*Article* **Synthesis of Two Novel Copper (II) Complexes as Potential Inhibitors of HIV-1 Protease Enzyme: Experimental and Theoretical Investigations**

**Meriem Hamlaoui 1,\*, Ikram Hamlaoui 2, Maamar Damous 1,3, Youghourta Belhocine 3,\*, Najoua Sbei 4, Fatima Adam Mohamed Ali 5, Mashael A. Alghamdi 5, Sarra Talab 6, Seyfeddine Rahali <sup>7</sup> and Hocine Merazig 1,\***


**Abstract:** In this study, we report the synthesis of two new copper complexes: [Cu(C11H7O2)(SCN)(C10H8N2)], denoted as (C-1), and [Cu(C11H7O2) (C12H8N2) Cl]·H2O, denoted as (C-2). They are based on 2,2 -bipyridine or 1,10-phenanthroline and 2-hydroxy-1-naphtaldehyde ligands. The obtained complexes were characterized by FT-IR, UV-visible spectroscopy, and singlecrystal X-ray diffraction analysis. Molecular docking was employed to predict the binding mode involved in the interaction between the two synthetic copper (II) complexes and HIV-1 protease enzyme. The X-ray structural analysis revealed that the crystal structures of both complexes are mainly stabilized by several intra- and intermolecular hydrogen bonds. The fingerprint plots associated with the Hirshfeld surfaces of both complexes clearly show that H···H interactions provide the largest contributions. According to the docking results, the synthesized complexes exhibit promising features which enable them to be bound to the HIV-protease enzyme.

**Keywords:** copper (II) complex; synthesis; HIV-1 protease enzyme inhibitor; crystal structure; non-covalent interactions; Hirshfeld surface; molecular docking

## **1. Introduction**

In recent years, there have been several reports dedicated to the development of transition metal complexes that are based on the 2-hydroxy-1-naphthaldehyde C11H8O2 ligand [1]. Indeed, 2-hydroxy-1-naphthaldehyde ligands can be used as a starting block for the synthesis of new ligands [2,3], or as a potential chelating agent for ions; however, only a few studies were focused on the synthesis of copper (II) complexes of bidentate ligands [4,5].

Moreover, the 2,2 -bipyridine C10H8N2 and 1,10-phenanthroline C12H8N2 bidentate chelating ligands are widely used as complexing moieties to form stable coordination

**Citation:** Hamlaoui, M.; Hamlaoui, I.; Damous, M.; Belhocine, Y.; Sbei, N.; Ali, F.A.M.; Alghamdi, M.A.; Talab, S.; Rahali, S.; Merazig, H. Synthesis of Two Novel Copper (II) Complexes as Potential Inhibitors of HIV-1 Protease Enzyme: Experimental and Theoretical Investigations. *Crystals* **2022**, *12*, 1066. https:// doi.org/10.3390/cryst12081066

Academic Editor: Waldemar Maniukiewicz

Received: 29 June 2022 Accepted: 22 July 2022 Published: 30 July 2022

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complexes with miscellaneous transition metal ions [6]. Transition metal complexes that have these ligands are recognized in the literature as potent antibacterial and antimicrobial agents [7–9]. It was also reported that copper complexes are good candidates for the design of novel types of protease enzyme inhibitors; here, the main goal is to treat the acquired immunodeficiency syndrome (AIDS) infection induced by the human immunodeficiency virus (HIV). Since the protease of the human immunodeficiency virus is key to the replication of the virus, the enzyme is therefore considered an attractive target for antiviral therapy; thus, the treatment strategy for AIDS involves the inhibition of HIV-1 protease [10]. To date, real progress in terms of therapy for AIDS has been achieved by using peptide-based therapeutics; however, the toxicity of peptides reduces the effectiveness of this treatment [11,12]. The copper complexes, which contain non-peptide ligands that have therapeutic potential, could be an alternative methodology for the treatment of AIDS-induced diseases.

In this contribution, we describe the synthesis of two new complexes, namely, the [(2,2 -bipyridine-K2N,N ) (1-formyl-2-naphtholato-K2O,O ) (Thiocyanate-KN) copper(II)], denoted as C-1, and [(1-formyl-2-naphtholato-K2O,O ) (1,10-phenanthroline-K2N,N ) chloride copper], denoted as C-2. FT-IR, UV–visible spectroscopy, and single-crystal X-ray diffraction analysis were used to characterize and analyze the structure of the two synthesized complexes. DFT calculations using the B3LYP [13–16] functional were conducted on the molecular structures of C-1 and C-2, and the findings were compared with the X-ray crystal data. The docking of C-1 and C-2 into the HIV-1 protease active site was assessed through the calculation of binding energy with the Autodock vina program [17], and the obtained results were compared with those of the (dimethanol)bis[N-(4-methyl-2-pyridyl)- 2,3,6-trimethoxybenzamide] copper (II) dimethanol diperchlorate complex as a reference (complex-ref), which was found to be a non-peptide HIV-1 protease inhibitor with an IC50 of 1.5 μM [18]. It is well known that the most effective inhibitor is the one that forms a bond deep in the hydrophobic cavity of the HIV-1 protease active site, and is positioned in the axes of an octahedron with the four subsites S1/S2 and S 1/S 2 forming the equatorial plane. The binding pocket of this enzyme includes the Asp25 (25 )-Thr26 (26 )-Gly27(27) catalytic triad and flap regions, which are most likely involved in the binding process of substrates or inhibitors [19,20].

#### **2. Experimental**

#### *2.1. Materials and Instruments*

All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, St. Louis, MI, USA), and they were used in the form in which they were received.

Infrared spectra were recorded in the range of 4000–400 cm−<sup>1</sup> on a FT-IR Bruker ATR Vertex 70 Spectrometer. UV-visible spectra were recorded on a UV–visible Spectrophotometer Optizen 1220. The absorbance of ligands and their metals complexes were measured in DMSO in a concentration of 10−<sup>5</sup> mol·L<sup>−</sup>1, in a wavelength range of 280–440 nm.

Crystals suitable for single crystal X-ray diffraction were selected, and the lattice parameters were determined using an APEXII Bruker diffractometer.

#### *2.2. General Procedure for the Synthesis of C-1 and C-2*

2.2.1. Synthesis of [(2,2 -Bipyridine-K2N,N ) (1-formyl-2-Naphtholato-K2O,O ) (Thiocyanate-KN)-Copper (II)] (C-1)

For the synthesis of C-1, Cu(CH3COO)2. H2O (0.25 mmol) and 2-hydroxy-1-naphthaldehyde (0.125 mmol) were added and stirred into a DMSO solution containing 0.25 mmol of 2,2 -bipyridine and 0.125 mmol of KSCN. The mixture was stirred for 1 h at T = 25 ◦C (Scheme 1a). After several days of slow evaporation, light green crystals appeared. This complex is stable in the air and soluble only in DMSO. Yield: 61%. IR (cm−1): 2063.83(s), 1604.77(s), 1577.77(s), 1531.48(s), 1423.47(s), 1392.61(s), 1365.60(s), 1311.59(m), 1184.29(m), 1141.86(w), 1014.56(s), 972.12(w), 948.98(m), 829.39(m), 721.38(s), 636.51(w), 563.21(w), 497.63(w). UV–Vis (DMSO) (λmax, nm): 290, 300 (π-π\*) 305, 315.

**Scheme 1.** Synthesis of (**a**) C-1, (**b**) C-2.

2.2.2. Synthesis of [(1-Formyl-2-naphtholato-K2O,O ) (1,10-Phenanthroline-K2N,N ) Chloride Copper] (C-2)

A mixture of CuCl2.2H2O (0.167 mmol), 2-hydroxy-1-naphthaldehyde (0.167 mmol), 1,10-phenanthroline (0.167 mmol), and 4-dimethyl aminopyridine (0.167 mmol) was dissolved in ethanol. The solution was refluxed for 2 h at 60 ◦C (Scheme 1b). After the slow evaporation of the solution, green single crystals were obtained and characterized by X-ray diffraction. Yield: 68%. IR (cm<sup>−</sup>1): 1608.63(s), 1581.63(s), 1539.20(m), 1423.47(s), 1392.61(s); 1365.60(m), 852.54(s), 825.53(s), 752.24(m), 717.52(s), 543.93(s), 520.78(s), 493.78(s). UV–Vis (DMSO) (λmax, nm): 290, 300 (π-π\*) 310, 325.

#### *2.3. X-ray Diffraction*

Data collection was performed using Mo*K*α radiation with a BRUKER APEX2 diffractometer [21]. Cell refinement and data reduction were performed through the SAINT program. SHELXS and SHELXL [22] were used for structure solution and refinement. Structure refinement and crystal data details are reported in Table 1. All hydrogen atoms were refined in geometrically idealized positions, with C-H = 0.93 Å for aromatic rings (2,2 -bipyridine, 1,10-phenanthroline), and they were allowed to ride on their parent atoms, with Uiso(H) = 1.2 Ueq(C). The molecular structures of the two complexes are illustrated in Figure 1.

#### *2.4. Computational Methodology*

The structural optimization of C-1 and C-2 was performed using the framework of the density functional theory (DFT). The B3LYP hybrid functional was used with a 6–31 + G(d) basis set for C, H, N, Cl, O, S, and SDD for Cu. The DFT computations were conducted using the Gaussian 09W software package [23]. The PDB file of the ligand was generated using OpenBabel, version 2.4.1 [24,25]. The HIV-1 protease–ligand interaction properties were assessed by performing docking studies using AutoDock vina software. The threedimensional structure of HIV-1 protease (PDB ID: 3TLH) was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb (accessed on 8 March 2022)), and it was used to prepare the target site for docking calculation. The water molecules and ligand were removed from the protein, and the missing hydrogen atoms and charge were corrected. The search grid of the HIV-1 protease was identified as center\_x: −6.118, center\_y: 23.382, center\_z: 36.63 with dimensions size\_x: 62, size\_y: 82, and size\_z: 76. The best-scoring conformation was visualized and analyzed using Autodock vina tools.


**Table 1.** Crystallographic data and refinement parameters of C-1 and C-2.

**Figure 1.** An ORTEP view of the asymmetric unit of the two complexes. (**a**) [Cu (C11H7O2) (SCN) (C10H8N2)] (C-1), (**b**) [Cu(C11H7O2) (C12H8N2) Cl]·H2O (C-2).

#### **3. Results and Discussions**

#### *3.1. Crystal Structure of C-1 [Cu (C11H7O2) (SCN) (C10H8N2)]*

The single-crystal X-ray diffraction shows that the [Cu (C11H7O2) (SCN) (C10H8N2)] complex crystallizes in the monoclinic space group *P*21*/n* with Z = 4. The asymmetric unit of this structure is formed by a Cu(II) that coordinates with a thiocyanate ligand in an apical position, together with a nitrogen atom (N3). The coordination sphere of Cu(II) is completed by one 2,2 -bipyridine molecule acting as a bidentate ligand (through the two nitrogen atoms (N1, N2)), and one 2-hydroxy-1-naphthaldehyde (through the two oxygen atoms (O1, O2)). The Cu(II) atom has five coordinates, and it displays a square-pyramidal coordination geometry, as quantified by the value of the structural index [26] τ<sup>5</sup> = 0.019 (τ<sup>5</sup> = (β − α)/60, where β = N1-Cu1-O2 = 164.11◦ and α = N2-Cu1-O1 = 162.97◦. (Perfect coordination geometries of square pyramidal and trigonal bipyramidal arrangements are associated with the τ<sup>5</sup> values of zero and unity, respectively). The basal plane is formed by (O1, O2, N1, N2) and N3 atoms in the axial site (Figure 2a).

**Figure 2.** The copper square-pyramidal environment (**a**), and the dihedral angle between the two ligands (**b**).

The dihedral angle between the two planes that contain ligands is 3.65◦. The Cu-N and Cu-O distances in the basal plane are [(Cu1-N1 = 2.005 Å, Cu1-N2 = 2.001 Å, Cu1-O1 = 1.908 Å, Cu1-O2 = 1.943 Å], and the apical Cu1-N3 bond 2.188 Å is longer than those of the basal plane (Table S1); this is in accordance with the data in the literature [27]. These parameters were also calculated in the gas phase using the B3LYP functional, and the results indicate that the calculated bond lengths in the copper coordination sphere agree with the corresponding experimental values within a maximum deviation of 2.3% (Table S1). The bond angles exhibit a maximum deviation of 10%. This discrepancy between the experimental and theoretical values may be due to environmental effects; indeed, the theoretical values were obtained from gas-phase DFT calculations, whereas the experimental values were taken from crystallographic data.

The crystalline structure of C-1 consists of four hydrogen bond networks (Table 2). Two intermolecular hydrogen bonds are observed between H7 of C11 with S1 (½ − *x*, ½ + *y*, ½ − *z*), and H14 of C20 with S1(*x* + 1, *y*, *z*). Moreover, two intramolecular hydrogen bonds of the C-H···O type are established between 2,2 -bipyridine and 2-hydroxy-1 naphthaldehyde, subsequently generating S(5) ring motifs [28,29]; thus, the presence of hydrogen-bond networks points to the significant role of intra and intermolecular hydrogen bonding in terms of the stabilization of complex structures. Indeed, the networks facilitate intramolecular contact and they hold the chains in a zig-zag arrangement along the a-axis (Figure 3a).


**Table 2.** Hydrogen-bond geometry (Å, ◦) in C-1 and C-2 complexes.

Symmetry codes: (i) −*x* + 1/2, *y* + 1/2, −*z* + 1/2; (ii) *x* + 1, *y*, *z*; (I): 1 − *x*, −*y*, 1 − *z*; (II): −*x*, 1 − *y*, −*z*; (III): −*x*, −*y*, −*z*; (IV): −1 + *x*, *y*, *z*; (V): 1 + *x*,1+ *y*, *z*.

**Figure 3.** (**a**) Zig-zag chains of C-1 formed by hydrogen bonds (dashed lines), (**b**) the aromatic ring organization with centroid–centroid distances (Å).

(**a**)

In addition, the C-1 complex exhibits three π···π stacking interactions; the first is between the centroid of the 2-hydroxy-1-naphthaldehyde ring Cg1 (C4 to C9) and the centroid of the 2,2 -bipyridine ring Cg2 (N2 and C17 to C21), with a Cg1 ... Cg2 distance of 3.601 Å, the second is between the Cg3 (C1/C2/C3/C4/C9/C10) rings of the 2-hydroxy-1-naphthaldehyde, with a Cg3···Cg3 distance of 3.919 Å, and the third is between Cg2 and Cg3, with a distance of 3.887 Å (Figure 3b).
