Thermal and spectroscopic studies of some metal complexes with a new enaminone ligand 3-chloro-4-((4-methoxyphenyl)amino)pent-3-en-2-one and their investigation as anti-urease and cytotoxic potential drugs

2.1 Materials

In this paper, the solvents and chemicals were provided by the commercial sources with the reagent quality, and they are utilized directly. They included 3- Chloro-2,4-pentanedione, p- Toluidine , p-Toluenesulfonic acid monohydrate, Cobalt(II) acetate tetrahydrate., Cadmium chloride monohydrate, Molybdenum hexacarbonyl.

2.2 Synthesis of ligand (PA) (Martin et al., 1961)

A mixture of 3-chloro-2,4-pentanedione (1.13 mL,0.01 mol), p-anisidine (1.23 g, 0.01 mol), p- toluene sulfonic acid (0.5 g) was refluxed in toluene (100 mL) for 8 h. As the reaction proceeded, water produced as a by-product and removed using Dean-Stark apparatus. The advancement of reaction was noted by TLC (hexane: ethyl acetate; 8:1). On the completion of reaction excess solvent was eliminated under reduced pressure. Shiny crystals of pure product (1.44 g) were obtained after recrystallization (Scheme 1a).

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Scheme 1 Scheme of reaction. (a) Synthesis of (PA) [3-chloro-4-((4-methoxyphenyl)amino)pent-3-en-2-one] . (b) Synthesis of Metal complexes.

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(PA) Molecular formula: C₁₂H₁₄ClNO₂, Pale yellow solid, m.p: 42 °C, Yield: 60%, Molecular weight: 239.7 g/mole ; IR (cm-¹): 3014 (NH), 1587 (C=O), 1557 (C=C) ; ¹H NMR (300 MHz, CDCl₃, δ ppm): 2.13 (s, 3H, CH₃), 2.36 (s, 3H, CH₃), 3.82 (s, 3H-OCH₃), 6.92–6.87 (m, 2H, - Ar-H), 7.05–7.00 (m, 2H, - Ar-H), 12.64 (s, 1H, NH); ¹³C NMR (75 MHz, CDCl₃) δ: 18.10, 28.53, 55.48, 103.74, 114.41, 127.04, 131.25, 158.09, 159.33 and 194.13; FAB-MS: m/z = 240.1 [M + H] ⁺; Anal. Calcd. for C₁₂H₁₄ClNO₂, Theoretical: C, 60.13; H, 5.89; N, 5.84% Found: C, 58.78; H, 4.92; N, 4.82%.

2.3 Synthesis of metal complexes

2.4 Physical measurements

The Infra-Red spectra were taken on (Agilent Technologies) FT-IR instrument (4000–400 cm⁻¹). TMS was utilized as an internal reference to find the ¹H NMR spectrum on a Bruker Ascend NMR spectrometer. The JEOL JMS-600H Mass Spectrometer was used to find (FAB) mass spectrum. ICP-OES Optima 2100DV Perkin Elmer instrument was used to find the metal ion concentration with the help of already reported method in literature (Ahmed et al., 2017). Magnetic moment of all the complexes were calculated using magnetic susceptibility balance (Sherwood Scientific. Cambridge, UK). Thermogravimetric analysis of complexes was carried out to find the stability and decomposition pattern utilizing SDT-Q 600 V20.9 Build 20 instrument up to 800 °C in the presence of an inert gas. Conductivity measurements were made with the inoLab Cond 720 conductometer at room temperature.

2.5 Biological evaluation

2.6 Molecular docking studies

2D assemblies of all the synthesized compounds were first generated using Marvin Sketch then imported into Discovery studio client for making 3D assemblies and then energy minimization (Elmer, 2017). Ligand preparation tool in Discovery studio client creates molecular structures of minimum energy with their optimized stereoisomers, ring conformations, tautomers, and ionization states to make broad structural and chemical diversity from a single input compound.

Three dimensional X-rays crystallographic structure of target protein urease were retrieved from protein data bank utilizing PDB ID: 4UBP which has high resolution of 1.55 Å. To prepare the protein structure, Discovery Studio Client software was utilized which eliminates the heteroatoms, water molecules; inserted the hydrogens and missing residues and assign charges (if any) (Biovia 2018). Once targets structures were prepared, active position was identified utilizing co-crystal acetohydroxamic acid ligands.

3.1 Ftir spectra

The attachment of the metal ions with the ligand to form complexes was determined by comparison of the FTIR spectra of both the ligand with metal complexes (Table 1).

A strong peak at 3014 cm⁻¹ in the spectrum of the ligand (PA) was allocated to υ(NH) group (Shi et al., 2004). While spectra of all complexes showed absorption peaks in the range (3325–3150 cm ¹). This change showed the involvement of –NH group in coordination (Li et al., 2013). It can be attributed to the flow of electron density from nitrogen of NH group to the metal ion causing a greater ionic character of N-H bond and a rise in frequency. Further it is verified by the occurrence of a new band in all the spectra of metal complexes in the range of 483–473 cm⁻¹ assigned to the formation of metal and nitrogen bond (Pansuriya and Patel, 2007).

The stretching vibration at 1599 cm⁻¹ (conjugated carbonyl group) of compound PA showed hypsochromic shift in Cd (PA-Cd) and Mo (PA-Mo) complexes, indicating involvement of oxygen of CO group in the coordination (Tyagi et al.,2015). This is further reinforced by the presence of band in region of (587–573 cm⁻¹), which confirms the formation of (M−O) bond (Pansuriya and Patel, 2007). However, the blue-shift and a broad band in Co complex (PA-Co) recommended the Coordination of an acetate group. In fact, the band for the coordinated acetate anion and coordinated carbonyl group of the ligand are being overlapped (Shi et al., 2008).

The IR spectra of Mo complex (PA-Mo) showed the existence of bands related to four terminal carbonyl groups in the range 2010–1710 cm⁻¹ (Saleem et al., 2012). All this data suggested that the ligand (PA) is attached to the metal ions in bidentate form (neutral) through with the nitrogen atom of amino group and the oxygen of carbonyl (Jeragh and Elassar, 2015). The proposed structures of complexes are shown in Figs. 5, 6 and 7.

3.2 ¹H NMR

¹H NMR of ligand (PA) is shown in Fig. 1. It indicated the existence of N–H proton at 12.64 ppm (Shi et al., 2005). Two multiplets were also observed in the range 7.05–7.00 and 6.92–6.87 ppm indicating the presence of aromatic protons. Methyl protons of the aminopent-3-en-2-one chain (aliphatic chain) revealed two singlets at 2.36 and 2.13 ppm. Furthermore, methoxy protons also revealed a singlet at 3.82 ppm. All these chemical shifts matched perfectly by the suggested structure of ligand.

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Fig. 1 ¹H NMR spectrum of PA.

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3.3 ¹³C NMR

The structure of the ligand was further verified by ¹³C NMR spectrum. The peak of carbonyl carbon appeared at 194.13 ppm. Two major peaks at 159.33 and 158.09 ppm belonged to the olefinic carbon atoms. The carbon nuclei of aromatic ring showed peaks in the range 131–114 ppm and the aliphatic carbons emerged at 28.53 ppm and 18.10 ppm respectively.

3.4 Mass spectrum

FAB-MS of ligand (PA) was also determined in which it showed peak at 240.1 as [M + H] ⁺.

3.5 Thermogravimetry (TG)

Thermal analysis was performed to acquire significant information about the thermal stability of the prepared complexes and to investigate the nature of solvent molecules (if present) to be outside or inside the inner coordination sphere of the metal (Shebl et al., 2012). The TG curves of all the compounds are shown in Fig. 2. The temperature ranges, %age weight losses together with removed units are provided in Table 2.

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Fig. 2 Thermogravimetric curves of PA and its complexes.

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The TG curve of (PA) exhibited one step decomposition which occurs at higher temperatures relative to its complexes. This may be ascribed to the existence of intramolecular H-bonding in the ligand (Seleem et al., 2007). All the complexes go through three stages of breakdown, but Co (II) complex undergoes four stage decomposition. The PA-Co complex with molecular formula [C₂₈H₃₄Cl₂CoN₂O₈] displayed four stages of mass loss. The first stage is between 80 and 160 °C and showed a mass loss of 19 %, resulted in the loss of two acetate groups (calc., 17.9 %). The second step occurs between 160 and 300 °C, with a mass loss of 13.8 %, leading to the loss of [C₃H₃ClO] (calc., 14%). The third stage of decomposition takes place between 300 and 360 °C, with a mass loss of 33.9 %, leading to the loss of [C₁₂H₁₃ClON] (calc., 33 %). The fourth step of decomposition take place above 360 °C, with 34.4 % residue as [C₉H₁₁CoNO₂] (calc., 36%).

The other two complexes PA-Cd and PA-Mo go through three stages of breakdown. In the first stage, the PA-Cd complex with the molecular formula [C₂₄H₂₈Cl₄CdN₂O₄], showed a mass loss of 28.4 % between 220 and 410 °C, resulted in the loss of two chlorides and [C₅H₆ClO] (calc., 29 %). The second step occurs between 410 and 620 °C, with a mass loss of 53 %, leading to the loss of [C₁₈H₂₁ClN₂O₃] (calc., 54.6%). The third stage of decomposition takes place above 650 °C, with 16.9 % residue [C₁₂H₁₃ClON] as (calc., 18%).

The PA-Mo complex with molecular formula [C₁₆H₁₄ClMoNO₆] displayed three stages of mass loss. The first stage is between 100 and 270 °C and showed a mass loss of 25.1 %, resulted in the loss of four carbonyls groups (calc., 24 %). The second step occurs between 270 and 800 °C, with a mass loss of 33.3 %, leading to the loss of [C₉H₁₁NO] (calc., 35%). The third stage of decomposition takes place above 800 °C, with 41.6 % residue as [C₃H₃ClMoO](calc., 41 %)(Tyagi et al.,2015).

3.6 Magnetic moment and electronic spectral data

Electronic absorption spectrum was obtained in UV–visible zone. Charge transfer absorption bands were observed for all the complexes between 260 and 385 nm (Yang et al., 2016) Table 3.

The PA-Co complex revealed a band of mild intensity at 570 nm, which may be due to 4T1g (F) → 4T1g (P) transition in an octahedral geometry (Shebl et al., 2021). Its magnetic moment value (4.70B.M) also confirmed octahedral geometry (Abd El-Wahab, 2007). Only charge transfer bands were found for complexes PA-Cd and PA-Mo and they did not indicate any, d-d transition in their spectrum. Unsurprisingly, both are diamagnetic. Octahedral Geometry is recommended for both complexes based on FTIR, conductance and analytical data (Omar and Mohamed, 2005; Mohamed et al., 2002).

3.7 Biological activity

All the compounds were assessed for anti-urease and cytotoxic activities and findings are given in Table 4.

3.7.2

3.7.2 Cytotoxic activity

The cytotoxicity of the ligand and complexes against human breast cells (MCF-7) were also assessed. Cis-platin was used as a standard drug with an IC₅₀ of 20 µg/mL. Result suggested that the tested free ligand (PA) (IC₅₀: 78.04 ± 0.56 µg/ml) show higher cell viability than standard drug. This indicates that cytotoxicity of PA is less than the standard drug. On the other hand, PA-Co with IC₅₀ of 24.03 ± 0.82 µg/ml exhibited higher cytotoxicity than parent compound and very close cytotoxic value to standard drug Cis-platin. This indicates the potential of complex PA-Co to induced cytotoxicity in cancer cells. The IC₅₀ concentration PA-Co when applied on normal cells (3T3) exhibits non-significant cytotoxicity. The Mo (0) complex (PA-Mo IC₅₀: 150.57 ± 2.61) showed weak activity as its concentration of 50 % inhibition was very high than that of standard drugs while the Cd (II) complex (PA-Cd) was inactive to induce the cytotoxicity. Analysis of cellular viabilities exhibited by ligand PA and its metal complexes are shown in Fig. 3.

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Fig. 3 Analysis of cellular viabilities exhibited by series of PA complex. (A) cell viabilities of free ligand PA, A1) the cell viability values by different concentrations of PA; A2) IC₅₀ concentration of PA; A3) cell viability of PA on normal cells (3 T3). (B) viability values of complex PA-Co, B1) cell viability by different concentrations of PA-Co; B2) IC₅₀ concentration of PA-Co; B3) cell viability of PA-Co on normal cells (3 T3). (C) viability values of complex PA-Cd, C1) cell viability by different concentrations of PA-Cd; C2) IC₅₀ concentration of PA-Cd; C3) cell viability of PA-Cd on normal cells (3 T3). (D) viability values of complex PA-Mo, D1) cell viability by different concentrations of PA-Mo; D2) IC₅₀ concentration of PA-Mo; D3) cell viability of PA-Mo on normal cells (3 T3). All values are stated as SEM and p > 0.05 is considered to be significant while * indicate the degree of significance.

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3.8 Molecular docking studies

Molecular docking study was conducted using Autodock4 implemented in AMDOCK v 1.5.0 (Assisted Molecular Docking with AutoDock4 and AutoDock Vina) (Valdes-Tresanco et al.,2020). Default parameter in AMDOCK was used for the Molecular Docking calculation. Based on lowest binding energies, best pose was selected for each compound and subjected to Discovery Studio Visualizer for depicting the binding interaction as shown in Fig. 4. Estimated Free Binding Energy (kcal/mol) of all tested compounds are shown in Table 4 while detailed binding interaction are also summarized in Table 5. Comparative analysis of free binding energy and invitro urease activities showed as very good correlation in predicting the most active and least active compounds affinity. As shown in Table 4, most potent compound. PA-Co has lowest free binding energy (−8.37 kcal/mol) which is most stable complex among all other tested compounds. Similarly, least active compound against urease, PA-Mo having highest free binding energy (-2.30 kcal/mol) has least stable complex formation with urease.

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Fig. 4 Three dimentionl binding interaction of metal complexes with target urease proteins in color represenation. Hydrogen and attractive charge bonding are shown in green and gold dotted lines, respectively. (colored).

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3.9 Validation of Molecular docking studies

Validation of molecular docking studies was performed by redocking of cocrystal ligand and docking of reference compound thiourea within the active site of urease. Redocking of crystal ligand give a RMSD of less than 1 Å which decipher the acceptability of molecular docking protocol.

3.10 Binding analysis of metal complexes with amino acid residue of urease

Analysis of biding interaction between metal complexes and amino acid residue of urease were performed to decipher the differences in the binding pattern of different ligand and metal complexes with urease. Detailed binding interactions are summarized in Fig. 4 and Table 5. As shown in Table 5, ligand PA has only two bonding with amino residue of urease. While in case of metal complexes of Cu (II), Cd (II) and Mo (0), there were substantial increase in the total number of binding interactions as shown in Table 5. These interactions show differences and significant binding of the synthesized metal complex than ligand without metal complex against the urease contributing to a favorable invitro inhibition activity.