Synthesis, Characterization, and Anticancer Bioactivity of a Novel Nano-Schiff Base Ligand (NPTIPPE) Derived from 4-Aminoantipyrine and Its Palladium Complex

INTRODUCTION 
1-phenyl-2,3-dimethylpyrazol-5-one is known as 4-aminoantipyrine or 4-aminophenazone. Pyrazole, a five-membered ring with two nitrogen atoms and a double bond, is important. Its substituted derivatives are biologically active [1, 2]. It also makes ion-selective electrodes [3] and suppresses enzymes [4]. The biological action of 4-aminoantipyrine against bacteria makes it notable. It is a strong analgesic, anti-inflammatory, and fever reducer in circumstances where aspirin is unsuccessful, such as Hodgkin’s disease and salicylate-resistant fevers. It also helps identify aromatic chemicals and metal ions utilizing various analytical methods. It helps make antipyrinyl azo dyes, which are essential metal ion colorimetric indicators. Under acidic circumstances, these compounds produce metal chelate complexes. Certain pyrazole compounds have also shown promise against certain cancers [5]. Over 100 types of cancer result from the uncontrolled, fast, and deadly development of abnormal cells in organs and tissues [6, 7]. This disease is caused by DNA mutations in cells, which are organized into many genes with instructions for cellular functioning, growth, and division [7]. Incorrect instructions can cause the cell to stop working, causing numerous malignancies, including breast cancer, the most frequent disease in women after skin cancer [8]. Breast cancer usually starts in the milk-producing ducts (invasive ductal carcinoma), but it can also occur in the lobules or other breast tissues [9]. The new Schiff base ligand (NPTIPPE) generated from 4-aminoantipyrine and a pd (II) complex was synthesized in this study. These compounds were characterised spectroscopically and physically and tested for anticancer efficacy.

MATERIALS AND METHODS
Reagents and instruments 
The chemicals used in this study were obtained from Merck, Sigma-Aldrich, and BDH. Breast cancer cell lines (MCF-7) and normal cell lines (WRL-68) were also obtained from the Educational Research Center, Division of Cellular, Molecular, Microbial, and Animal Services / Roshdy Azmeh Company in Tehran. UV-Vis spectra in the range of 200–1000 nm were acquired using a Shimadzu UV-165 PCS spectrophotometer. 1H and 13C-NMR spectra were recorded at a frequency of 300 MHz using tetramethylsilane as an internal standard reference and DMSO-d6 as a solvent. The FTIR spectra were recorded using a Shimadzu FTIR 8400S spectrophotometer in the range of 400-4000 cm−1. Melting points of the nano ligand and the prepared complex were determined using a Stuart melting point apparatus. Magnetic susceptibility measurements at room temperature were performed using a magnetic susceptibility balance model MSB-MKI. Flame atomic absorption spectra were recorded using a Shimadzu AA-6300. Elemental analysis for C, H, and N was conducted using an EA-300 elemental analyzer.

Synthesis procedure 
Synthesis of the heterocyclic Schiff base nano ligand (NPTIPPE) 
The ligand (NPTIPPE) was synthesized in two steps. The first step involved the preparation of (4-(((2E,3E) -3- (hydroxy imino) butan-2-ylidene) amino) -1,5-dimethyl -2-phenyl- 1,2-dihydro -3H-pyrazol -3 - one (compound-A). This was achieved by reacting benzil (1.05 g, 0.005 mol) in 25 mL of absolute ethanol, with the addition of 4-5 drops of glacial acetic acid. Simultaneously, 4-aminoantipyrine (2.03 g, 0.01 mol) was dissolved in 25 mL of absolute ethanol under continuous stirring. The mixture was then refluxed for 8 h, followed by cooling and collection. Recrystallization was performed by removing unreacted material using 98% ethanol. The resulting product was collected and weighed for use in the second step, yielding an 85% productivity. The nano ligand (1E,2E)-N1, N2-bis((Z)-1,5-dimethyl-2-phenyl-3-(thiazol-5-ylimino)-2,3-dihydro-1H-pyrazol-4-yl)-1,2-diphenylethane-1,2-diimine (NPTIPPE) was then prepared by dissolving compound-A (2.86 g, 0.01 mol) in 25 mL of absolute ethanol with continuous stirring and adding 4-5 drops of glacial acetic acid. Separately, 2-aminothiazole (2 g, 0.02 mol) was dissolved in 25 mL of absolute ethanol. The solutions were mixed together and refluxed for 8 h. The mixture was cooled, and the resulting precipitate was filtered and dried. Recrystallization from absolute ethanol yielded a product with an 81% yield and a melting point of 120℃, as illustrated in Fig. 1. 

Synthesis of the complex 
The complex was prepared in a 1:1 ratio using a standard method. A solution of the nano ligand (NPTIPPE) (0.37 g, 0.001 mol) in 10 mL of ethanol was mixed with palladium chloride (PdCl2) (0.18 g, 0.001 mol) in 10 mL of absolute ethanol. The mixture was refluxed for 2 h with continuous stirring. It was then cooled, filtered, and dried before being recrystallized using absolute ethanol to obtain a pure complex. The yield was 72%, with a melting point of 173℃ (Fig. 2). 

Cell toxicity tests: cell lines and cultivation of MCF-7 breast cancer cell line  
In this study, two cell lines were utilized: the breast cancer cell line (MCF-7) and the normal human liver cell line (WRL-68). The cell lines were stored in liquid nitrogen, maintained and tested at the Division of Cellular, Molecular, Microbial, and Animal Services / Roshdy Azmeh Company in Tehran. After preparing the cancer cell line suspensions (MCF-7) at a concentration of 1×10-5 cells/well, the cell suspension was placed in a 96-well flat-bottom plate and incubated under appropriate conditions in an incubator containing 5% carbon dioxide (CO2) at 37℃ for 24 h. Subsequently, 100 μL of this suspension was added to each well. The prepared concentrations of the ligands and the Pd (II) complex—10, 25, 50, 100, 250, and 500 μg/mL—were then added to the wells, with three wells per concentration. The plate was then incubated for 24 h at 37℃. Following this incubation, 10 μL of MTT solution at a concentration of 0.45 mg/mL was added to each well, and the plate was incubated for an additional 4 h at 37℃. Afterward, 100 μL of a DMSO solution was introduced into each well and left to incubate for duration of 5 min. Ultimately, the samples’ absorbance was measured at a wavelength of 570 nm utilizing an ELISA reader. Subsequent statistical analysis was conducted on the optical density measurements in order to get the IC50 value.

RESULTS AND DISCUSSION  
The novel nano ligand (NPTIPPE) was synthesized through the sequential reaction of benzil with 4-aminoantipyrine and 2-aminothiazole. The Pd (II) complex was prepared by reacting the nano ligand (NPTIPPE) with palladium chloride dissolved in ethanol. Table 1 presents the physical properties and elemental analysis of both the ligand and its complex. The palladium complex was prepared in a 1:1 of metal:ligand ratio.

NMR spectrum of the novel Schiff base ligand (NPTIPPE)     
Fig. 3 illustrates the 1H-NMR spectrum of the ligand (NPTIPPE). The 1H-NMR spectrum of the ligand (NPTIPPE) exhibited two singlets at δ = 2.13 ppm (S, 3H) and δ = 3.16 ppm (S, 3H), corresponding to the protons of the methyl groups (C-CH3 and N-CH3) on the pyrazole ring, respectively [10]. Additional multiple signals were observed at δ = 7.18 - 7.69 ppm (M, 10H), attributed to the protons of the phenyl rings on the pyrazole and benzyl moieties [11]. The protons on the thiazole ring produced a doublet at δ = 7.72 and 7.75 ppm (d, 2H) [12]. 

13C-NMR spectrum of the ligand (NPTIPPE)
The 13C-NMR spectrum of the ligand (NPTIPPE) showed a signal at 40.35 ppm due to DMSO. A signal at 10.43 ppm was observed (Fig. 4), corresponding to the carbon atoms (C8 and C39) of the methyl groups (-CH3) attached to the pyrazole ring. The carbon C6 and C37 atoms gave a signal at 35.29 ppm, indicating the methyl group attached to the nitrogen of the pyrazole ring [13]. A signal at 106.49 ppm was attributed to the carbon atoms C3 and C31 of the pyrazole ring. Additionally, a signal at 138.63 ppm was associated with the carbon atoms C4 and C35 of the pyrazole ring containing the methyl group (-CH3) [14]. Signals at 116.34 ppm were attributed to the carbon atoms C45 and C50, while signals at 137.16 ppm corresponded to the carbon atoms C46 and C54, and signals at 192.81 ppm were associated with the carbon atoms C48 and C52 of the thiazole ring [15, 16]. The following carbon atoms: C7, C13, C14, C16, C18, C20, C21, C22, C23, C24, C25, C27, C28, C29, C30, C38, C40, C42, and C44, gave values ranging between 124.12 ppm and 135.46 ppm, indicating the phenyl rings attached to the azomethine group and the phenyl ring attached to the nitrogen of the pyrazole ring [16]. A signal at 152.08 ppm was attributed to the carbon atoms C12 and C11, while another signal at 154.35 ppm corresponded to the carbon atoms C2 and C35 of the azomethine groups [17-20].  

FT-IR results
The ligand (NPTIPPE) had several IR bands (Fig. 5). The bands seen at 3055 cm-1 and 3031 cm-1 can be attributed to aromatic (C-H) groups, while the bands at 2908 cm-1 and 2815 cm-1 correspond to aliphatic (C-H) groups [21-24]. A well-defined absorption band at 1658 cm-1 indicated ligand production and confirmed the azomethine (C=N) group. The 1589 cm-1 band indicated the azomethine (C=N) group in the thiazole ring [25, 26]. The aromatic (C=C) groups and thiazole ring (C-S) are indicated by absorption bands at 1558 cm-1, 1488 cm-1, and 1134 cm-1 [27-29]. 
The FTIR spectrum of the palladium (II) complex (Fig. 6) shows that coordination and complex formation shifted several absorption bands in the free ligand spectrum to lower frequencies. This shift strongly suggests complex development. The azomethine group changed from 1658 cm-1 in the ligand spectrum to 1635 cm-1 in the complex spectrum [30-34]. The M-N group also produced a 594 cm-1 [35-37]. Table 2 shows the ligand (NPTIPPE) and palladium (II) complex FTIR bands.

UV-visible results
The UV-Vis spectrum of the nano ligand (NPTIPPE) exhibited two peaks, as shown in Fig. 7 and Table 3. The first peak, centered at 243 nm (41152 cm-1), corresponds to the (π-π*) transition, while the second peak, at 320 nm (31153 cm-1), is attributed to the (n-π*) transition of the azomethine group [35, 38, 39]. In the UV-Vis spectrum of the palladium (II) complex (Fig. 8), several absorption peaks were observed at 252 nm (39683 cm-1) and 341 nm (29326 cm-1), indicating intra-ligand transitions. Three additional absorption peaks appeared at 478 nm (20921 cm-1), 508 nm (19685 cm-1), and 536 nm (18657 cm-1), corresponding to the electronic transitions 1A1g → 1Eg, 1A1g → 1B1g, and 1A1g → 1A2g, respectively, confirming the square planar geometry of the complex [40]. Magnetic susceptibility measurements indicated that the complex exhibits diamagnetic properties [41]. 

Molar conductivity measurements
Molar conductivity measurements were prepared using absolute ethanol at a concentration of 1x10-3 M at room temperature. These measurements indicated that the prepared complex has a molar conductivity value of 73.98 ohm-1 cm2 mol-1, suggesting that the complex possesses ionic characteristics with a 1:2 ratio [42].

    
XRD analysis
A solid-state XRD study examined the crystal structure [22, 23] of NPTIPPE and its metal complexes in the 2θ=5-80 range. This investigation measured crystal structure, crystallite size, macrostrains, and dislocation density to determine purity and defects when the ligand is transformed into the palladium complex. Micro-strains such lattice deformation and crystal fracture due to crystal distortions, crystallite size, and distribution cause some diffraction peaks [43-47]. The synthesized ligand’s XRD pattern displayed strong peaks, indicating a crystalline network. In contrast, the palladium complex had broad peaks, indicating amorphous structures. The sharpness of the peaks depends on the crystalline order, lattice properties, and crystal planes. Debye-Scherrer’s equation was used to calculate the crystallite size of the nano ligand (NPTIPPE) and the palladium complex:

To calculate the dislocation density, the following equation is commonly used:

By analyzing the XRD patterns, significant differences in crystallite size, dislocation density, and interplanar spacing (d-spacing) between the nano ligand (NPTIPPE) and the prepared palladium complex were observed. These differences confirm the coordination process between the ligand and the palladium ion [48, 49]. In Fig. 9 and Table 4, a comparison of the intensity and positions of the obtained peaks with standard reference cards indicates that these peaks correspond to the original compounds from which the complexes were derived. However, there were also unusual peaks that did not match any known substances, suggesting that the compounds are new and have not been compared with various global standards [50]. Utilizing the XRD data, it was concluded that the prepared materials exhibit nanomaterial characteristics [51-53]. 

Scanning electron microscopy (FE-SEM)
As shown in Fig. 10, the surface investigations of resulting NPTIPPE and pd(II) complex possess smooth shapes. It should be noted that Pd-complex reveals an interconnected structures as compared with NPTIPPE ligand. 

Anticancer activity
The synthesized NPTIPPE and Pd(II) complex were evaluated for MTT on the most prevalent cancer cell line (MCF-7) (Figs. 11-14). To determine their therapeutic potential, NPTIPPE and Pd(II) complex were evaluated on healthy WRL-68 liver cells. Following 24 h of incubation at 37°C, the MTT assay was performed at doses of 0, 10, 25, 50, 100, 250, and 500 µg/mL. The NPTIPPE effectively inhibited MCF-7 cancer and WRL-68 healthy cell growth, with the lowest inhibition at 10 µg/mL and the maximum at 500 µg/mL. The proportion of viable MCF-7 and WRL-68 cells after ligand contact ranged from 4.35924% to 92.94909% and 60.57843% to 99.06862%, respectively. Maximum MCF-7 cell inhibition was 95.64072% at 500 µg/mL, while WRL-68 cells showed 39.42157% inhibition. The ligand produced an IC50 value of 78.56 µg/mL for MCF-7 cells and 220.84 µg/mL for WRL-68 cells. The proliferation of MCF-7 cancer cells and WRL-68 healthy cells was inhibited by the Pd(II) complex of the NPTIPPE at concentrations ranging from 10 µg/mL to 500 µg/mL. After interaction with the Pd(II) complex, MCF-7 cells had 7.98046% to 93.89225% vitality and WRL-68 cells 65.07944% to 97.18371%. The maximum inhibition of MCF-7 cells was 92.01954% at 500 µg/mL, while WRL-68 cells showed 34.92056% inhibition. IC50 values for palladium complex interaction with MCF-7 and WRL-68 cells were 87.37 and 125.94 µg/mL, respectively (Table 5 and Table 6).

 
CONCLUSION
In this study, a novel ligand (NPTIPPE) derived from antipyrine was synthesized in two steps, followed by the preparation of its palladium complex. Various methods were employed, including spectroscopic techniques (FTIR spectroscopy, UV-Vis. spectroscopy, and NMR spectroscopy), atomic absorption, FESEM, XRD, and physical methods (melting point, molar conductivity, elemental analysis) to confirm the structure of the synthesized NPTIPPE and its complex. The FTIR spectrum showed that the ligand coordinates with the Pd (II) ion through the four nitrogen atoms of the azomethine groups, indicating that the ligand acts as a tetradentate ligand. This coordination results in a square planar geometry for the complex. MTT assays were conducted for both NPTIPPE and its palladium complex on cancerous and healthy cells. The palladium complex was found to be more effective against breast cancer cells (MCF-7) than the nano ligand itself, suggesting that the palladium complex holds significant potential as a therapeutic agent.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.