Synthesis,crystal structure,the Hirshfeld surface analysis,and antimicrobial activity of a three-dimensional Cu(II) complex with N-substituted biguanide

Abstract

A new antibacterial complex C₄H₁₅CuN₅O₇S, which is derived from the non-insulin-dependent diabetes mellitus drug metformin hydrochloride (MH) and cupric sulfate pentahydrate, is prepared and characterized. The complex shows that a three-dimensional supramolecular structure and the intermolecular interactions are analyzed by the Hirshfeld surface analysis. More importantly, compared with the MH free ligand, the present complex shows excellent antimicrobial activity, especially for Gram-negative bacteria.

Introduction

The development of metal–drug complexes has attracted significant attention over the past decades. Metal–drug complexes show high diversity of possible structures due to their dynamic nature,^1,2 and the wide application of coordination of metal ions to biologically active agents in many fields has inspired numerous scientific studies toward constructing drugs.^3,4 Research on metal–drug complexes mainly focuses on anticancer, antimicrobial, and antiviral activity, and altering the biological and pharmacological properties of the free drugs.^5–9 Furthermore, the utilization of metal–drug complexes has been considered as a promising method to treat challenging diseases,^10,11 including cancer and HIV.^12,13 Metformin hydrochloride (MH), as an example of a bioligand containing a biguanide skeleton, is a first-line drug for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) throughout the world.^14,15 MH has a relative 50%–60% bioavailability and its short and variable biological half-life is 0.9–2.6 h. ¹⁶ The absorption of MH is limited to the upper gastrointestinal (GI) tract. In the pharmaceutical field, it is also found that metal complexes of MH show obvious antimicrobial activity bial activity compared to the free bioligand, due to its good chelating properties because of the close proximity of its two =NH functional groups.

The free drug of MH has two different polymorphs, both of which have monoclinic structures: stable form I and metastable form II.^17,18 The structure of MH indicates that it can coordinate with cations or anions. Herein, we are concerned with the attractive properties of MH coordinated with metal ions and the new promising opportunities they may bring in the development of metal–drug complexes. This study constructs a novel three-dimensional (3D) metal–drug complex by self-assembly of MH with hydrated copper sulfate. The crystal structure was analyzed by means of single-crystal X-ray diffraction, and molecular Hirshfeld surface analysis was performed for the purpose of investigating intermolecular interactions. In addition, evaluation of the antimicrobial activity of the title compound has been performed against Gram-positive and Gram-negative bacteria (e.g. Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa).

Results and discussion

Description of the crystal structure of complex 1

Single-crystal X-ray diffraction analysis revealed that complex 1 crystallizes in the monoclinic system with space group P21/n and exhibits 3D interpenetrating units. The unit of complex 1 consists of one Cu(II) ion, one MH ligand, three coordinated water molecules, and one ${\mathrm{SO}}_4^{2-}$ counterion. As shown in Figure 1, the Cu(II) ion is penta-coordinated with three oxygen atoms (O1, O2, and O3) from coordinating water molecules and two nitrogen atoms (N0 and N1) from the MH ligand, which forms a six-element ring. The lengths of the Cu–O bonds varied from 1.974(5) to 2.471(4) Å, while the Cu–N bond lengths are 1.922(5) (Cu–N0) and 1.916(4) Å (Cu–N1). In the crystal structure of complex 1, each structural unit is connected to another by hydrogen bond (Cu–O. . .O–S) interactions between the coordinating water molecules and ${\mathrm{SO}}_4^{2-}$ counterions in the primary level forming an infinite one-dimensional zigzag chain structure (Figure 2(a)). As shown in Figure 2(b), the one-dimensional chain structure is further connected via a 3D structure through hydrogen bonds (Cu–O. . .O–S) between coordination water molecules and ${\mathrm{SO}}_4^{2-}$ counterions and hydrogen bonds (N. . .O. . .N) between ${\mathrm{SO}}_4^{2-}$ counterions and MH ligands. The crystal data and refinement parameters of complex 1 are shown in Table 1 (The higher R1 factor and goodness-of-fit values were caused by lattice water, but this does not affect the accuracy of the data.). Selected bond lengths (Å) and angles (°) are presented in Table 2.

Figure 1.

Crystal structure of complex 1.

Figure 2.

(a) 2D and (b) 3D crystal structures of complex 1.

Table 1.

Crystal data and structure refinement for complex 1.

Identification code
Formula	C4H15CuN5O7S
Formula weight	340.81
Crystal system	Monoclinic
Space group	P21/n
a (Å)	7.02170(10)
b (Å)	18.6879(3)
α (°)	90
B (°)	98.1510(10)
γ (°)	90
V (Å3)	1205.58(3)
Z	4
Dcalc (Mg m−3)	1.878
T (K)	293(2)
μ (mm−1)	1.54184
Crystal dimensions	0.2 × 0.1 × 0.1
Reflections collected	8946
Reflections unique	2391
Parameters	170
Goodness-of-fit on F2	1.500
R1, wR2 (I > (σI))	0.1247, 0.3053
R1, wR2 (all data)	0.1337, 0.3372
CCDC number	2009030

Table 2.

Selected bond lengths (Å) and angles (°) for complex 1.

Bond length (Å)
Cu1–O1	1.974(5)
Cu1–O2	1.916(4)
Cu1–O3	2.471(4)
Cu1–N0	1.922(5)
Cu1–N1	1.916(4)
N1–C1	1.303(8)
N0–C2	1.304(8)
N4–C1	1.376(6)
N4–C2	1.392(6)
Angle (°)
N1–Cu–N0	89.9(2)
N1–Cu–O3	106.5(2)
N0–Cu–O3	93.5(2)
N1–Cu–O1	89.6(2)
N0–Cu–O2	91.6(2)

Hirshfeld surface analysis

The Hirshfeld surface analysis is a method for the study of the intermolecular interactions as illustrated by the colours red, blue, and white represented on the surfaces. The color intensity can be used for visualizing the strength of the interactions, with the three colors representing the relative strengths of the intermolecular interactions. Red regions represent the hydrogen bond interaction, blue regions represent the longer contacts, and white regions represent the contacts exactly equal to the van der Waals separation.

The Hirshfeld d_norm surfaces, shape index, and curvedness of asymmetric unit complex 1 are illustrated in Figure 3. In general, hydrogen donors and hydrogen acceptors are identified from the red areas of the d_i and d_e plots. The surfaces are displayed as transparent to permit visualization of the complex 1. The d_norm surface is the sum of d_i and d_e, which was mapped over the distance range from −0.6813 to 1.0106 Å. The prominent red areas on the d_norm Hirshfeld surfaces of complex 1 around the metal ion are due to significant intermolecular O–H. . .H, O–H. . .O, O–H. . .N, and O–H. . .C hydrogen bonding interactions, which are attributed to the participation of three coordinating water molecules and represent the closest intermolecular interactions in the crystal. Also, the two prominent red spots observed around the N₃ and N₄ atoms are due to the participation of these two atoms in the formation of N–H. . .O intermolecular interactions. The O–H. . .O close contacts appear as pale red areas, which are attributed to the participation of the ${\mathrm{SO}}_4^{2-}$ counterion.

Figure 3.

Hirshfeld d_norm surfaces, shape index, and curvedness for complex 1.

The two-dimensional (2D) fingerprint plot of complex 1 is shown in Figure 4, and reveals that the main intermolecular interactions in the complex are H. . .H, O. . .H/H. . .O, and O. . .O contacts. The highest share of the total Hirshfeld surface is related to H. . .H close contacts with 42.2%, which focus on the middle part of the scatter points and cover most of the area in the 2D fingerprint plots, and have a greater contribution to the total Hirshfeld surfaces. The O. . .H/H. . .O interactions comprise 40.5% of the total Hirshfeld surface, which are characteristic sharp large spikes in the bottom left and bottom right parts of fingerprint plots. O. . .O close contacts comprise 5.0% of the total surface, and the primary interactions are quantitatively summarized in Figure 4.

Figure 4.

Fingerprint plots for complex 1.

In vitro antimicrobial studies

The antibacterial activity of metal–drug complexes has been the focus of continued attention.^19–21 Thus, we have investigated complex 1 and evaluated its antibacterial activities. Complex 1 was assessed for its antibacterial activities against eight bacterial strains by measurement of the minimal inhibitory concentrations (MICs)—four Gram-positive bacteria (Bacillus subtilis, S. aureus, Streptococcus agalactiae, and S. agalactiae) and four Gram-negative bacteria (E. coli, P. aeruginosa, Salmonella typhimurium, and Aeromonas hydrophila) as indicator strains. Table 3 collects the results of the antibacterial studies as MIC values in μg mL⁻¹. The results indicate that complex 1 exhibited higher antibacterial activity compared to the free MH ligand. The MIC values (μg mL⁻¹) of complex 1 were 512, 256, 512, and 512 μg mL⁻¹ against B. subtilis, S. aureus, S. agalactiae, and S. agalactiae; 128, 32, 128, and 64 μg mL⁻¹ against E. coli, P. aeruginosa, S. typhimurium, and A. hydrophila. The MIC values of complex 1 against Gram-positive bacteria were more significant than those against Gram-negative bacteria.

Table 3.

MIC values of complex 1 and MH (μg mL⁻¹).

	Bacterial strain	Complex 1	MH
Gram-positive strains	Bacillus subtilis (ATCC 6051)	512	–
	Staphylococcus aureus (ATCC 29213)	256	–
	Streptococcus agalactiae (ATCC 13813)	512	–
	Streptococcus agalactiae (A909)	512	–
Gram-negative strains	Escherichia coli (ATCC 25922)	128	–
	Pseudomonas aeruginosa (ATCC 27853)	32	–
	Salmonella typhimurium (ATCC 14028)	128	–
	Aeromonas hydrophila (ATCC 35654)	64	–

MH: metformin hydrochloride.

Conclusion

A novel 3D Cu(II) metal–drug complex based on an MH ligand has been synthesized. We have investigated the crystal information through structural investigations. The space group was determined to be monoclinic space of P21/n by single-crystal X-ray diffraction. The crystal packing was studied by the Hirshfeld surface analysis, which revealed the supramolecular structure and hydrogen bonding networks. In addition, we have studied the antibacterial properties of complex 1, the results indicated that it exhibited significantly higher antibacterial activity compared to the free MH ligand. The MIC values of complex 1 against Gram-positive bacteria were more prominent than those against Gram-negative bacteria.

Experimental

Materials and methods

The MH ligand was obtained from Shanghai Macklin Biochemical Co., Ltd and used as received. All other chemicals were of reagent grade quality obtained from commercial sources and were used without further purification. Single-crystal X-ray diffraction data of the product crystals were measured using monochromated MoKα radiation (λ = 0.071073) at 298 K on a Rigaku SCXmini diffractometer. The crystal data were collected and processed using SAINT, and the structures were solved and refined by full-matrix least-squares on F² using SHELXL. The structure of the title complex was resolved with the MERCURY program. ²²

Synthesis of complex 1

The ligand MH (166 mg and 10 mmol) was dissolved in methanol 10 mL and was adjusted to pH 6 with triethylamine, and then slowly added dropwise to a 10-mL aqueous solution containing CuSO₄·5H₂O (500 mg and 20 mmol) and stirred for 30 min and filtered. Blue block transparent crystals of complex 1 were obtained in about 73% yield (based on Cu) after evaporation of the filtrate at room temperature. Pure phase single crystals were isolated by filtration, washing with mother liquid, and drying under ambient conditions over a few weeks. Elemental analysis for C₄H₁₅CuN₅O₇S: calcd (%): C, 14.08; N, 20.53; H, 4.40; S, 9.38; found (%): C, 14.06; N, 20.49; H, 4.34; S, 9.34.

Single-crystal X-ray diffraction

The single-crystal X-ray diffraction data of complex 1 was collected at 298 K with graphite-monochromated MoKα radiation (λ = 0.071073) using a Rigaku SCXmini diffractometer by applying the ω-scan technique. ²³ The lattice parameters were integrated using vector analysis and refined from the diffraction matrix; the absorption correction was carried out using a Bruker SADABS program with the multiscan method. The structures were solved by full-matrix least-squares methods on all F2 data using the SHELX-2014 and SHELXL-2014 program for structure solution and structure refinement, ²⁴ respectively.

Hirshfeld surface analysis

Molecular Hirshfeld surface calculations were mapped using the CrystalExplorer by reading the cif files of the complexes. During the analysis, all bond lengths to hydrogen were automatically modified to typical standard neutron values (C–H = 0.93 Å and N–H = 0.86 Å). The normalized contact distance (d_norm) is defined as d_e and d_i. In this study, the Hirshfeld surfaces were generated using a standard (high) surface resolution. The 3D d_norm surfaces are mapped over a fixed color scale of 0.42 (red) to 1.6 Å (blue). The 2D fingerprint plots are displayed using the standard 0.6–2.6 Å view with the d_e and d_i distance scales displayed on the graph axes.

In vitro antibacterial activity studies

The antibacterial activity of the complex 1 was investigated on four Gram-positive strains (B. subtilis ATCC 6051, S. aureus ATCC 29213, S. agalactiae ATCC 13813, and S. agalactiae A909) and four Gram-negative strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. typhimurium ATCC 14028, and A. hydrophila ATCC 35654). Microbial suspensions of 10⁸ CFU/mL⁻¹ obtained from 24-h bacterial cultures developed in Lysogeny broth (LB) were used. The antimicrobial activity was tested using the MüllereHinton (MH) medium. The complex was dissolved in water with an initial concentration of 2048 μg mL⁻¹.

The MIC was the lowest concentration of antimicrobial agent showing complete inhibition of growth, which was studied by the liquid microdilution method, and performed by preparing twofold dilutions of complex 1 in water and in MH dispensed in 96-multiwell plates. For this purpose, serial dilution of the concentrations ranging from 2048 to 4 μg mL⁻¹ were prepared, and compared to the free MH ligand. All tests were performed in triplicate.