Synthesis, Characterization and Cytotoxicity of Ni (II), Pd (II), Pt (II) Complexes with 6-Methoxy-2, 3, 4, 5-Tetrahydropyridine (MTP)

Introduction

N-heterocyclic (NHC) compounds constitute a new type of ligands in coordination chemistry and organometallic chemistry¹, they belong to a new family of ligands with electronic characteristics similar to those of the phosphines². The ligands of heterocyclic rings contain nitrogen atom as a donor set the have π-acidity properties and they form many color complexes with transition elements in various oxidation state³. Recently mixed ligand of dithizone metal ion complexes were reported to have anticancer activity in vitro⁴. The current work reports synthesis of new complexes contains nitrogenic ligand of general structure [M(MTP)₂Cl₂] and their characterizations as tetra coordination number as square planar geometry. Also, the study of their anticancer ability versus MCF-7 cell line.

Experimental

Materials and Instrumentation

All chemicals used in this work are in of reagent grade and used without farther purification as supplied from Sigma, Solarbio, Fluka, Scharlue, BDH, Capricorn, Santacruze Biotechnology, and Bio-world. Electro molar conductivities for the 10^-3 Msolution of the synthesized complexes in (DMSO) using [Senz µSiemen tester 4200] college of education / Salahaddin University /Erbil. Elemental analysis (C.H.N) was measured using [EuroEA 3000/Italy] in the service laboratory at Ibn al-Haitham /college of education / University of Baghdad. FT-IR spectra were recorded using [Ftir-600 FTIR Spectrometer Biotech Engineering Management.UK] in the service laboratory at Ibn al-Haitham college of education/University of Baghdad. The UV/Vis were recorded using, AE-UV1609 Spectrometer (UK) Co.] in the college of education/Salahuddin University/Erbil. ¹HNMR and ¹³CNMR spectra were recorded on Brucker 300MHZ with tetramethylsilane as an internal standard in DMSO-d6 Measurements were made at water, environment and arid regions research center/ Al- a Bayt University/ Jorden. 

Synthesis of [M(MTP)₂Cl₂]

To a methanol (25 ml) solution of (MTP) (0.23g, 2mmol), an equivalent methanol (25 ml) solution of NiCl₂.6H₂O (0.24g, 1mmol), PdCl₂ (0.18g, 1mmol) and K₂PtCl₄ (0.42, 1mmol) was added in dropwise. Thereafter, the mixture was stirred for 9-14 h at room temperature. The resultant solution was filtered and set aside for a few days to give colored precipitates. The precipitates were filtered off and dried under vacuum.

Cytotoxicity Assay

Cell Lines

MCF-7 cells were provided from the Iraqi Cell Bank and kept and treated as in⁵

Cytotoxicity Assay

To emphasize the effect of cytotoxicity, methyl thiazolyl tetrazolium (MTT)⁵ viability test was measured using 96-well plates. MCF-7 were sowed at 1 × 10⁴ cells/well. Monolayer achievement has been occurred after 24 h. The cell viability assay has been taken after 72 h by addition 28 µL of 2 mg/mL of MTT solution, the cells were incubated for 1.5 h at 37 °C. The crystals which formed on the wells, during the incubating were dissolved by the addition of 130 µL of DMSO⁶. Absorbances were recorded at 492 nm. All tests were performed in triplicate and he inhibition rate of cell growth was obtained as follows:

Inhibition growth rate = A-B/A x 100 (where A and B are absorbances of control and tested compounds respectively).

Results and Discussion

A synthesized divalent metal complexes (Scheme 1) were obtained from reaction of Ni (II), Pd (II) and Pt (II) chloride salts with (MTP) in (2:1) ratio produce neutral-colored complexes.  All complexes have good solubility in dimethylsulphoxide. The conductivities in 10^-3M dimethylsulphoxide solutions are between (10-20) Ω^-1 cm² mol^{-1 7}. This is inconsistent with the stoichiometry suggested complexes. The analytical data of synthesized complexes are shown in the (Table 1), were agreement with the calculated values. The suggested molecular formulas were also supported by spectral and magnetic moment measurements.

Scheme 1: Formation of [M(MTP)2Cl2] complexes Click here to View Scheme

Magnetic Susceptibility

The magnetic moments were taking at 25⁰C. The results proved square planar structures for all complexes ⁸

Table 1: Physical analytical data for synthesized complexes

Elemental analysis, calc. (found) %				M%	Yield %	Melting point	Color	µeff	Complexes	No.
N	H		C
7.63 (7.87)	6.19 (6.23)	40.25 (40.50)		15.68 (16.49)	72	–	Blue- green	Dia.	[Ni(MTP)2Cl2]	1
6.74 (6.94)	5.49 (5.47)	35.13 (35.71)		26.10 (26.37)	77	143-148	Yellow-orang	Dia.	[Pd(MTP)2Cl2]	2
5.66 (5.69)	4.26 (4.50)	28.36 (29.28)		38.11 (39.63)	68	–	Dark Brown-	Dia.	[Pt(MTP)2Cl2]	3

Electronic Transitions Spectra

The electronic transition spectra of the ligand and its synthesized complexes in the 10^-3 M (DMSO) solution were illustrated in (Table 2). The electronic transition band at 37450 cm^-1 is for π- π* transition within ligands. A given spins allowed transitions at (21978) cm^-1 (υ₁), (22883-24875) cm^-1 (υ₂), (26109-28571) cm^-1 (υ₃) were assigned to ¹A₁g→¹A₂g (υ₁), ¹A₁g→¹B₁g (υ₂) and ¹A₁g→¹Eg (υ₃) transitions respectively. The strong bands at (29850-31847) cm^-1 are due to (MLCT) and d→d transitions. The transition band at 32786- 36363 cm^-1 is due to the n→p* transitions (LMCT) of the pyridine ring. These transitions value is indicated to square planar geometry ^9-11.

Table 2: UV/Vis transition for the synthesized complexes

	Transitions	Absorption band		Complexes	No.
		nm	cm-1
Sp.	1A1g ® 1B1g 1A1g ® 1Eg C.T	437 350 314	22883 28571 31847	[Ni(MTP)2Cl2]	1
Sp.	1A1g ® 1B1g 1A1g ® 1Eg C.T	402 383 305	24875 26109 32786	[Pd(MTP)2Cl2]	2
Sp.	1A1g ® 1A2g 1A1g ® 1B1g C.T C.T	455 411 335 275	21978 24330 29850 36363	[Pt(MTP)2Cl2]	3

Infrared Spectral

IR frequencies of the free ligand and complexes are tabulated in (Table 3). A peak at (3056) cm^-1 is for υ(CH) stretching structure¹², a frequency band at (2960) cm^-1 which may be due to the υ(CH) of methoxy group, generally it has been observed at lower frequencies than the normal υ(CH) for (CH₃-) group ¹³. The frequency band at 1625 cm^-1 is related   to υ(C-C) in the pyridine ring. A υ(C-O-C) is at 1260 Cm^-1. A frequency band at 1480 cm^-1 which are corresponding to υ(CN) stretching vibrations, all complexes showed a negative shift in ν(CN) confirmed the participate of nitrogen in coordination ¹⁴.

Table 3: IR band in cm^-1 for the ligand and the complexes

Compound	n(CH) ring	n(CH) methoxy	n(CN)	n(CC)	n(C-O-C)
MTP	3056	2960	1260	1625	1480
[Ni(MTP)2Cl2]	3100	2962	1256	1655	1442
[Pd(MTP)2Cl2]	3036	2954	1247	1666	1438
[Pt(MTP)2Cl2]	3085	2955	1256	1630	1448

NMR Spectra

¹H and ¹³C NMR bands were obtained using (DMSO-d₆) as a solvent (Table 4) and (Figure 1-3). The spectra showed two sets of bands which attributed to the pyridine part and substituted aliphatic of the MTP ligand. The ¹H NMR bands of coordinated ligand were shifted more than free ligand and they agree with complexion with metal ions through N of pyridine ring¹⁵. The ¹³C NMR data in all complexes show. No observed change in this signal in complexes formation and this due to uncoordinated aromatic carbon in the metal ion complexes¹⁶.

Table 4: NMR spectral for band in δ(ppm) for the ligand and their complexes

Compound	Atom	1HNMR	Atom	13CNMR
MTP	1H	3.53	1C	45.4
	2H	1.3	2C	22.2
	3H	1.77	3C	16.4
	4H	2.11	4C	29.7
	5H	3.47	5C	161.7
			6C	53.2
[Ni(MTP)2Cl2]	1H	2.6	1C	38.66
	2H	1.3	2C	22.24
	3H	1.96	3C	16.58
	4H	2.1	4C	37.65
	5H	3.53	5C	162.14
			6C	54.15
[Pd(MTP)2Cl2]	1H	2.82	1C	38.67
	2H	1.3	2C	21.65
	3H	1.77	3C	18.2
	4H	2.23	4C	29.74
	5H	3.59	5C	159.41
			6C	51.2
[Pt(MTP)2Cl2]	1H	2.94	1C	39.39
	2H	1.3	2C	21.36
	3H	1.6	3C	18.23
	4H	2.53	4C	31.34
	5H	3.88	5C	162.47
			6C	51.620

Figure 1: 1HNMR for [Ni(MTP)2Cl2] (A)  and  13CNMR for [Ni(MTP)2Cl2](B) Click here to View figure

Figure 2: 1HNMR for [Pd(MTP)2Cl2] (A) and 13CNMR for [Pd(MTP)2Cl2] (B) Click here to View figure

Figure 3: 1HNMR for [Pt(MTP)2Cl2 (A) and 13CNMR for [Pt(MTP)2Cl2] (B) Click here to View figure

Figure 4: A: IC50 on MCF-7 cell line for [Ni(MTP)2Cl2]   B: Histograph of five  different  concentration  of  MCF-7 versus % cytoxicity Click here to View figure

Figure 5: A: IC50 on MCF-7 cell line for [PT(mtp)2CI2] B: Histograph of five different concentration of MCF-7 versus % cytoxicity Click here to View figure

Figure 6: A: IC50 on MCF-7 cell line for [Pd(MTP)2Cl2]   B: Histograph of five  different  concentration of  MCF-7  versus % cytoxicity Click here to View figure

Cytotoxicity Results

Our study on synthesized Ni (II), Pd (II) and Pt (II) metal ion complexes were evaluated against human MCF-7 cells. Cisplatin was employed as in a reference [6]. It is found that the [ Pt (MTP)₂Cl₂] complex has the highest IC₅₀ cell viability and p-value¹⁷. The results included (cell growth %) or (% of cell treatment). The low concentration kills about 19% of MCF-cell line (Table 5) and (Figure 4- 6). Whilst Ni (II), Pd (II) complexes were less active than cisplatin is in the micromolar range. On the basis of existence of methoxy moiety on both pyridine rings play important role in inhibition linking of N of DNA with central metal, thus leading to increase cytotoxicity meta lion from the complex ¹⁸.

Table 5: Cytotoxic abilities of the synthesized complexes on the MCF-7 cells

Sample	Con.	6.25    µg/mL	12.5 µg/mL	25 µg/mL	50 µg/mL	100 µg/mL
Cisplatin	Mean	24.67	51.80	71.87	88.10	94.00
	P value	0.0343	0.0148	0.0100	0.0065	0.0068
	Significant (alpha=0.05)	Yes	Yes	Yes	Yes	Yes
[Pt(MTP)2Cl2]	Mean	13.66	30.71	53.87	68.00	74.98
	P value	0.0024	0.0004	0.0001	0.0094	0.0001
	Significant (alpha=0.05)	Yes	Yes	Yes	Yes	Yes
[Ni(MTP)2Cl2]	Mean	9.091	23.97	42.84	53.69	61.89
	P value	0.1318	0.0008	0.0024	0.0036	0.0011
	Significant (alpha=0.05)	No	Yes	Yes	Yes	Yes
[Pd(MTP)2Cl2]	Mean	10.40	24.18	33.79	46.91	68.91
	P value	0.0367	0.0215	0.0228	0.0147	0.0101
	Significant (alpha=0.05)	Yes	Yes	Yes	Yes	Yes

All complexes have been synthesized and characterized; the results indicate that all of them have square planer geometry in which the metal ions coordinate with the ligand through a nitrogen atom. The cytotoxicity effects study of the prepared complexes versus MCF-7 cell line in different concentrations showed that [Pt (MTP)₂Cl₂] has good cytotoxicity in comparison with the other complexes.

Acknowledgement

The authors are thankful to the Chemistry Department, College of Education/ Salahuddin University for assisting this work. The authors also thank the University of Mosul, College of Science for a partial help of this study.

Conflict of Interests

No conflict of interest.

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