Synthesis of a Novel Unexpected Cu(II)–Thiazolidine Complex—X-ray Structure, Hirshfeld Surface Analysis, and Biological Studies

An unexpected trinuclear Cu(II)–thiazolidine complex has been synthesized by mixing CuCl2·2H2O with the Schiff base ligand, 1-(((4,5-dihydrothiazol-2-yl)ethylidene)hydrazono)methyl)phenol L, in ethanol. Unexpectedly, the reaction proceeded via the hydrolysis of the Schiff base L, followed by cyclization to afford 3-methyl-5,6-dihydrothiazolo[3,2-c][1,2,3]triazole (La), then complexation with the Cu(II) salt, forming the trinuclear [Cu3(La)4(Cl)6] complex. The complex was characterized by means of FTIR spectra, elemental analysis, and X-ray crystallography. In the trinuclear [Cu3(La)4(Cl)6] complex, there are two crystallographically independent hexa- and penta-coordinated Cu(II) sites, where the thiazolidine ligand La units act as a monodentate ligand and a linker between the Cu(II) centers. The crystal packing of the [Cu3(La)4(Cl)6] complex is primarily affected by the weak non-covalent C-H∙∙∙Cl interactions. In accordance with Hirshfeld surface analysis, the Cl∙∙∙H, H∙∙∙H, S∙∙∙H, and N∙∙∙H percentages are 31.9%, 27.2%, 13.5%, and 9.9%, respectively. X-ray photoelectron spectroscopy confirmed the oxidation state of copper as Cu(II), as well as the presence of two different coordination environments around copper centers. The complex showed interesting antibacterial activity against the Gram-positive bacteria S. subtilis, with MIC = 9.7 µg/mL compared to MIC = 4.8 µg/mL for the control, gentamycin. Moreover, the Cu(II) complex showed an equal MIC (312.5 µg/mL) against C. albicans compared to ketoconazole. It also exhibits a very promising inhibitory activity against colon carcinoma (IC50 = 3.75 ± 0.43 µg/mL).


Introduction
Copper is one of the most widely used metals [1,2], with a very low toxic limit [3,4]. It acts as a vital micronutrient and plays various functions in biological systems. Recently, copper compounds have been recommended as therapeutic agents against cancer [5], microbial diseases [6][7][8][9], chronic lung inflammation [10], influenza A [11], neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases, in addition to disorders related to copper homeostasis, such as Wilson's and Menkes disorders [12][13][14][15]. Moreover, the copper coordination complexes were considered as promising multi-functional materials due to their success in photophysical and photoelectrochemical applications. These complexes were considered as better alternatives to other transition metal complexes (Pt(II), Ir(III) and Ru(II)) which are expensive, toxic, and of low abundance [16,17]. In contrast, the Schiff bases have become an encouraging choice as functional ligan chemistry because of their synthetic flexibility, structural diversity, va sensitivity, and selectivity towards metal ions. Predominantly, the hyd azomethine group (-C=N-N=C-) has been considered as powerful ligan ity to stabilize various metal ions, with different oxidation states prod molecular designs and geometries [20][21][22][23][24][25][26][27][28]. In addition, the aryl hyd plexes were found to have interesting electrical and magnetic proper lized as novel heterogeneous catalysts in redox, chemical, and photoch several industrial applications [29][30][31].
On the other hand, the thiazolidine nucleus is well-known in the f tical chemistry. This group of compounds has played a crucial role in or and medicinal chemistry [32]. This is because most of the antimicrobi as penicillin, cephalosporin, narcodicins, and thienamycins, were syn zolidines [33,34]. Various pharmacological activities, including antipr protective, antidiabetic, antihypertensive, platelet activating factor a antagonist, anti-inflammatory, antipyretic, analgesic, anthelmintic, an anticancer, mucolytic, and anti-HIV activities, are associated with com a thiazolidine nucleus [32]. In this work, the reaction of CuCl2·2H2O w type ligand, 1-(((4,5-dihydrothiazol-2-yl)ethylidene)hydrazone)methy 1) in ethanol is presented. The structure of the resulting Cu(II)-thiazoli unexpected product was investigated using different spectroscopic tec crystal X-ray analysis. Its antimicrobial, antioxidant, and anticancer a investigated.

Chemistry and Characterizations
The designed ligand L was synthesized according to the method d 1. The acetyl derivative reacted with hydrazine, followed by reaction w to give the corresponding Schiff base L. The reaction of CuCl2·2H2O w drothiazol-2-yl)ethylidene)hydrazone)methyl)phenol L did not procee of the corresponding Cu(II)-hydrazone complex. Unexpectedly, the hy derwent oxidative hydrolysis, followed by cyclization, affording the c zolidine L a , which proceeded to complexation with the Cu(II) ion, affor [Cu3(L a )4(Cl)6] complex (Scheme 1). Spectral characterizations of the pounds are presented in Figures S1-S6 (Supplementary Materials). T novel Cu(II) complex was confirmed using elemental analysis, diffe measurements, and X-ray crystallography. The antimicrobial, anticanc

Chemistry and Characterizations
The designed ligand L was synthesized according to the method depicted in Scheme 1. The acetyl derivative reacted with hydrazine, followed by reaction with salicylaldehyde to give the corresponding Schiff base L. The reaction of CuCl 2 ·2H 2 O with 2-(1-(4,5dihydrothiazol-2-yl)ethylidene)hydrazone)methyl)phenol L did not proceed to the formation of the corresponding Cu(II)-hydrazone complex. Unexpectedly, the hydrazone ligand underwent oxidative hydrolysis, followed by cyclization, affording the corresponding thiazolidine L a , which proceeded to complexation with the Cu(II) ion, affording the trinuclear [Cu 3 (L a ) 4 (Cl) 6 ] complex (Scheme 1). Spectral characterizations of the synthesized compounds are presented in Figures S1-S6 (Supplementary Materials). The structure of the novel Cu(II) complex was confirmed using elemental analysis, different spectroscopic measurements, and X-ray crystallography. The antimicrobial, anticancer, and antioxidant activities of the [Cu 3 (L a ) 4 (Cl) 6 ] complex were also presented.

X-ray Structure Description of [Cu3(L a )4(Cl)6] Complex
The X-ray crystallographic measurements confirmed the structure of the trinuclear [Cu3(L a )4(Cl)6] complex, which is formed via Cu(II)-mediated hydrolysis and cyclization of the hydrazone ligand L to the corresponding thiazolidine L a , followed by complexation with the cupric ion. Table 1 lists the crystallographic data of the trinuclear [Cu3(L a )4(Cl)6] complex.

X-ray Structure Description of [Cu 3 (L a ) 4 (Cl) 6 ] Complex
The X-ray crystallographic measurements confirmed the structure of the trinuclear [Cu 3 (L a ) 4 (Cl) 6 ] complex, which is formed via Cu(II)-mediated hydrolysis and cyclization of the hydrazone ligand L to the corresponding thiazolidine L a , followed by complexation with the cupric ion. Table 1 lists the crystallographic data of the trinuclear [Cu 3 (L a ) 4 (Cl) 6 ] complex.
The X-ray structure of the trinuclear Cu(II) complex [Cu 3 (L a ) 4 (Cl) 6 ] is shown in  (Table 2). Of course, the bond angles of all the trans bonds are 180 • , while the angles between the cis bonds are in the range of 85.63(3) to 97.12 (4) for Cl1-Cu1-Cl2 and Cl1-Cu2-Cl3, respectively. Hence, the structure of the coordination environment could be described as an elongated octahedron, where the two Cl(1) and the two N(2) atoms represent the base, and the two Cl(2) are located as apical. The coordination sphere of the Cu(2) is completely different. The Cu(2) is penta-coordinated, with five coordination interactions. There is one interaction with the terminal Cl(3) atom, in addition to two interactions with the bridged Cl(1) and Cl(2) atoms, which are already coordinated to the Cu(1). The corresponding Cu-Cl distances are 2.2720(12), 2.6178 (11), and 2.3140(13) Å, respectively. The coordination environment of the Cu(2) is completed by two interactions with the N(1) and N(4) atoms from two trans L a ligand units. The corresponding Cu-N distances are 2.024(3) and 1.987(3) Å, respectively. The distortion in the CuCl 3 N 2 coordination sphere of Cu(1) was described based on the criterion of Addison [35]. The largest angles are Cl2-Cu2-Cl3 (β = 171.35 • ) and N1-Cu2-N4 (α = 166.56 • ), giving a τ = {(β − α)/60} value of only 0.08. Hence, the coordination geometry is more like to be a distorted square pyramid, where the Cl(1) donor would be regarded as apical. It is worth noting that the structure of this complex comprised two crystallographically independent L a ligand units, with one of them acting as a terminal ligand, coordinating only the Cu(2) metal site via the significantly short Cu2-N4 bond, and the other L a unit acting as a connector between the two Cu sites. Hence, this ligand unit and the two Cl1 and Cl2 ions act as a bridging ligand, connecting the Cu(II)-sites leading to the formation of the trinuclear [Cu 3 (L a ) 4 (Cl) 6 ] complex. with one of them acting as a terminal ligand, coordinating only the Cu(2) metal site the significantly short Cu2-N4 bond, and the other L a unit acting as a connector betw the two Cu sites. Hence, this ligand unit and the two Cl1 and Cl2 ions act as a bridg ligand, connecting the Cu(II)-sites leading to the formation of the trinuclear [Cu3(L a )4(C complex. The structure of [Cu3(L a )4(Cl)6] is stabilized by an intramolecular C1-H1A···Cl1 in action, with hydrogen-acceptor and donor-acceptor distances of 2.72 and 3.427(5) Å spectively. Its packing is dominated by the weak non-covalent C-H···Cl interactions picted in Table 3 and shown as a red dotted line in the upper part of Figure 3. A view the packing through the bc plane, showing the complex units connected by the Cl-H interactions, is presented in the lower part of the same illustration. The donor-accep interaction distances range from 3.609(5) Å (C6-H6A···Cl1) to 3.692(5) Å for C7-H7B··· respectively.  The structure of [Cu 3 (L a ) 4 (Cl) 6 ] is stabilized by an intramolecular C1-H1A···Cl1 interaction, with hydrogen-acceptor and donor-acceptor distances of 2.72 and 3.427(5) Å, respectively. Its packing is dominated by the weak non-covalent C-H···Cl interactions depicted in Table 3 and shown as a red dotted line in the upper part of Figure 3. A view of the packing through the bc plane, showing the complex units connected by the Cl-H···O interactions, is presented in the lower part of the same illustration. The donoracceptor interaction distances range from 3.609(5) Å (C6-H6A···Cl1) to 3.692(5) Å for C7-H7B···Cl1, respectively.

Hirshfeld Analysis
Hirshfeld surface analysis is a powerful tool for visualizing interactions in m crystals. Crystal Explorer 17 is used to create the Hirshfeld analysis, analyze the structure of the synthesized complex, and represent intermolecular interactions surface [36]. The dnorm surfaces are mapped in the range of −0.05 to 0.80 Å, while th index, curvedness, and fragment patch are mapped over the ranges −1.0 to 1.0 Å 0.4 Å, and 0 to 15 Å, respectively Figure 4. The dnorm surface detected the very clo molecular interactions presented as red spots, indicating short H···H, N···H, S·· Cl···H interactions. The shape index shows the shape of the surface (concave (−1.0 vex (+1.0)), while curvedness clarifies the flatness of surface indicated as flat (−4.0 gular (+0.4). A fragment patch is often used to divide the surfaces into patches, sug interactions between neighboring molecules. This mapping color patch allows th fication of the closest neighbor coordination environment of a molecule.

Hirshfeld Analysis
Hirshfeld surface analysis is a powerful tool for visualizing interactions in molecular crystals. Crystal Explorer 17 is used to create the Hirshfeld analysis, analyze the crystal structure of the synthesized complex, and represent intermolecular interactions on that surface [36]. The d norm surfaces are mapped in the range of −0.05 to 0.80 Å, while the shape index, curvedness, and fragment patch are mapped over the ranges −1. The fingerprint plots are demonstrated in Figure 5. The complementary regions in these plots are visualized, where one molecule behaves as a donor (d e > d i ), while the other acts as an acceptor (d e < d i ). The fingerprint plots highlight the close contacts of specific atom pairs. Thus, selected contributions can account for the stability of the complex crystal structure. The Cl···H contacts have the largest contribution in the molecular packing and also have short Cl···H distances of 3.49, 3.39, 3.036, 2.87, and 2.81 Å, corresponding to Cl2···H1B, Cl1···H6B, Cl3···H7B, Cl2···H6B, and Cl1···H7B, respectively. The S···H contacts have a relatively strong contribution to the stability of the crystal structure, within the range of 3.22 to 3.43 Å. The proportion of Cl···H, H···H, S···H, and N···H interac-tions comprises 31.9%, 27.2%, 13.5%, and 9.9% of the total Hirshfeld surface, respectively ( Figure 6). These are the important interactions which stabilize the crystal structure of the [Cu 3 (L a ) 4 (Cl) 6  The fingerprint plots are demonstrated in Figure 5. The complementary regions in these plots are visualized, where one molecule behaves as a donor (de > di), while the other acts as an acceptor (de < di). The fingerprint plots highlight the close contacts of specific atom pairs. Thus, selected contributions can account for the stability of the complex crystal structure. The Cl···H contacts have the largest contribution in the molecular packing and also have short Cl···H distances of 3.49, 3.39, 3.036, 2.87, and 2.81 Å, corresponding to Cl2···H1B, Cl1···H6B, Cl3···H7B, Cl2···H6B, and Cl1···H7B, respectively. The S···H contacts have a relatively strong contribution to the stability of the crystal structure, within the range of 3.22 to 3.43 Å. The proportion of Cl···H, H···H, S···H, and N···H interactions comprises 31.9%, 27.2%, 13.5%, and 9.9% of the total Hirshfeld surface, respectively ( Figure 6). These are the important interactions which stabilize the crystal structure of the [Cu3(L a )4(Cl)6] complex.  The fingerprint plots are demonstrated in Figure 5. The complementary regions in these plots are visualized, where one molecule behaves as a donor (de > di), while the other acts as an acceptor (de < di). The fingerprint plots highlight the close contacts of specific atom pairs. Thus, selected contributions can account for the stability of the complex crystal structure. The Cl···H contacts have the largest contribution in the molecular packing and also have short Cl···H distances of 3.49, 3.39, 3.036, 2.87, and 2.81 Å, corresponding to Cl2···H1B, Cl1···H6B, Cl3···H7B, Cl2···H6B, and Cl1···H7B, respectively. The S···H contacts have a relatively strong contribution to the stability of the crystal structure, within the range of 3.22 to 3.43 Å. The proportion of Cl···H, H···H, S···H, and N···H interactions comprises 31.9%, 27.2%, 13.5%, and 9.9% of the total Hirshfeld surface, respectively ( Figure 6). These are the important interactions which stabilize the crystal structure of the [Cu3(L a )4(Cl)6] complex.

XPS Studies
X-ray photoelectron spectroscopy is a quantitative measurement for the elemental composition of the material surface, as well as its valence state. Based on single-crystal Xray structure, the investigated trinuclear copper(II) complex has two distinct coordination environments around copper(II) centers, with coordination numbers 5 and 6.  Additionally, the XPS showed two peaks assigned to N1sA and N1s, with B.E. of 401.42 and 400.33 eV, respectively, indicating the existence of two binding types for nitrogen atoms, which are the sp 3 and sp 2 ( Figure 7B). In addition, the presence of a doublet

XPS Studies
X-ray photoelectron spectroscopy is a quantitative measurement for the elemental composition of the material surface, as well as its valence state. Based on single-crystal X-ray structure, the investigated trinuclear copper(II) complex has two distinct coordination environments around copper(II) centers, with coordination numbers 5 and 6. Figure 7 shows the XPS diagram for the [Cu 3 (L a ) 4 (Cl) 6

XPS Studies
X-ray photoelectron spectroscopy is a quantitative measurement for the elemental composition of the material surface, as well as its valence state. Based on single-crystal Xray structure, the investigated trinuclear copper(II) complex has two distinct coordination environments around copper(II) centers, with coordination numbers 5 and 6.  Additionally, the XPS showed two peaks assigned to N1sA and N1s, with B.E. of 401.42 and 400.33 eV, respectively, indicating the existence of two binding types for nitrogen atoms, which are the sp 3 and sp 2 ( Figure 7B). In addition, the presence of a doublet Additionally, the XPS showed two peaks assigned to N1sA and N1s, with B.E. of 401.42 and 400.33 eV, respectively, indicating the existence of two binding types for ni-trogen atoms, which are the sp 3 and sp 2 ( Figure 7B). In addition, the presence of a doublet peak at 197.89 and 199.47 eV, corresponding to Cl2p 1/2 and Cl2p 3/2 , respectively (∆B.E. = 1.58 eV), indicated its coordinating behavior ( Figure 7C) [39,40]. The detailed XPS spectrum showed the two sulfur peaks S2p 3/2 and S2p 1/2 [41] at 163.86 and 164.99 eV, corresponding to S2p 3/2 and S2p 1/2 , respectively. The small value of ∆B.E. (1.13 eV) indicates a non-coordinating S-atom.

Antimicrobial Activity
The bio-activities of the [Cu 3 (L a ) 4 (Cl) 6 ] complex as an antimicrobial agent against Gram-positive bacteria (S. aureus and B. subtilis), Gram-negative bacteria (E. coli and P. vulgaris), and the injurious fungi (A. fumigatus and C. Albicans) were examined and compared with the antibacterial and antifungal control, gentamycin and ketoconazole, respectively. Table 4 summarizes the zone of inhibitions for the complex. The inhibition zone diameters range from 15-20 mm and 13-30 mm against fungi and bacteria, respectively. Generally, the [Cu 3 (L a ) 4 (Cl) 6 ] complex has higher activity against Gram-positive bacteria than Gram-negative bacteria. The complex has higher activity against the Gram-positive bacteria B. subtilis (30 mm) than S. aureus (14 mm). Interestingly, the Cu(II) complex also has a higher activity against B. subtilis than the antibacterial control, gentamycin, and equal activity against C. albicans when compared with the control compound, ketoconazole. In addition, the antibacterial activity of the Cu(II) complex against Gram-negative bacteria (E. coli and P. vulgaris) is relatively lower than for the control, gentamycin.

Microbe [Cu 3 (L a ) 4 (Cl) 6 ] Control
A. fungimatus Moreover, the minimum inhibitory concentrations (MIC) in micrograms/mL were determined and depicted in Table 5. The Cu(II) complex showed equal MIC values against C. albicans compared to ketoconazole. For the antibacterial activity, the best MIC value for the complex is found against B. subtilis (9.7 µg/mL).

Cytotoxic Activity against Colon Carcinoma (HCT-116 Cell)
The [Cu3(L a )4(Cl)6] complex was tested for its in vitro cytotoxicity against colon car cinoma (HCT-116 cell line). The results of the cytotoxicity experiments are presented graphically in Figure 9. These results showed an extraordinary cytotoxicity against th examined cell line (HCT-116). The percentage of the cell viability is only 0.93 µg/mL at 500 µg/mL, while the IC50 value is 3.75 ± 0.43 µg/mL. For doxorubicin as a positive control and under the same experimental conditions, the IC50 value is 0.49 ± 0.07 µg/mL. Conse quently, this Cu(II) complex has a very promising inhibitory activity against colon carci noma.

Materials
All chemicals were purchased from Aldrich chemical company and were used with out further purifications.   The [Cu3(L a )4(Cl)6] complex was tested for its in vitro cytotoxicity against colon carcinoma (HCT-116 cell line). The results of the cytotoxicity experiments are presented graphically in Figure 9. These results showed an extraordinary cytotoxicity against the examined cell line (HCT-116). The percentage of the cell viability is only 0.93 µg/mL at 500 µg/mL, while the IC50 value is 3.75 ± 0.43 µg/mL. For doxorubicin as a positive control and under the same experimental conditions, the IC50 value is 0.49 ± 0.07 µg/mL. Consequently, this Cu(II) complex has a very promising inhibitory activity against colon carcinoma.

Materials
All chemicals were purchased from Aldrich chemical company and were used without further purifications.

Instrumentations
Details of the instrumentations are described in the Supplementary Material.

Materials
All chemicals were purchased from Aldrich chemical company and were used without further purifications.

Instrumentations
Details of the instrumentations are described in the Supplementary Material.
Then, 2-(1-hydrazonoethyl)dihydrothiazole (1.143 g, 8 mmol) and salicylaldehyde (0.976 g, 7 mmol) were mixed in 50.0 mL ethanol and 2 drops of AcOH. The reaction mixture was further sonicated for 60 min at 60 • C. Then, the solution was cooled to room temperature, and light-yellow block shaped crystals were formed by slow evaporation of the solution in air after a few days. The yield of the isolated yellow solid was 0.62 g. (90%) [45][46][47].
Anal. Calc. for C 12

X-ray Structure Determination
Details of the single crystal structure determination are are described in the Supplementary Material [48][49][50].

Biolological Studies
The biological antimicrobial, anticancer and antioxidant activities of the Cu(II) complex were studied.
Details of the biological analysis are described in the Supplementary Materials [51][52][53].

Conclusions
A novel [Cu 3 (L a ) 4 (Cl) 6 ] complex was synthesized by the reaction of 2-((E)-(((E)-1-(4,5dihydrothiazol-2-yl)ethylidene)hydrazone)methyl)phenol L with CuCl 2 .2H 2 O in Ethanol. This reaction occurs through a Cu(II) mediated hydrolysis of L, followed by cyclization to afford the 3-methyl-5,6-dihydrothiazolo[3,2-c] [1][2][3]triazole L a , which undergoes complexation with Cu(II). The structural aspects of the trinuclear [Cu 3 (L a ) 4 (Cl) 6 ] complex were determined using single crystal X-ray diffraction, Hirshfeld surface analysis, and XPS. The complex consists of two crystallographically independent L a ligand units, with one of them acting as a terminal monodentate ligand coordinating only the Cu(2) metal sites, and the other L a unit acting as a connector between the two Cu(1) and Cu(2) sites. Using Hirshfeld analysis, the Cl···H and H···H intermolecular contacts are the most prevailing interactions. XPS analysis confirmed the divalent oxidation state of the copper ion. The [Cu 3 (L a ) 4 (Cl) 6 ] complex has higher activity against Gram-positive bacteria than Gram-negative bacteria, especially against B. subtilis. Additionally, it showed promising antifungal activity against C. albicans. Interestingly, the [Cu 3 (L a ) 4 (Cl) 6 ] complex has an unexpected cytotoxic activity against the HCT-116 cell line (IC 50 =3.75 ± 0.43 µg/mL).