Synthesis and Anticancer Activity of Mixed Ligand, Cobalt (II), Nickel (II), Manganese (II) Complexes of Tertiary Diphosphines with Dithizone (H₂dz)

Introduction

Extensive study of metal complexes as chemotherapeutic and drug agents is widely related to employment of cisplatin as an anticancer drug a above thirty years ago.¹ But all the cisplatin is suffering from several affects like individual drug resistance. Above six years ago the status of cisplatin has been updated.² The studies of N-heterocyclic Pt (II) complexes as reproducible of cisplatin have been showed that the planar substituted pyridine ligands in platinum complexes can be the mode of action of cisplatin.^3,4

The medicinal uses and applicable of metals and metal coordination compounds are increasing drug and chemotherapeutic agents.^5,6 The anticancer abilities of the 10 elements; As, Sb, Bi, Au; V, Fe, Rh, Ti, Ga, Pt have been reported.⁶

Dithizone has Thione-Thiol tautomer (Scheme 1) and dithizonate has isomeric form (Scheme 2). Dithizone has been reported as a good ligand because of its ability to form colored complexes with different metal ions. Dithizone is also used in medical applications for the treatment of prostate cancer and pain due to metastases.^7,8

Scheme 1: Thione –Thiol tautomers1, 5-diphenylthiocarbazone (dithizone;H2dz) Click here to View scheme

 

Scheme 2: Isomeric forms of dithizonate ion and its metal complexes. Click here to View scheme

 

This work reports that the mixed ligand dithizone containing phenyl phosphine’s with Cobalt(II), Nickel(II), Manganese(II) formulas; [Co(H₂dz)(k²-dppf)]Cl, [Ni(H₂dz)(k²-dppf)]Cl, [Ni(H₂dz)(k²-dppe)]Cl and[Mn(H₂dz)(k²-dppf)]Cl. The anticancer evaluation activities were obtained on Human breast cancer cells, using cisplatin as a reference.

Experimental

Materials and Instrumentation

The compounds CoCl₂.2H₂O, NiCl₂.6H₂O, MnCl₂.6H₂O are obtained from BDH. Whereas dppm, dppp, dppf, were commercially available and obtained from Yacoo chem. China. Dithizone from Sigma Aldrich Company.

Micro elemental analyses were obtained using EuroEA 3000 instrument. The infra-red spectra in range (4000-400 cm^-1) were measured as potassium bromide discs on Shimadzu, FT-IR spectroscopy Mod IR Affinity-1CE spectrophotometer. The ¹H-NMR spectra were recorded on a Bruker 400 MHZ Ultra-shied. The electronic transition spectra were taken  by using AE-UV1609 (UK) CO., LTD Shimadzu, in the range (200-800) nm. The magnetic  moment values of the synthesized complexes were measured at 25°C using Bruker Magnet BM6. The metal analyses were obtained by using a Py-Unicm SP9 atomic absorption spectrophotometer. The molar conductivities were recorded using a pH/Conductivity/Meter 901 (0.93 cell constant) (Turkey). Melting points of the complexes  were carried out using Melting Point-MPD-100 Pixel Technology CO., Limited apparatus.

Human breast cancer cell line CAL5 was maintained by minimum essential media (MEM) (US Biological, Salem, MA, USA) afforded with 10% fetal calf serum, penicillin and streptomycin at 37°C. Rat Embryo Fibroblast (REF) cells were kept in RPMI-1640 (US Biological, Salem, MA, USA) gained   by penicillin/streptomycin and 10% fetal Bovine serum (Capricorn scientific, Germany). All cell lines were obtained from cell culture research lab, Iraqi Biotechnology Company, IRAQ Biotech (Baghdad, Iraq).

Synthesis of [Co (Hdz) (k²-dppf)] Cl (1)

The solutions of a (0.0256 g, 0.1 mmol) of H₂dz in 10 cm³ of methanol, (0.1 mmol, 0.0273g) of CoCl₂.6H₂O in a10 cm³ of methanol) and (0.0554 g, 0.1mmol of 1,1′-bis (diphenylphosphine) ferrocene (dppf) were mixed and refluxed at 55°C for 2 hrs. to give brown colored precipitate and the solutions was filtered and finally it was dried in an open air. Yield= 76%, d.p= 288°C. Anal. Calc.% for C₄₇ H₄₁ Cl N₄ P₂ Co S Fe:C, 62.30; H, 4.56; N, 6.18, S,3.54, Co, 6.50,Cl,3.91. Found %: C, 61.95; H, 4.62; N, 6.48, S; 3.85, Co; 6.73,Cl, 4.01. ¹H-NMR (d6-DMSO) δ:7.78(4H,d) ,7.42( 4H,t ), 7.28 – 7.38(mH), 11.61(1H). The proposed geometrical structure is shown in Figure 1.

Synthesis of [Ni (Hdz)(k²-dppf)]Cl (2)

The solutions of a (0.0256 g, 0.1 mmol)of H₂dz in 10 cm³ of methanol,(0.1 mmol, 0.0273g) of NiCl2.6H₂O in 10 cm³ of methanol) and (0.0554 g, 0.1mmol of 1,1′-bis(diphenyl phosphine) ferrocene (dppf) were mixed and refluxed at 55ºC for 2 h. to give red-brown colored precipitate and the solutions was filtered and finally it was dried in an open air. Yield= 72%, d.p= 265°C. Anal. Calc. % for C₄₇ H₄₁ Cl N₄ P₂Ni S Fe: C, 62.32; H, 4.56; N, 6.19, S, 3.54, Ni, 6.48, Cl,3,91. Found %: C, 61.83.7; H, 4.72; N, 5.94, S; 2.89,Ni; 6.93, Cl, 3.98. The proposed geometrical structureis is shown in Figure 1.

Figure 1: The suggested Geometrical Shapes of, [M (Hdz)(k2-dppf)]Cl Complex. Click here to View figure

 

Synthesis of [Ni (Hdz)(k²-dppe)]Cl (3)

The solutions of (0.0256 g, 0.1 mmol) of H₂dz in 10 cm³ of methanol, (0.1 mmol, 0.0273g) of NiCl₂.6H₂O in 10 cm³ of methanol and (0.0398 g, 0.1mmol of 1,2-bis(diphenylphosphino) ethane (dppe) were mixed and refluxed at 55 Cº for 2 h. to give violet colored precipitate and the solutions was filtered and finally it was dried in an open air. Yield= 78, d.p= 198°C. Calc.% for C₄₇ H₄₁ Cl N₄ P₂Ni S:C, 66.41; H, 4.86; N, 6.59, S, 3.77, Ni, 6.90,Cl,4.71, Found %: C, 66.85; H, 4.92; N, 6.84, S; 3.87, Ni; 7.03,Cl, 4.83. ¹H-NMR (d6-DMSO) δ:7.82(4H,d), 7.46 (4H,t), (mH)7.36 – 7.44. The proposed geometrical structure is shown in Figure 2.

Figure 2: The Proposed Geometrical Structure of, [Ni(Hdz)(k2-dppe)]Cl Complex. Click here to View figure

 

Synthesis of [Mn(Hdz)(k²-dppf)]Cl (4)

The solutions of (0.0256 g, 0.1 mmol) of H₂dz in 10 cm³ of methanol, (0.1 mmol, 0.0233g) of MnCl2.6H₂O in 10 cm³ of methanol) and (0.0554 g, 0.1mmol of 1,1′-bis(diphenyl phosphine) ferrocene (dppf) were mixed and refluxed at 55ºC for 2 h. to give brown colored precipitate and the solutions was filtered and finally it was dried in an open air. Yield=81%, d.p= 238°C. Anal. Calc. % for C₄₇ H₄₁ Cl N₄ P₂ Mn S Fe:C, 62.58; H, 4.58; N, 6.21, S, 3.55, Mn, 6.09, Cl, 3,93. Found %: C, 62.87, H, 4.82; N, 6.53, S; 3.88, Mn; 6.27, Cl.4.11. The proposed geometrical structure is shown in Figure 1.

Results and Discussions

The complexes were prepared by one pot- reaction (Scheme 3). This reaction consist of mixing of the solutions of H₂dz ligand in methanol, MCl₂.6H₂O where M= Ni, Mn and Co in methanol and 1,1′-bis(diphenyl phosphine) ferrocene (dppf) or 1,2-bis(diphenylphosphino) ethane (dppe) and refluxed at 55ºC for 2 h. The properties of complexes are tabulated in Table (1), the proposed structural formula are approved by IR, ¹H-NMR, UV-Visible spectra, CHNS analysis, magnetic susceptibility and molar conductivity measurement. The synthesized complexes have biological activity against breast cancer IC50 cell line that have a significant differences from cisplatin.

Scheme 3: one pot reaction for the synthesis of complexes. Click here to View scheme

 

Table 1: physical characteristics of synthesized metal Complexes.

Complex	Formula	Yield%	d.P(°C)	Color
[Co(Hdz)(k2-dppf)]Cl	C47 H41Cl N4 P2 Co S Fe	76	288	Brown
[Ni(Hdz)(k2-dppf)]Cl	C47 H41Cl N4 P2 Ni S Fe	72	265	Red-Brown
[Ni(Hdz)(k2-dppe)]Cl	C47 H41Cl N4 P2 Ni S	78	198	Violet
[Mn(Hdz)(k2-dppf)]Cl	C47 H41Cl N4 P2Mn S Fe	81	238	Brown

 

IR Spectral Studies

The IR spectra of complexes exhibited a medium intensity peaks in the range 423 – 432 cm^-1 these bands are related to ν(M-N), The a bands between 507 – 510 cm^-1 are attributed  to ν(P-Ph) and bands in (744 – 750) cm^-1 region are related to ν(C-S) and many other bands that shown in the⁹ Table 2.

Table 2: Selected IR stretching vibration bands in (cm^-1) for the prepared complexes.

Complex	v(N-H)	v(C-H) arom.	v(C-H) aliph.	ν(N=N)	ν(C=N)	ν(C-S)	ѵ(P-Ph)	ѵ(M-N)
1	3437w	3053w	–	1479m	1600m	746m	510m	423w
2	3439w	3053w	–	1463m	1591m	750s	508w	428w
3	3437w	3029w	3952w	1496m	1593m	744m	507w	432w
4	3410w	3103w	–	1489m	1602m	752s	515w	425w

 

¹H-NMR Spectra

The ¹H-NMR spectra for, [Ni(Hdz)(k²-dppf)]Cl (2) and, [Ni(Hdz)(k²-dppe)]Cl (3) Complexes showed many signals which attributed to aromatic rings¹⁰. See Table 3.

Table 3: ¹H-NMR signal for [Ni(Hdz)(k²-dppf)]Cl and [Ni(Hdz)(k²-dppe)]Cl

	4 protons
Complex	Doublet (d)	Triplet (t)	Multiple protons	N-H
[Ni(Hdz)(k2-dppf)]Cl	7.78	7.42	7.28 – 7.38	11.61
[Ni(Hdz)(k2-dppe)]Cl	7.82	7.46	7.36 – 7.44	–

 

Electronic Spectra

The electronic transition spectra in 10^-3 M in methanol (Table 4) gave two peaks of complex (1), at ( 1764, 30674 )cm^-1, which are assigned to the ⁴A₂→ ¹T₁(P), and charge transfer (C.T) transitions respectively, which  are indicated  a tetrahedral geometry.¹¹ The spectrum of complex (2), exhibited two transitions at 15974 cm^-1, and 29411 cm^-1 which are related to the ³T₁→ ¹T₁(p) and C.T respectively, these bands indicated tetrahedral geometry.¹² The spectrum of complex (3), gives  two peaks at (16155, 30674) cm^-1 which are assigned to ³T₁→ ¹T₁(p) and C.T transitions respectively. These transitions value are approved the tetrahedral geometry.¹³ The spectrum of complex(4), exhibits  three peaks at (15384,22371 , 31250) cm^-1. These transitions values approved a tetrahedral geometry.¹⁴

Magnetic Moments

The magnetic moments of complexes [Co (Hdz) (k²-dppf)] (1), [Ni (Hdz)(k²-dppf)]Cl (2), [Ni (Hdz)(k²-dppe)]Cl (3) and [Mn(Hdz)(k²-dppf)]Cl (4) are between (4.0- 5.20) B.M. (Table 4). These data are agreed with the tetrahedral geometrical complexes. The Cl% has been determined after digestion 0.1g of the complexes in (3-5) cm³ of 6M of HNO₃ and their titrations against 5% AgNO₃.¹⁶

Conductivity Measurement

The molar conductivity of complexes [Co (Hdz) (k²-dppf)] (1), [Ni (Hdz)(k²-dppf)]Cl (2), [Ni (Hdz)(k²-dppe)]Cl (3) and [Mn(Hdz)(k²-dppf)]Cl (4) in methanol are 46, 22, 28 and 60 (Ω^-1 cm² mol^-1) respectively indicating that they are electrolytes complexes in the 1:1 ratio.¹⁷ See Table 4.

Table 4: Electronic spectra, magnetic moments and molar conductivities  of Complexes.

Complex	λmax (nm)/	assignments	μeff (BM)a	ʌm (cm2. Ohm-1. Mol-1)/
	Methanol		Methanol
1	586	4A2 → 4T1(P)	4	46
	326	C.T	Td
2	273	C.T	4.2	22
	626	3T1 → 3T1(p)	Td
3	326	C.T	4.22	28
	619	3T1 → 3T1(p)	Td
4	320	C.T
	477	6A1 → 6T2(G)	5.2	60
	650	6A1 → 6T1(G)	Td

 

Methylthiazolyltetrazolium (MTT) Solution

Methylthiazolyltetrazolium (MTT) (0.2g) (Bio-world, USA) was dissolved in 100 ml of PBS in order to prepare a 2 mg/ml concentration of the dye. The resulted solution was filtered by 0.2 µm syringe filter to remove any produced blue formazan, and then kept in sterile, dark, screw-capped bottles at 4°C. The solution was used within no longer than two weeks of preparation.

Cytotoxicity Essay

MTT cell viability assay was joined on 96-well plates (Santacruz Biotechnology, USA), CAL5 cells were planted at 10000 cells/well, 200μl of cells in growing medium were added to each well of a sterile 96-well micro titration plate. The plates were placed with a self-adhesive film, then put  in incubator  at 37°C.

The achievement of monolayer has been obtained  after 24h the medium was removed and serial dilutions of the extract were added to the wells at 2-fold serial dilutions (1000ug, 500ug, 250ug and 125ug). Triplicates were used for each. Control cells treated with Serum Free Media only, and then the plates were put again in incubator at 37°C for 72 h (see Table 5).

Table 5:  Cytotoxic activities of the prepared complexes on the CAL5 cell line.

T/Ccorr. [%]^b+(S.D)^a

Complex	1000 µg	500 µg	250 µg	125 µg
1	20.01± 0.007	12.05 ± 0.017	10.29 ± 0.009	8.18 ± 0,06
2	24.10± 0.013	20.89 ± 0.011	18.11 ± 0.51	17.45 ± 0.016
3	25.56± 0,0051	15.85 ± 0.000	14.75 ± 0.0035	13.51 ± 0.0014
4	28.12 ± 0.0056	10.05 ± 0.023	10.29± 0.0097	8.18 ± 0.0023
MLc	28.12 ±0.018	31.48 ±0.015	27.17± 0.01	24.76 ± 0.096
Cisplatin	34.91 ± 0.094	28.56 ± 0.00	27.83 ± 0.011	26.07 ± 0.05

 

^aS.D standard Deviation, ^bCytocidal effect, ^cML Mixed ligand

Cell viability was recorded after 72 h of exposure by removing the medium, addition of 28 µl of 2 mg/ml of MTT solution (Bio-World, USA) and then placing in incubator for 1.5h. at 37°C. The MTT solution was removed then  the formed crystals in the wells was solubilized by the addition of 50 µl of DMSO (Santacruz Biotechnology, USA) sequenced  by 37°C incubation for 15 min with shaking. The absorbence was recorded on a micro plate reader (Biochrom, UK) at 584 nm .The process  was carried out in 5 replicates.

The mentioned procedure  leads to calculation of: 1- % of cell viability or % of cell proliferation (PR) = mean of treatment / mean of control. 2- Lowest concentration that kills 50% of cells (CAL5).

Cytotoxicity Results

The protocol described was according to.^18,19 Human Breast cancer cell line CAL5 was used for evaluation anticancer activities of prepared complexes using cisplatin as a reference, the results indicated that the Co(II) complex exhibited the highest cell viability Table 5. Table 6 showed the cytocidal effects of complexes were exhibited as adjusted T/C regarding to the applicable relationship (1).²⁰

T/C_corr.[%] = [(T-C_o)/(C-C_o)] x100———-(1)

Cytocidal effect [%] =[(Co –T)/Co]x100 ——-(2);

if T< Co. (Where T: mean of the cell absorbance C: mean of the control Co: The absorbance of cell when t=0).The significant differences from cisplatin have been observed for all treatment complexes especially for [Co(Hdz)(K²-dppf)]Cl and [Ni(Hdz)(K²-dppe)]Cl, Table 6 ,Figures .(3- 8).

Table 6:% Cell viability of selected compounds versus IC50 concentration.

Compounds	125µg	250µg	500µg	1000µg
[Co(Hdz)(k2-dppf)]Cl	91.8	89.7	87.94	79.5
[Ni(Hdz)(k2-dppf)]Cl	82.54	81.88	79.10	75.96
Ni(Hdz)(k2-dppe)]Cl	89.55	88.10	80.42	75.96
[Mn(Hdz)(k2-dppf)]Cl	86.4	85.24	84.10	74.40
Mixed ligand(ML)	75.20	72.80	68.50	71.80
Cisplatin	73.90	72.00	71.43	65.00

 

Figure 3: Breast cancer cells 72 hours MTT assay for [Co (H2dz)(dppf) ]Cl. Click here to View figure

 

Figure 4: Breast cancer cells 72 hours MTT assay for [Ni(H2dz)(dppe)]Cl. Click here to View figure

 

Figure 5: Breast cancer cells 72 hours MTT assay for [Ni(H2dz)(dppf) ]Cl. Click here to View figure

 

Figure 6: Breast cancer cells 72 hours MTT assay for [Mn(Hdz)(dppf)]Cl. Click here to View figure

 

Figure 7: Histograph of four concentration (125 µg,250 µg, 500 µg,1000 µg) of IC50 versus % cell viability. Click here to View figure

 

Figure 8: Histograph of treatment compounds at 125µg IC50 cell Click here to View figure

 

Conclusion

All complexes have been synthesized from one- pot reaction. The characterization of the structure of the complexes shows a tetrahedral geometry around the metal center, Figure (1,2). Cytocidal effect study versus different concentrations of breast cancer IC50 cell line showed significant differences from cisplatin.

Acknowledgement

The author is acknowledger to the Department of Chemistry, College of Education, at Salahaddin University for supporting this work. Nelson Mandela Metropolitan University, Faculty of Science for their helps. Baghdad, Anticancer Research Center for anticancer evaluation of the complexes.

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