coefficients (logPo/w) were in the range of 0.00−6.15.
The logP value, a measure of lipophilicity that determines the overall quality of a drug molecule, is of great importance [39,40], and it can be evaluated by various druglikeness filters, i.e., MLogP for Lipinski, WlogP for Ghose and Egan, and XlogP for Muegge filters, as well as their mean values (consensus logP).The recommended range of lipophilicity (logP) for a drug is from −0.4 to 5.6 [31].All logP values for the HL and compounds 1 and 2 are in the recommended range (except XLOGP and WLOGP in the case of 1 and 2 which are very slightly higher than the limit).
The estimated aqueous solubility (ESOL) of a compound is an important parameter in drug discovery, and the ideal range of the ESOL is from 5 to 8 [41].The ESOL values for the screened compounds are −2.34 (HL) and −13.25 (1 and 2), which fall in the class of soluble to insoluble.
A bioavailability score (BS) is expressed as the probability of a compound with >10% bioavailability in rat or Caco-2 permeability [42], and the recommended value is 0.11 for compounds with tPSA > 150 Å 2 , while the recommended value is 0.85 for compounds with tPSA < 75 Å 2 .The BS values for the screened compounds are 0.85 (HL) and 0.55 (1 and 2).
The human gastrointestinal absorption values were high for the HL and the complexes 1 and 2 [43].P-glycoprotein and cytochromes P450 can be evaluated in terms of pharmacokinetics.The HL does not act as the P-glycoprotein substrate which detects that Drug likeness of a compound indicates the resemblance between the compound and effective drugs [36,37], and it is determined from its Fsp 3 value which corresponds to the percentage of sp 3 -carbon atoms in the molecule.About 84% of commercially available drugs have Fsp 3 ≥ 0.42 [38].For the synthesized compounds, the Fsp 3 values are 0.12 (HL) and 0.14 (1, 2).The molar refractivity values for the screened compounds are 37.90 (HL) and 102.50 (1,2).The predicted octanol/water partition coefficients (logP o/w ) were in the range of 0.00−6.15.
The logP value, a measure of lipophilicity that determines the overall quality of a drug molecule, is of great importance [39,40], and it can be evaluated by various drug-likeness filters, i.e., MLogP for Lipinski, WlogP for Ghose and Egan, and XlogP for Muegge filters, as well as their mean values (consensus logP).The recommended range of lipophilicity (logP) for a drug is from −0.4 to 5.6 [31].All logP values for the HL and compounds 1 and 2 are in the recommended range (except XLOGP and WLOGP in the case of 1 and 2 which are very slightly higher than the limit).
The estimated aqueous solubility (ESOL) of a compound is an important parameter in drug discovery, and the ideal range of the ESOL is from 5 to 8 [41].The ESOL values for the screened compounds are −2.34 (HL) and −13.25 (1 and 2), which fall in the class of soluble to insoluble.
A bioavailability score (BS) is expressed as the probability of a compound with >10% bioavailability in rat or Caco-2 permeability [42], and the recommended value is 0.11 for compounds with tPSA > 150 Å 2 , while the recommended value is 0.85 for compounds with tPSA < 75 Å 2 .The BS values for the screened compounds are 0.85 (HL) and 0.55 (1 and 2).
The human gastrointestinal absorption values were high for the HL and the complexes 1 and 2 [43].P-glycoprotein and cytochromes P450 can be evaluated in terms of pharmacokinetics.The HL does not act as the P-glycoprotein substrate which detects that there is no effect for such protein on its bioactivity, while the complexes 1 and 2 act as the P-glycoprotein substrate which shows that there is an effect for such protein on the bioactivity of these compounds.
The value of skin permeation is expressed as log(Kp), and for compounds with a molecular weight (MW) in the range from 10 to >750, the value of log(Kp) is from −3 to +6 cm/s.A high negative value of log(Kp) indicates that the compound has less penetration into the skin [44].The values of log(Kp) for the screened compounds are −6.01 (HL) and −4.98 (1 and 2).Thus, the free ligand acid HL has greater skin penetration ability than its complexes [45].
Synthetic accessibility of drug-like molecules is needed in many areas of the drug discovery process.Its values for the evaluated compounds (1.54 for HL and 4.97 for 1 and 2) fall in the range of commercially available drugs [46].

Materials and Methods
Copper(II) chloride dihydrate; 3,4-difluorophenylacetic acid; 2-methylpyridine; 3-methylpyridine; 2,2-diphenyl-1-picrylhydrazyl radical (DPPH); ascorbic acid; iron(II) sulfate; hydrogen peroxide; nitric acid; sodium salt of the salmon sperm DNA (SS-DNA); methanol; and DMSO were purchased from Sigma-Aldrich, St. Louis, MO, USA.These chemicals were used without any further treatment.Distilled water was used for the experimental work.The melting points of the synthesized complexes were recorded in a capillary tube using a digital electro-thermal melting point apparatus.Elemental analysis was performed on a Leco CHNS 932.A Perkin Elmer atomic absorption spectrometer A analyst 700 was used to determine the percentage of copper.The electronic absorption spectra (200-800 nm) of the complexes were recorded using a Perkin Elmer UV/Vis spectrometer Lambda 25 in DMSO solvent.A nicolet-6700 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to record FT-IR spectra in the range of 4000-400 cm −1 , adopting the attenuated total reflectance (ATR) technique.

Syntheses of the Complexes
For the synthesis of complexes 1 and 2, 3,4-difluorophenylacetic acid (3.44 g, 20 mmol) dissolved in methanol (15 mL) was added dropwise to the constantly stirred solution of copper(II) chloride dihydrate (1.70 g, 10 mmol) in methanol (15 mL).The reaction mixture was refluxed for 3 h followed by the addition of 2-methylpyridine (1.94 mL, 20 mmol) for complex 1 and 3-methylpyridine (1.93 mL, 20 mmol) for complex 2. The resulting reaction mixture was refluxed for a further 3 h.Then, the reaction mixture was cooled to room temperature.The solution was transferred to a beaker and kept undisturbed.Sea green color crystals were obtained after a few days, and they were separated by filtration and analyzed by different analytical and spectroscopic methods.

Single-Crystal X-ray Analysis
Diffraction data were collected by the ω-scan technique, at 100(1) K, on a Rigaku XCalibur four-circle diffractometer with an Eos CCD detector, equipped with a graphitemonochromatized MoK α radiation source (λ = 0.71073 Å).The data were corrected for Lorentz polarization as well as for absorption effects [47].The structures were solved with SHELXT [48] and refined with the full-matrix least-squares procedure on F 2 by SHELXL-2013 [49].All non-hydrogen atoms were refined anisotropically, hydrogen atoms were placed in idealized positions and refined as 'riding model' with isotropic displacement parameters set at 1.2 (1.5 for methyl groups) times the U eq of appropriate carrier atoms.In structure 2, one of the difluorophenyl rings was disordered over two positions, rotated approximately by 180 • .The site occupation factors for these positions converged at 65.8(4)%:34.2(4)%.Weak restraints were applied to the shapes of displacement ellipsoids in disordered fragments.

Hirshfeld Surface Analysis
The X-ray single-crystal cif files were used as input files to generate the Hirshfeld surfaces as well as 2D-fingerprint plots using Crystal Explorer 17.50 [50].The following default settings were applied: property, none and resolution, high (standard).For fingerprint generation (di vs. de plot) we applied: range = standard and filter = by elements, whereas fingerprint filter options were both in-and outside elements including reciprocal contacts.The interactions with normalized contact distance in crystal structure that are shorter than the sum of the corresponding van der Waals radii of the atoms are shown as red spots, while those with longer contacts having the positive d norm value are highlighted in blue color.White regions correspond to the distance of contacts that are equal to the van der Waals separation having a d norm value of zero [51].

DNA Binding Study
The complexes were screened for their binding potential to salmon sperm DNA (SS-DNA) by using UV-visible absorption spectroscopy and viscometry.

DNA Binding Study by UV-Visible Absorption Spectroscopy
Sodium salt of the SS-DNA (10 mg) was dissolved in distilled water.The solution was continuously stirred for 24 h.The solution was found sufficiently free of protein impurities as indicated by the nucleotide to protein (N/P) ratio of ~1.9, observed at 260 and 280 nm (A 260 /A 280 ) [52].The SS-DNA solution (ε = 6600 M −1 cm −1 at 260 nm) concentration was determined via absorption spectroscopy [53].The complex solutions (200 µM) were made in aqueous DMSO (1:4).The absorption titrations were performed with fixed concentrations of the complex solutions (200 µM) against varying concentrations (5-30 µM) of SS-DNA solutions.To eliminate the absorbance of DNA, equivalent SS-DNA solutions were added to the complex and reference solutions.The complex-DNA solutions were incubated for one hour at room temperature before absorption titrations.For the absorption spectra, cuvettes of 1cm path length were used at room temperature (25 ± 1 • C).

DNA Binding Study by Viscometry
The viscosity of the SS-DNA solution (87.72 µM) in the absence and the presence of complexes 1 and 2 (5-30 µM) was measured using an Ostwald viscometer.For accuracy, the readings were made in triplicate for each sample, and then an average flow rate was calculated.Data are presented as (η/η o ) 1/3 vs. binding ratio ® of [complex]/[DNA], where η is the relative viscosity of the complex-DNA adduct, and η o is the relative viscosity of DNA alone.The relative viscosity values for SS-DNA in the presence (η) and the absence (η o ) of the complexes were calculated using the relation η = (t − t o )/t o , where t is the observed flow time in seconds [54].

DPPH Free Radical Scavenging Activity
The in vitro DPPH free radical scavenging activities of the free ligand acid and complexes 1 and 2 were assessed, as per a reported procedure with slight modification in the protocol [55].
Different concentrations (15,30,60,120, and 240 µM) of the free ligand acid and complexes were mixed with 1000 µL of 255 µM DPPH solution made in ethanol.The mixture solutions were shaken and incubated in the dark for 30 min at room temperature.The absorbance of the solutions was measured at 518 nm using a UV-VIS spectrophotometer.Ascorbic acid of fixed concentration was used as a positive control.The readings were made in triplicate.Percentage inhibition of the test compounds was evaluated by using the formula: %scavenging = A c − A s A c × 100 where A c and A s are the observed absorbance values of the control and the sample, respectively.

Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activities of the free ligand acid and synthesized complexes (1 and 2) were evaluated according to a reported method [56].Different concentrations (15,30,60,120, and 240 µM) of the free ligand acid and complexes were mixed with 0.4 M phosphate buffer (pH 6.6) (1000 µL), 0.1% hydrogen peroxide (100 µL), 3.7 mM o-phenanthroline (200 µL), and 7.5 mM ferrous sulfate (100 µL).The mixtures were diluted with distilled water to 3000 µL before incubating for 30 min at room temperature.The absorbance of mixtures was recorded at 510 nm using ascorbic acid as a standard.
The %scavenging was calculated using the following formula: where A s , A c , and A b are the observed absorbance values of the sample, control, and blank, respectively.

Amylase Enzyme Inhibition Study
A reported protocol with slight modification was used to perform the α-amylase enzyme inhibition assay [57].A stock solution (1 unit/mL) was prepared by dissolving α-amylase solution from Saccharomyces cerevisiae in 50 mM potassium phosphate buffer (pH 6.8), and for the substrate, in the same buffer 20 mM p-nitrophenyl-α-Dglucopyranoside (PNG) was prepared.The assay was conducted in triplicate using 96-well plates.Sample solutions (62.5, 125, and 250 µM) were made in DMSO solvent.For the experiments, each well contained phosphate buffer 65 µL (50 mM, pH 6.8), PNG 25 µL, α-amylase enzyme 5 µL (0.05 U/mL), and test sample 5 µL.The plates were allowed to incubate at 37 • C for 30 min, followed by the addition of 100 µL sodium bicarbonate (0.5 mM) as a stopping agent.Acarbose and DMSO were used as positive and negative controls, respectively.
By using a microplate reader (BioTek Elx-800, Winooski, VT, USA), absorbance was recorded at 405 nm.The percent inhibition was calculated using the formula: where A control is the control absorbance and A sample is the sample absorbance.

Molecular Docking Studies
The molecular docking study was performed using the MOE (2016) software.The 3D structure of alpha-amylase with PDB ID (3BAJ) and salmon sperm DNA (1BNA) was retrieved from the protein databank (PDB) database [58,59].All the water molecules were removed prior to energy minimization.Energy minimization was performed up to 0.05 gradients with MMFF94s force field.The 3D structure coordinates of the free ligand acid, synthesized complexes, and control (acarbose) were built and saved in a new MOE database.The substrate-binding site of alpha-amylase was specified for molecular docking,

Figure 1 .
Figure 1.Perspective view of complex 1; ellipsoids are drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radii.The unlabeled part is related to the labeled one by the symmetry operation 1-x, 1-y, 1-z.

Figure 1 .
Figure 1.Perspective view of complex 1; ellipsoids are drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radii.The unlabeled part is related to the labeled one by the symmetry operation 1-x, 1-y, 1-z.Pharmaceuticals 2023, 16, 1462 4 of 21

Figure 2 .
Figure 2. Perspective view of one of the symmetry-independent molecules of complex 2; ellipsoids are drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radii.The unlabeled part is related to the labeled one by the symmetry operation -x, -y, 2-z.

Figure 2 .
Figure 2. Perspective view of one of the symmetry-independent molecules of complex 2; ellipsoids are drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radii.The unlabeled part is related to the labeled one by the symmetry operation -x, -y, 2-z.
Pharmaceuticals 2023, 16, 1462 10 of 21DNA adduct viscosity show intercalation and groove binding as the possible mode of complex-DNA interactions.

Figure 5 .
Figure 5. Effects of increasing the amounts of free ligand acid HL and the complexes (1 and 2) on the relative viscosity of SS-DNA.

Figure 5 .
Figure 5. Effects of increasing the amounts of free ligand acid HL and the complexes (1 and 2) on the relative viscosity of SS-DNA.

Figure 6 .
Figure 6.Docked conformations of the HL, the compleses 1 and 2, and the control: (A) The surface representation of the substrate binding pocket of alpha-amylase; (B) the 3D binding mode of 1, (C) 2, (D) HL, and (E) control (acarbose), against alpha-amylase.

Figure 7 .Table 7 .Figure 7 .
Figure 7. 3D representation of the binding poses of: (A) Complex 1; (B) complex 2; (C) free ligand acid HL, with SS-DNA.Table 7. Docking score interactions report for the free ligand acid HL and the complexes (1 and 2) against SS-DNA.Compound Interaction Type Interacting Residues Distance Energy (Kcal/mol) Docking Score HL H-donor DC 9 3.55 −0.5 −5.1124 is used to determine the drug transport parameters [35] and compounds with a smaller tPSA value have greater drug transport potential.The tPSA values for the synthesized compounds are as follows: HL (37.00 Å 2 ) and 1-2 (73.21 Å 2 ).The HL and complexes follow the Lipinski Rule of five (MW < 500, LogP < 5, No. H-bond acceptors < 10, No. H-bond donor < 5) [35].Figure 8 represents the bioavailability radar image of HL and complex 1. Pharmaceuticals 2023, 16, 1462 14 of 21 the H2O molecules in an aqueous solution were 1 (HL) and 0 (complexes 1 and 2) and 4 (HL) and 8 (complexes 1 and 2), respectively.The predicted numbers of rotatable bonds were in the range of 2-10.The total surface area (tPSA) due to polar atoms of a molecule is used to determine the drug transport parameters [35] and compounds with a smaller tPSA value have greater drug transport potential.The tPSA values for the synthesized compounds are as follows: HL (37.00 Å 2 ) and 1-2 (73.21 Å 2 ).The HL and complexes follow the Lipinski Rule of five (MW < 500, LogP < 5, No. H-bond acceptors < 10, No. H-bond donor < 5) [35].Figure 8 represents the bioavailability radar image of HL and complex 1.

Table 1 .
Crystal data, data collection, and structure refinement for complexes 1 and 2.

Table 3 .
DPPH free radical scavenging activity of the free ligand acid HL and the complexes (1 and 2).

Table 4 .
Hydroxyl radical scavenging activity of the free ligand acid HL and the complexes (1 and 2).

Table 5 .
% α-Amylase activity and IC 50 values of the free ligand acid HL and the complexes (1 and 2).

Table 6 .
Docking score interactions report for the free ligand acid HL, the complexes (1 and 2), and acarbose, towards α-amylase.

Table 6 .
Docking score interactions report for the free ligand acid HL, the complexes (1 and 2), and acarbose, towards α-amylase.

Table 7 .
Docking score interactions report for the free ligand acid HL and the complexes (1 and 2) against SS-DNA.

Table 8 .
In silico properties for HL and the complexes (1 and 2) determined by SwissADME.