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Original article
2021
:14;
202103
doi:
10.1016/j.arabjc.2021.102993

New metal complexes derived from diacetylmonoxime-n(4)antipyrinylthiosemicarbazone: Synthesis, characterization and evaluation of antitumor activity against Ehrlich solid tumors induced in mice

, , , , ,
a Biochemistry Division, Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Koom 32512, Egypt
b Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama 7008530, Japan
c Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom 32512, Egypt
d Department of Biochemistry, College of Medicine, Qassim University, PO 6655-51425, Saudi Arabia
e Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91, Stockholm, Sweden
f Pharmacognosy Group, Department of Medicinal Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden
g International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China
h Al-Rayan Research and Innovation Center, Al-Rayan Colleges, Medina 42541, Saudi Arabia

⁎Corresponding authors at: Biochemistry Division, Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Koom 32512, Egypt (B. El-Aarag) and Pharmacognosy Group, Department of Medicinal Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden (H.R. El-Seedi). bishoy.yousef@gmail.com (Bishoy El-Aarag), hesham.el-seedi@ilk.uu.se (Hesham R. El-Seedi)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

The present study aimed to synthesize new metal complexes of diacetylmonoxime-N(4)antipyrinylthiosemicarbazone ligand and evaluate their antitumor activity. New complexes with ferric, cobalt, nickel and copper ions were prepared. Elemental, 1H Nuclear magnetic resonance, Mass spectroscopy, Electron paramagnetic resonance, Fourier Transform Infrared Spectroscopy, Ultraviolet–visible and thermal gravimetric analysis were used to characterize the obtained complexes 1–11. An in vivo tumor model was established to investigate the effect of the naked ligand and its metal complexes 2, 5 and 8. Ehrlich ascites carcinoma solid tumor was induced in mice through subcutaneous inoculation of Ehrlich ascites carcinoma cells. The volumes of the formed solid tumors, the alanine transaminase, aspartate transaminase, albumin concentration in the serum, as well as the levels of Ki67 and p53 proteins in tumor and liver tissues were detected. All the tested complexes, especially complex 5, possessed proliferative inhibition manifested as the reduction of the tumor volume, Alanine aminotransferase & Aspartate aminotransferase activity, and the level of the Ki67 protein. Additionally, they restored the albumin concentration to normal levels as well increased the level of pro-apoptotic p53 protein. In conclusion, the antitumor activity of the newly synthesized metal complexes against Ehrlich ascites carcinoma solid tumors was proved to be mediated by the inhibition of Ki67 and induction of p53 proteins.

Keywords

Metal complexes
Thiosemicarbazone
Antitumor
Ehrlich tumor
Ki67
P53
1

1 Introduction

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and is the second main reason of death worldwide with the majority percent arises from the developing countries (Bray et al., 2018). The estimated worldwide rate of cancers will be increased to reach 12 million case within the next decade (WHO). On the same hand, Ehrlich Ascites Carcinoma (EAC) is a murine mammary adenocarcinoma (Mishera et al., 2018) and considered as a tumor model commonly used for the investigations of anticancer activity of newly synthesized chemical compounds (El-Sonbaty, 2013, Saad et al., 2017, Rageh et al., 2018, Aldubayan et al., 2019). A transcription factor p53 possesses important roles in activation of many target genes that mediated the apoptosis (programmed cell death) in cancer cells (Vousden and Lu, 2002, Lacroix et al., 2006). Additionally, the nuclear protein Ki67, expressed in proliferating cells, has essential functions in cancer cell proliferation (Schluter et al., 1993, Miller et al., 1994). Therefore, the new compounds that can trigger the cancer cells apoptosis and prevent their proliferation are attractive candidates in cancer treatment (Reed, 2003).

Organometallic compounds achieved a great success in medicine and attracted a significant interest as potential chemotherapeutic agents for the treatment of several types of diseases including cancer through inhibition of the targeted biomolecules (Benjamin and Paul, 2020, Dik-Lung et al., 2020). Metal complexes derived from oximes are very promising entities due to their numerous biological activities (Park et al., 2005, Dodof et al., 2009, El-Gamal et al., 2010) and their biodiversity was attributed to the coordination ability of oxime (Baghlaf et al., 1987, Kukushkin et al., 1992) as well as the binding of metal ions to oximato group (Ruiz et al., 1993, Coacio et al., 1995). In addition, antipyrine and thiosemicarbazones related complexes exhibited a wide-range of biological and chemotherapeutical impact Leovac et al., 2011, Refata and El-Metwaly, 2012).

In continuation to our previous investigations on the biologically active metal complexes (El-Sawaf et al., 1998, El-Saied et al., 2019), the current study aimed to synthesize, characterize and evaluate the antitumor activity of new metal complexes derived from diacetylmonoxime-N(4)antipyrinylthiosemicarbazone (DAMATSC) ligand on solid EAC tumors induced in mice. Also, the effect of complexes on the size of solid tumors as well as the expression levels of Ki67 and p53 in both hepatic and tumor tissues was evaluated.

2

2 Materials and methods

2.1

2.1 Chemistry

2.1.1

2.1.1 Synthesis of DAMATSC ligand (H2L)

The synthesis of DAMATSC ligand (H2L) was illustrated in Scheme 1. The ligand (H2L) was synthesized by mixing equimolar quantities of N(4)-antipyrinylthiosemicarbazide (El-Saied et al., 2019), and syn-methyl anti-acetylmonoxime in anhydrous EtOH and then the mixture was heated (80 °C) under reflux for 2 h. Two drops of sulfuric acid (H2SO4) were added to the mixture in order to accelerate the reaction rate.

Preparation of diacetylmonoxime-N(4)antipyrinylthiosemicarbazone (DAMATSC) ligand (H2L).
Scheme 1
Preparation of diacetylmonoxime-N(4)antipyrinylthiosemicarbazone (DAMATSC) ligand (H2L).

H2L·H2O (ligand): Yield (91%); color: white; M.P. = 210–212; C16H20N6O2S, (F.W. = 378.0): Found (calcd) %C 50.24(50.79), %H 5.59(5.82), %N 21.86(22.22), %S 8.90(8.47); IR (KBr, cm−1), 3447a, 3421b ν(H2O)a/(OH)b, 3303, 3123ν(NH), 1635 ν(C = O), 1613,1585,1524 ν(C = N)imine/(C = N)oxime, 882 ν(C = S), 1032, 960 ν(N-O); UV–Vis. (Nujol mulls) (nm) 280, 309, 315, 340 π → π*/n → π*(nm) 1H NMR signals, 11.591 δ(s-OH),10.624δ(s-N(2)-H,9.047δ(s-N(4)-Hδ, 7.507, 7.279 (m-Ar-H), 3.068δ(sN-CH3), 2.135δ(s-C-CH3), 2.034δ(s,N = C-CH3) (oxime).

2.1.2

2.1.2 Synthesis of metal complexes (1–11)

The metal complexes of the ligand with Fe(III), Co(II), Ni(II) and Cu(II) ions were synthesized by mixing 30 cm3 of a hot ethanolic solution of the metal salts: FeCl3·6H2O, CoCl2·6H2O, Co(OAc)2·4H2O, Co(NO3)2·6H2O, NiCl2·6H2O, Ni(OAc)2·4H2O, Ni(NO3)2·6H2O, CuCl2·2H2O, CuBr2, Cu(OAc)2·H2O and Cu(NO3)2·3H2O with the appropriate amount of hot (75 °C) ethanolic solution of the ligand (as shown in Table 1). The mixture was refluxed for about 3 hrs. The precipitated complexes were filtered and washed with ethanol many times followed by diethyl ether. The complexes were dried under vacuum over anhydrous calcium chloride.

Table 1 Physicochemical analysis of ligand and complexes 1–11.
No. Molar ratio /compound Colour F.W. M.P. (°C) TGA Analysis (%) / Found (calcd.) Molar Conductance
Temp. range (°C) C H N S M
H2L·H2O White 378.0 210–212 50.60
(50.79)		 5.59
(5.82)		 21.86
(22.22)		 8.90
(8.47)		 – –
1 H2L + FeCl3 (1:1) [Fe2(HL)2Cl4].H2O Green 990 Over 300 27–170

170–226
227–900		 38.62
(38.79)		 3.81
(4.04)		 16.63
(16.97)		 6.56(6.46) 11.53 (11.31) 30.24
2 H2L + CoCl2 (1:1) [Co(HL)Cl] Yellowish brown 453.4 Over 300 42.20
(42.35)		 4.35
(4.20)		 18.33
(18.50)		 6.99
(7.10)		 13.12 (12.99) 18.2
3 H2L + Co(OAc)2 (1:1) [Co (HL)(OAc)].H2O Reddish brown 494.9 280 43.46
(43.68)		 4.64
(4.85)		 17.31
(17.00)		 6.40
(6.47)		 11.73 (11.90) 20.6
4 H2L + Co(NO3)2 (1:1) [Co2(HL)2].(NO3)2·H2O Orange 977.8 Over 300 31–65

65–175 175–550		 39.22
(39.30)		 4.42
(4.10)		 19.82
(20.04)		 6.90
(6.60)		 11.92 (12.05) 125
5 H2L + NiCl2 (1:1) [Ni(HL)Cl].2H2O Brown 489.2 Over 300 31–90

90–210
225–780
780		 39.60
(39.30)		 4.9
(4.7)		 17.25
(17.17)		 6.40
(6.54)		 12.23 (12.00) 5.4
6 H2L + Ni(OAc)2 (1:1) [Ni(HL)(OAc)].H2O Reddish Brown 494.7 280 31–100
100–252

252–890
890		 43.39
(43.66)		 4.63 (4.85) 17.23
(17.00)		 6.13
(6.50)		 11.71 (11.87) 1.7
7 H2L + Ni(NO3)2 (1:1) [Ni2(HL)2].(NO3)2·H2O Beige 986.4 Over 300 39.14
(38.90)		 4.30 (4.15) 20.13
(19.87)		 6.65
(6.49)		 11.62 (11.90) 128
8 H2L + CuCl2 (1:1) or H2L + CuCl2 + NaOH (1:1:1)

[Cu(HL)Cl]		 Green 458.0 220 27–189

189–899		 41.95
(41.92)		 4.24 (4.14) 18.24
(18.34)		 7.38
(7.00)		 13.91 (13.86) 5.77
9 H2L + CuBr2(1:1) [Cu(HL)Br] Dark green 502.0 220 38.30
(38.25)		 3.63 (3.78) 16.50
(16.73)		 6. 61
(6.37)		 12.81 (12.65) 4.00
10 H2L + Cu(OAc)2 (1:1) [Cu(HL)(OAc)].2H2O Green 517.5 208–210 25–135
135–899

 42.01
(41.73)		 5.32 (5.02) 16.03
(16.23)		 6.31
(6.18)		 12.53 (12.27) 2.30
11 H2L + Cu(NO3)2 (1:1) [Cu(HL)(NO3)] Green 484.5 210 27–165
165–900

 39.77
(39.63)		 3.57 (3.92) 19.94
(20.22)		 7.00
(6.60)		 12.83 (13.11) 2.42

[Fe2(HL)2Cl4].H2O (complex 1): Yield (75%); color: green, M.p. = over 300 °C; ΛM: 30.24 O−1cm2 mol−1; Elemental analysis, C34H42N12O4S2Fe2Cl4, (F.W. = 990): Found (calcd) %M 11.53 (11.31), %C 38.12 (38.79), %H 3.76 (4.04), %N 16.44 (16.97), %S 6.79(6.46); IR (KBr, cm−1), 3445a, 3358b ν(H2O)a/(OH)b, 3191 ν(NH), 1634 ν(C = O), 1566, 1531, 1490 ν(C = N)imine/(C = N)oxime, 847ν(C = S), 1008.915 ν(N-O), 501ν(MN), 589ν(Fe-O); UV–Vis. (Nujol mulls) (nm) 270, 290, 348, 368, π → π*/n → π*(nm), 390, 450 Charge transfer(nm), 500(br), 580(Sh), 904(s)(br) d → d bands(nm); µeff (B.M) = 4.1.

[Co(HL)Cl] (Complex 2): Yield (74%); color: Yellwish brown; M.P. = over 300 °C; ΛM: 18.2 O−1cm2mol−1; Elemental analysis for, C17H21N6O2SCoCl, (F.W. = 453.4): Found (calcd) %M 13.12 (12.99), %C 42.19(42.35), %H 4.45(4.20), %N 17.80 (18.50), %S 6.99(7.10); IR (KBr, cm−1), 3410bν(H2O)a/(OH)b, 3280ν(N-H), 1637ν(C = O), 1613,1525,1495 ν(C = N)imine/(C = N)oxime, 815ν(C = S), 1074.970ν (N-O), 499ν(M−N); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 277, 292, 325 (br), charge transfer(nm) 397, 460, 497, d → d bands(nm) 530, 610, 680; µeff (B.M) = 3.87.

[Co(HL)(OAc)].H2O (Complex 3): Yield (75%); color: Reddish brown; M.P. = 280 °C; ΛM: 20.6 O−1cm2mol−1; Elemental analysis for, C19H24CoN6O4S, (F.W. = 494.9): Found (calcd) %M 11.73 (11.90), %C 44.19(43.68), %H 4.54(4.85), %N 17.59(17.00), %S 6.40(6.47); IR (KBr, cm−1), 3445a(sh), 3420b(OH)ν(H2O)a/(OH)b, 3325, 3260ν(N-H), 1632ν(C = O), 1591, 1545, 1495ν(C = N)imine/(C = N)oximen, 846ν(C = S), 1085,980ν(N-O), 503ν(M−N), 1652, 1380ν(NO3); UV–Vis. (Nujol mulls) π → π*/ n → π*(nm) 280, 292, 350(br), charge transfer(nm) 396, 450(br), d → d bands(nm) 623 (s,br), 690(Sh); µeff (B.M) = 3.91.

[Co2(HL)2].(NO3)2·H2O (Complex 4): Yield (66%); color: orange; M.P. = over 300 °C; ΛM: 125 O−1cm2mol−1; Elemental analysis for, C34H44Co2N14O11S2, (F.W. = 977.8): Found (calcd) %M 11.92 (12.05), %C 40.02(39.30), %H 4.79(4.10), %N 19.30(20.04), %S 7.10(6.60); IR (KBr, cm−1), 3440a, 3388b (br), ν(H2O)a/(OH)b, 3222ν(N-H), 1638ν(C = O), 1591, 1533, 1495ν(C = N)imine/(C = N)oximen, 815ν(C = S), 1024,942ν(N-O), 500ν(M−N), 1383(s)ν(NO3), 590 ν(Co-O), UV–Vis. (Nujol mulls) π → π*/ n → π*(nm) 260,300,338,353, charge transfer(nm) 400, 460, d → d bands(nm) 510, 545, 560; µeff (B.M) = 2.3.

[Ni(HL)Cl].2H2O (Complex 5): Yield (64%); color: brown; M.P. = over 300 °C; ΛM: 5.4 O−1cm2mol−1; Elemental analysis for, C17H21ClN6NiO2S, (F.W. = 489.2): Found (calcd) %M 12.23 (12.00), %C 40.01(39.30), %H 4.9(4.7), %N 17.25(17.17), %S 6.4 (6.54); IR (KBr, cm−1), 3446a, 3420b (br), ν(H2O)a/(OH)b, 3236ν(N-H), 1634ν(C = O), 1589, 1544, 1522ν(C = N)imine/(C = N)oximen, 813ν(C = S), 1073, 1025ν(N-O),501ν(M−N); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 278, 293, 313, 335, charge transfer(nm) 397, 430, 493, d → d bands(nm) 540(sh), 630(sh) µeff (B.M) = zero, 1H NMR signals, 11.591δ(s-OH),δ(s-N(2)-H, 9.052δ(s-N(4)-Hδ, 7.488,7.296(m-Ar-H), 3.058δ(s-N-CH3), 2.124δ(s-C-CH3), 2.0252 ,2.022δ(s, N = C-CH3) (oxime).

[Ni(HL)(OAc)].H2O (Complex 6): Yield (70%); color: reddishbrown; M.P. = 280 °C ; ΛM: 1.7 O−1cm2mol−1; Elemental analysis for, C19H24N6NiO4S, (F.W. = 494.7): Found (calcd) %M 11.71 (11.87), %C 43.19(43.66), %H 4.35(4.85), %N 17.59(17.00), %S 6.13(6.5); IR (KBr, cm−1), 3446a, 3413b (br), ν(H2O)a/(OH)b, 3262ν(N-H), 1641ν(C = O), 1591, 1522, 1495ν(C = N)imine/(C = N)oximen, 815ν(C = S), 1088, 1009ν(N-O), 501ν(M−N), 1650, 1382 ν(OAc); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 276(sh), 291, 321, 350, charge transfer(nm) 400, 500, 545, d → d bands(nm) 585, 635; µeff (B.M) = zero.

[Ni2(HL)2].(NO3)2·H2O (Complex 7): Yield (72%); color: beige; M.P. = over 300 °C ; ΛM: 128 O−1cm2mol−1; Elemental analysis for, C34H45N14Ni2O11.5S2, (F.W. = 986.4): Found (calcd) %M 11.62 (11.90), %C 39.75(38.90), %H 4.30(4.15), %N 20.13(19.87), %S 7.31(6.49); IR (KBr, cm−1), 3418a, 3380b (br), ν(H2O)a/(OH)b, 3192ν(N-H), 1635ν(C = O), 1591, 1556, 1493ν(C = N)imine/(C = N)oximen, 833ν(C = S), 1010, 944ν(N-O), 504, 471ν(M−N), 1383(s)ν(NO3), 588ν(Ni-O); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 280(sh), 296, 310 charge transfer(nm) 385, 400 d → d bands(nm) 444, 800(br); µeff (B.M) = 1.49.

[Cu(HL)Cl] (Complex 8): Yield (80%); color: green; M.P. = 220 °C; ΛM: 5.77 O−1cm2mol−1; Elemental analysis for, C17H21ClCuN6O2S, (F.W. = 458.0): Found (calcd) %M 13.91 (13.86), %C 41.95(41.92), %H 4.24(4.14), %N 18.24(18.34), %S 7.38(7.00); IR (KBr, cm−1), 3430b ν(H2O)a/(OH)b, 3352, 3173ν(N-H), 1640ν(C = O), 1614, 1541, 1541ν(C = N)imine/(C = N)oximen, 813ν(C = S), 1060 ,968ν(N-O), 525ν(M−N); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 280,294,330(br),354, charge transfer(nm) 385,400,446,467d → d bands(nm) 610(br); µeff (B.M) = zero.

[Cu(HL)Br] (Complex 9): Yield (77%); color: dark green; M.P. = 220 °C; ΛM: 4.0 O−1cm2mol−1; Elemental analysis for, C17H21BrCuN6O2S, (F.W. = 502.0): Found (calcd) %M 12.81 (12.65), %C 38.30(38.25), %H 3.10(3.78), %N 16.50(16.73), %S 6.80(6.37); IR (KBr, cm−1), 3427b ν(H2O)a/(OH)b, 3351, 3245, 3170ν(N-H), 1640ν(C = O), 1619, 1550, 1529ν(C = N)imine/(C = N)oximen, 813ν(C = S), 1063, 967ν(N-O), 535ν(M−N); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 277, 292(s),330(br), charge transfer(nm) 397, 443, 463(br), d → d bands(nm) 615(br); µeff (B.M) = 1.79.

[Cu(HL)(OAc)].2H2O (Complex 10): Yield (66%); color: green; M.P. = 208–210 °C; ΛM: 2.3 O−1cm2mol−1; Elemental analysis for, C19H24CuN6O4S, (F.W. = 517.5): Found (calcd) %M 12.53 (12.27), %C 42.25(41.73), %H 5.69(5.02), %N 17.01(16.23), %S 7.10(6.18); IR (KBr, cm−1), 3450a,3418bν(H2O)a/(OH)b, 3320,3220ν(N-H), 1638ν(C = O), 1620(sh), 1540, 1495ν(C = N)imine/(C = N)oximen, 845ν(C = S), 1085, 989ν(N-O), 540ν(M−N), 1648, 1373ν(OAc); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 277, 292, 330(br), charge transfer(nm) 401, 492(br), d → d bands(nm) 602, 650, 780(sh); µeff (B.M) = 2.1.

[Cu(HL)(NO3)] (Complex 11): Yield (67%); color: green; M.P. = 210 °C; ΛM: 2.42 O−1cm2mol−1; Elemental analysis for, C17H21CuN7O5S, (F.W. = 484.5): Found (calcd) %M 12.83 (13.11), %C 39.77(39.63), %H 3.37(3.92), %N 19.94(20.22), %S 7.00(6.60); IR (KBr, cm−1), 3427bν(H2O)a/(OH)b, 3370(sh), 3269ν(N-H), 1634ν(C = O), 1614, 1565, 1549ν(C = N)imine/(C = N)oximen, 811ν(C = S), 1071, 978ν(N-O), 531ν(M−N), 1416, 1383ν(NO3); UV–Vis. (Nujol mulls) π → π*/n → π*(nm) 278, 293, 340, 361, charge transfer(nm) 392, 500, d → d bands(nm) 582, 622, 700(sh); µeff (B.M) = 1.96.

2.1.3

2.1.3 Instrumentation and measurements

The determination of the elemental percentages of Carbon, Hydrogen, Nitrogen and Sulfur elements were done in the Micro-analytical Unit. The measurements of FT-IR spectra were performed using KBr discs on FT-IR (Shimadzu spectrophotometer model spirit Fourier Transform Infrared spectrophotometer). 1H NMR spectra were recorded at 300 MHz in DMSO‑d6 on Varian Gemini 200 NMR spectrophotometer (USA). Mass spectra of the solids were done employing direct probe controller intel part to single quadrupole mass analyzer (UK) using Thermo X-calibur software. Electronic spectra in solution and solid states were recorded in N,N-dimethylformamide (DMF) and Nujol nulls respectively, using Schimadzu UV–Vis 1800 spectrophotometer (UK).

A Tacussel type CD6NG conductivity bridge was used to measure molar conductance using 10-3 M DMF solutions (Chelmsford, UK). The molar conductivities were calculated according to the equation mentioned (El-Saied et al., 2019). The EPR spectra of the copper(II) complexes were recorded with X-band EMX spectrometer (Bruker, Berlin, Germany) using a standard rectangular cavity of ER 4102 operating at 9.5 GHz with 100 kHz modulation at 298 °K. A g-marker for the calibration of the spectra was diphenyl picryl hydrazide (DPPH). TGA was performed through Shimadzu DT-30 thermal analyzer in temperature range (room temperature to 1000 °C) with 10 °C/min heating rate. Johnson-Matthey magnetic balance (USA) was used to measure the magnetic susceptibilities along with a calibrating agent Hg[Co(CNS)4]. The diamagnetic corrections were performed using Pascal's constants (Sir and Wilkins, 1960) and the magnetic moments were calculated from the following equation: μ e f f = 2.84 X M corr . T

2.2

2.2 Biological assays

2.2.1

2.2.1 Experimental design

Six weeks old female Swiss albino mice (average 25 g) were kept under standard ventilation, 12:12-h light–dark cycle and provided with food and water ad libitum. After one-week of adaptation, the mice were subcutaneously (SC) injected into their lower limb with 0.2 mL (2 × 106/mL) EAC cells to induce Ehrlich solid tumor (Guirgis et al., 2010, Zahra et al., 2019). Seven days after tumor induction, the mice bearing Ehrlich solid tumors were haphazardly separated in sterile cages. The experiments of the current study were accepted by the Ethical Committee of Science Faculty, Menoufia University (No.: ECLA‐SFMU‐25015). The present study included the following groups:

Group 1 (normal control): mice didn’t receive any treatment.

Group 2 (vehicle group): mice bearing EAC tumors and treated with vehicle (DMSO).

Group 3: mice bearing EAC tumor and treated with ligand.

Group 4: mice bearing EAC tumor and treated with complex 2.

Group 5: mice bearing EAC tumor and treated with complex 5.

Group 6: mice bearing EAC tumor and treated with complex 8.

The treatments with vehicle (DMSO), ligand and only completely soluble complexes 2, 5 and 8 started one week after EAC cells inoculation. Mice were injected with freshly prepared solution of ligand and complexes 2, 5 and 8 at a dose of 0.36 mg/kg/day for two consecutive weeks. The selection of the administrated dose was based on the outcome of our previous study (El-Saied et al., 2019). After the treatment period was completed, mice were sacrificed. Blood samples were collected, centrifugated and the obtained sera samples were kept at − 20 °C until analysis. The tissues samples from livers and EAC solid tumors were sectioned. The tissues were fixed in formalin and then embedded in paraffin wax blocks for future immunohistochemical detection of ki67 and p53.

2.2.2

2.2.2 Evaluation of tumor volume

The volume of Ehrlich solid tumors in mice treated with DMSO, ligand and complexes 2, 5 and 8 was measured using the following equation: tumor volume (mm3) = 0.52 × A2 × B, where A and B are the minor and major tumor axes, respectively (Guirgis et al., 2010).

2.2.3

2.2.3 Evaluation of hepatic function tests

ALT and AST activities and albumin concentration in sera samples of normal mice and mice bearing solid tumors treated with ligand and complexes 2, 5 and 8 were determined by colorimetric methods according to the manufacturer's instructions using autoanalyzer apparatus.

2.2.4

2.2.4 Ki67 and p53 evaluation by immunohistochemical method

The expressions of Ki67 and p53 in Ehrlich tumor tissues and liver tissues of normal mice and mice bearing solid tumors treated with ligand and complexes 2, 5 and 8 were detected as previously reported (El-Saied et al., 2019). In brief, the dewaxing of paraffin sections was done followed by hydration and then the sections were immersed in antigen retrieval solution. The proteins of the samples were blocked and then incubated at 4 °C overnight with anti-ki67 and anti-p53. The slides were washed and then incubated with secondary antibodies (anti-mouse IgG). The samples were pictured and the percentage of labelling index was measured (El-Aarag et al., 2017).

2.3

2.3 Statistical analysis

The data are presented as mean ± SD and the statistical comparison among groups were analyzed using one-way ANOVA and Tukey post hoc test. A value of p<0.05 was considered statistically significant.

3

3 Results and discussion

The physicochemical analyses of the prepared complexes are shown in Table 1 and are found to be well-matched with their chemical structures (Scheme 2). The elemental analyses and the molar conductivity data confirmed the fact that complexes 2, 3, 5, 6 and 8–11 have the general formula of [M(HL)X], where M = Co(II), Ni(II), Cu(II) and X = Cl, (complexes 2, 5 and 8, respectively), M = Co(II), Ni(II), Cu(II) and X = OAc (complexes 3, 6 and 10, respectively), M = Cu(II), X = Br and NO3 (complexes 9 and 11, respectively). The data showed that complex 1 is a binuclear with a formula of [Fe2(HL)2Cl4] and complexes 4 and 7 are binuclear complexes of general formula of [M2(HL)2].(NO3)2, where M = Co(II), Ni(II), respectively.

Metal complexes 1–11 structures.
Scheme 2
Metal complexes 1–11 structures.
Metal complexes 1–11 structures.
Scheme 2
Metal complexes 1–11 structures.

3.1

3.1 1H NMR investigation

The assignments of 1H NMR are based on the former study that included thiosemicabazones derived from 4-aminoantpyrine (Refata and El- Metwaly, 2012) and the 1H NMR spectra of ligand and complex 5 are shown in the Supplementary Materials. The spectrum of the ligand displays three separate singlet signals at δ = 11.591 (1H), δ = 10.624 (1H) and δ = 9.047 (1H) ppm, attributed to the OH of oxime moiety, N-N(2)H of thiosemicarbazide, and CS-N(4)-H of thiosemicabazide, respectively (Ali et al., 2014). Multiple signals appear at δ = 7.507–7.279 ppm ascribed to aromatic protons. The other resonances for the ligand are found at δ = 3.068 (s, 3H), δ = 2.135 (s, 3H) and δ = 2.034 (s, 6H) ppm corresponding to N-CH3, C-CH3 of the antipyrine moiety and N = C-CH3 of the oxime moiety. The 1H NMR of complex 5 does not display the expected signal due to the presence of thiosemicarbazide N-N(2)H which is consistent with the loss of the N(2)-H proton and the existence of the ligand in its thiol form. The spectrum shows that the resonances attributed to the OH proton is at the same resonance as the free ligand, indicating that the ligand does not lose the OH proton upon coordination. In comparison to the free ligand, the N(4)-H protons of thiosemicabazide are shifted downfield as a result of the coordination of the thiol sulfur thus resulting in reduced electron density at N(4) (West et al., 1992).

3.2

3.2 Mass spectra measurement

The mass spectrum of the ligand gives a molecular ion peak at 378.34 amu, confirming that the molecular weight of the ligand is 378 g/mol of formula H2L·H2O (C16H22N6O3S). Additionally, the pattern of fragmentation reveals ion peaks at 304.33 (100%), 260.94 (28.96%), 201.89 (30.85%) and 125.29 (36.38%) amu, corresponding to, C14H16N5OS, C12H13N4OS, C11H12N3O and C5H7N3O, respectively. Complex 4 [Co2(HL)2].(NO3)2·H2O (C32H40N14O11S2Co2), F.W. = 977.8) is characterized by a molecular ion peak at 977.5 amu confirming a molecular weight of (977.8 g/mole). Complex 7 [Ni2(HL)2].(NO3)2·H2O (C32H38N14O10S2Ni2·H2O), F.W. = 986.4); exhibited a molecular ion peak at 987.9 amu, confirming the proposed complex structure. Supplementary Materials are including the mass spectra of ligand, complexes 4 and 7.

3.3

3.3 The molar conductance analysis

The values of the molar conductivity of the prepared complexes measured in DMF (10-3 M) solutions were listed in Table 1. The data illustrated that all complexes are non-electrolytes except for complexes 4 and 7 (West et al., 1999). This can be taken as an evidence that, there is a direct binding between the anions and metal ion. Complexes 4 and 7 gave molar conductivity values of 125 and 128 O-1cm2mol−1, respectively attributable to 1:2 electrolytes, and correspondence to the presence of ionic nitrate groups in the outer shell (West et al., 1998, El-Saied et al., 2009).

3.4

3.4 Infrared (IR) spectra measurement

The IR spectra are shown in Supplementary Materials. The data in Table 2 revealed the presence of bands at 3421, 3303, and 3123 cm−1 at the ligand infrared spectrum. The first band is assigned to ν(O-H) and the latter two bands are attributed to the absorptions of ν(N-H) that are distinguishing to –NH groups (El-Saied et al., 2019). Also, the band characteristic to ν(C = O) of antipyrine was displayed at 1635 cm−1. The spectrum showed three bands at 1613, 1585, and 1524 cm−1, assigned to ν(-S-C = N-), ν(C = N) (imine), and ν(-O-N = C-) (oxime), respectively. The presence of three bands distinctive to C = N groups, indicates that the ligand displays thione − thiol − tautomerism, additionally supported by the specific band at 2670 cm−1, given to ν(-SH) (Husain et al., 2008). The ν(N-O) bands were observed at 1032 and 960 cm−1. The band of ν(C = S) (thioamide IV) was observed at 882 cm−1 (El-Saied et al., 2019). The hydration water was assigned by a broad band at 3447 cm−1.

Table 2 IR spectral bands (cm−1) and assignments of ligand and complexes 1–11.
No. Ligand/ Complexes ν(H2O)a/(OH)b ν(N-H) ν(C = O) ν(C = N)imine/ (C = N)oxime ν(C = S) ν(N-O) ν(M−N) ν(OAc/ClO4/NO3
H2L·H2O 3447a, 3421b 3303, 3123 1635 1613, 1585, 1524 882 1032, 960 – –
1 [Fe2(HL)2Cl4].H2O 3445a, 3358b 3191 1634 1566, 1531, 1490 847 1008, 915 501 ν(Fe-O) 589
2 [Co(HL)Cl] 3410b 3280 1637 1613, 1525 1495 815 1074, 970 499 –
3 [Co(HL)(OAc)].H2O 3445a(sh), 3420b(OH), 3325, 3260 1632 1591 , 1545, 1495 846 1085, 980 503 1652 , 1380
4 [Co2(HL)2].(NO3)2·H2O 3440a, 3388b(br) 3222 1638 1591, 1533, 1495 815 1024, 942 500 1383 (S), ν(Co-O) 590
5 [Ni(HL)Cl].2H2O 3446a, 3420b 3236 1634 1589, 1544 , 1522 813 1073, 1025 501 –
6 [Ni(HL)(OAc)].H2O 3446a, 3413b 3262 1641 1591, 1522, 1495 815 1088, 1009 501 1650, 1382
7 [Ni2(HL)2].(NO3)2·H2O 3418a, 3380b 3192 1635 1591, 1556, 1493 833 1010, 944 504, 471 1383(s), ν(Ni-O) 588
8 [Cu(HL(Cl] 3430b 3352, 3173 1640 1614, 1541, 1541 813 1060, 968 525 –
9 [Cu(HL)Br] 3427b 3351, 3245, 3170 1640 1619, 1550, 1529 813 1063, 967 535 –
10 [Cu(HL)(OAc)].2H2O 3450a, 3418b 3320, 3220 1638 1620(sh), 1540, 1495 845 1085, 989 540 1648, 1373
11 [Cu(HL)(NO3)] 3427b 3370(sh), 3269 1634 1614, 1565, 1549 811 1071, 978 531 1416, 1383

Sh: shoulder, s: strong and br: broad, a : water moiety, b : oxime moeity, (-):no value available.

The infrared spectral data of the complexes were compared to that of the free ligand in order to deduce the binding mode of the ligand in complexes. The data in Table 2 showed that the infrared spectra of 2, 3, 5, 6, 8 and 9–11 complexes, display bands at 3450–3410 cm−1 ascribed to the υ(O-H) of oxime moiety. This indicates that the ligand does not lose the oximino proton during complex formation. The spectra of the complexes illustrated the υ(C = O) of antipyrine at the wavenumber 1641–1634 cm−1, which is the same or very close to that of the free ligand illustrating the non-participation of the carbonyl group in binding to the metal ion. The spectra reveal three bands at 1620–1566, 1565–1522 and 1549–1495 cm−1, given to υ(-N = C-S), υ(-C = N) and υ(-C = N-N), consecutively of oxime moiety. The appearance of these three bands is a good evidence that the presence of ligand in the thiol form and coordination has taken place through the oximino nitrogen and the azomethine nitrogen atoms in addition to the thiol sulfur atom. The υ(C-S) of metal complexes was observed at 846–811 cm−1, thus lowering wavenumber relative to that of the free ligand, and as a result of contribution to the bond formation. This is consistent with the described data of 1H NMR spectrum of complex 5. The above arguments indicate that the ligand in these complexes behaves as a monobasic tridentate ligand coordinated through the azomethine nitrogen, oximino nitrogen and the thiol sulfur atoms. Compared to the free ligand, the υ(N-O) bands in the spectra of these metal complexes was observed with higher wavenumbers, indicating that the binding has taken place via the nitrogen atom rather than oxygen atom. The spectra of complexes also reveal that υ(N-H) bands representative to –NH groups are perturbed by complex formation, due to the loss of the proton of the thiol group during complexation. The IR spectra of binuclear metal complexes [Fe2(HL)2Cl4]·H2O (complex 1), [Co2(HL)2](NO3)2·H2O (complex 4) and [Ni2(HL)2](NO3)2·H2O (complex 7) display the bands consistent to carbonyl of antipyrine moiety with identical wavenumbers relative to that of the ligand, due to its absence in the participation in coordination. The spectra of the three complexes show three bands attributable to υ(-N = C-S), υ(C = N-N) (azomethine) and υ(-C = N) of oxime moiety at 1591–1566, 1556–1531 and 1495–1490 cm−1 respectively. Moreover, the υ(C-S) band appeared at 847–815 cm−1. The existence of three –C = N bands, their positions and the C-S band at lower wavenumbers relative to the free ligand is a proof that the ligand reacts mainly in the thiol form and coordination has taken place by the thiol sulfur, azomethine nitrogen and the oxime nitrogen atoms. The υ(O-H) and υ(N-O) bands in the spectra of the three complexes were observed with lower wavenumbers compared to that of the free ligand. This is an indicator for binding of the hydroxo group without loss of the proton and binding of the oxime group via the oxygen atom to a metal ion in addition to the nitrogen atom to the other metal ion such as a bridge. This proved that these complexes are bi-nuclear rather than the mono-nuclear as shown in Scheme 2. This is also evidenced by the mass spectra of complexes and the values of abnormal magnetic moment.

The new IR spectral bands observed in the spectra of complexes 3, 6 and 10 at 1652–1648 and 1383–1373 cm-1, may be due to νas(COO–) and νs(COO–) of the acetate group, respectively (Seleem et al., 2007, Shebl, 2016). The difference between both bands was about 269–275 cm-1, indicating that the acetate group involved in the complexes coordinates as a monodentate ligand to the metal ion (Mikuriya et al., 1980, El-Saied et al., 2019). The infrared spectra of binuclear complexes 4 and 7 showed one strong band at 1383 cm−1 given to ionic nitrate indicating that the nitrate group is in the outer shell as evidenced by the value of molar conductivity (Shebl et al., 2017). The infrared spectrum of complex 11 showed two bands concerning to the nitrato ligand at 1416 and 1383 cm−1 assigned to the monodentate nitrato ligand (Jain et al., 1986) A wide-ranging band at 3450–3418 cm−1 was assigned to ν(OH) of water molecule in the hydrated complexes (Maurya and Rajput, 2007). A new band at 540–499 cm−1 was given to ν(M−N) in all complexes (Tossidis and Bolos, 1986, Ainscough et al., 1998, Sallam et al., 2002) as well as the complexes 1, 4 and 7 show a new band at 589, 590, and 588 cm−1, respectively pointed out to υ(M−O) (Ainscough et al., 1998).

3.5

3.5 Electronic spectra and magnetic moments analysis

Electronic spectral bands of the ligand and the complexes, in both solution and solid state, together with the values of magnetic moment are revealed in Table 3. Ligand spectrum in solution displays bands at 280 and 309 nm, owing to π → π* electronic transitions (Leovac et al., 2011, El-Saied et al., 2019) within the ligand. The recorded bands values are still stable in the metal complexes spectra (Nair and Radhakrishnan, 1996). Two bands appeared at 315 and 340 nm, pointed out to n → π* electronic transitions that is linked to thiosemicabazone's azomethine function, oxime's azomethine function, thiosemicabazone’s thione function, and the antipyrine moiety (West et al., 1996, El-Sawaf et al., 1998). Both bands usually shift to an advance energy due to the contribution of the thiosemicarbazone's azomethine, oxime's azomethine and thiol groups in coordination with the metal ion (Lever, 1968, Fouda et al., 2008). The Cl → metal(II, III) (Lever, 1968, Mikuriya et al., 1980, Downes et al., 1981) and O → metal(II, III) (Mikuriya et al., 1980) charge transfer bands appeared near 330 nm region while they are masked by the strong action of intra-ligand bands. New bands were detected in 390–500 nm range of the metal complexes spectra, owing to S(π) → metal(II, III) charge transfer bands (Suzuki et al., 1980, Kovala-Dermertzi et al., 1983, Suzuki et al., 1984) as well as Br → metal(II) charge transfer bands (Okawa et al., 1982) in the bromo complex 9.

Table 3 The electronic absorption spectral bands (nm) in DMF solutions and solid states along with magnetic moments of ligand and complexes 1–11.
No Compound π → π*/ n → π*(nm) Charge transfer (nm) d → d bands(nm) µeff (B.M)
H2L·H2O 280, 309, 315, 340 – – –
1 [Fe2(HL)2Cl4].H2O 270(20), 290, (25) 348(27), 368 (26) 390 (25), 450 (19.6)
[400, 450]		 500(br) (20), 580(Sh) (83), 904(s, br) (9)
[510 (br), 573(Sh), 869(br)]		 4.1
2 [Co(HL)Cl] 277(4.3), 292(4.7), 325(4.6) (br) 39 7 (4.8), 460 (4.6), 497 (4.7)
[395, 450, 495]		 530 (22), 610 (25), 680 (20)
[530(br), 600(Sh), 680(Sh)		 3.87
3 [Co(HL)(OAc)].H2O 280 (5.6), 292 (4.7), 350(br) (5.8) 396 (6.3), 450(br) (5.6)
[390, 445(br)]		 623(s, br) (24), 690(Sh) (26)
[620, 650]		 3.91
4 [Co2(HL)2].(NO3)2·H2O 260 (21), 300 (25),
338 (26), 353 (25)		 400 (24), 460 (20)
[395, 465]		 510 (20.3), 545 (20), 560 (18)
[515, 546, 570(Sh)]		 2.3
5 [Ni(HL)Cl].2H2O 278 (5.1), 293 (5.6), 313 (5.7), 335 (5.5) 397 (5.5), 430 (5.3), 493 (5.5)
[396, 435(br), 509]		 540(Sh) (4.7), 630 (Sh) (1.3)
[549, 630(Sh)]		 Zero
6 [Ni(HL)(OAc)]. H2O 276(Sh) (4.8), 291(5.2), 321(4.9), 350 (4.9) 400 (5.1), 500 (6.7), 545 (7)
[397, 506, 530(Sh)]		 585 (7), 635 (6.2)
[550(Sh), 600(Sh)]		 zero
7 [Ni2(HL)2].(NO3)2·H2O 280(Sh) (23), 296 (25), 310 (25) 385 (22), 400 (25)
[390, 425]		 444 (23), 800 (br) (2.1)
[466, 789(br)]		 1.49
8 [Cu(HL)Cl] 280 (4.75), 294 (5.4), 330 (br) (5.3), 354 (5.4) 385 (5.1), 400 (5.4), 446 (4.7), 467 (4.9)
[390, 465, 513]		 610(br) (2.9)
[615(Sh)]		 1.88
9 [Cu(HL)Br] 277 (5.5), 292(s) (6.04), 330(br) (6) 397(6), 443 (5.5), 463(br) (5.6) 615(br) (3.1) 1.79
10 [Cu(HL)(OAc)].2H2O 277 (4.6), 292 (5), 330(br) (4.9) 401(4.7), 492(br) (4.4) 602 (5), 650 (4.9),
780(Sh) (1.9)		 2.10
11 [Cu(HL)(NO3)] 278 (9.8), 293 (11.6), 340 (11.8), 361(12.2) 392 (11.9), 500 (9.4) 582 (9.6), 622 (10.1),
700 (Sh) (9.4)		 1. 96

Sh: shoulder, s: strong, br: broad, µeff: magnetic moment per one metal ion, B.M: boher magneton, (-): no value available. Values between square brackets represent the bands in solid state. Values between small parentheses are the molar extincition coefficient (∊ x10-2 mol−1 cm−1).

Iron(III) complex 1 exhibited magnetic moment value equal to 4.1B.M per iron ion. A lower magnetic value than the high spin d5 configuration Fe(III) complexes is explained by the presence of spin–spin connections between iron(III) ions in the binuclear complex (Shebl, 2009, Shebl, 2014). Complex 1 showed electronic spectrum characterized by the strong bands at 390, 450 and 500 nm that are associated to charge transfer from ligand to metal (LMCT). The spectrum displayed bands at 580 and 904 nm owing to the 6A1g → 4T2g and 6A1g → 4A1g (G) transitions in octahedral stereochemistry (Kumar et al., 2005).

Cobalt(II) complexes 2, 3 and 4 gave magnetic moment values 3.78, 3.91 and 2.30B.M, respectively, similar to those of high spin complexes and consistent with tetrahedral cobalt(II) species (West et al., 1999). The B.M. value of complex 4 might be clarified by the antiferromagnetic exchange between two cobalt(II) ions (El Saied et al., 2014) in the binuclear complex 4 which is in agreement with the complex mass spectrum. Cobalt(II) complexes 2, 3 and 4 possess d → d bands (Table 3) typical to a tetrahedral system with a strong broad band ranging from 690 − 510 nm that assigned to the 4A2 → 4 T1(P) (υ3) transition in a tetrahedral arrangement around the cobalt(II) ion [45].

Nickel(II) complexes 5 and 6 were diamagnetic. Nickel(II) complex 7 recorded magnetic moment values of 1.49B.M which is just under the spin value of two unpaired electrons (2.82B.M.) (Earnshaw, 1968) suggesting a different stereochemistry than the square planar. Ni(II) complexes 5 and 6 electronic spectra gave two bands at 558–540 and 635–630 nm ascribed to 3 T1 → 3 T2 and 3 T1(F) → 3 T2(P) transitions, respectively in a square planar (El-Saied et al., 2003). Complex 7 revealed electronic spectrum that involved two bands at 444 and 800 nm, owing to the transition of 3 T1(F) → 3A2(υ2) and 3 T1(F) → 3 T1(p)(υ3), respectively representative for a tetrahedral system (Sacconi, 1968).

Copper(II) complexes 8–11 have magnetic moment values of 1.88, 1.79, 2.10 and 1.96B.M., respectively. These values are near to the spin-only value for one unpaired spin (∼1.73B.M.). This is considered as an evidence that the molecular association of copper(II) ions in a square planar did not occur (Lever, 1968, Chohan et al., 2005, El-Tabl et al., 2009, . The electronic absorption spectra of the Cu(II) complexes 10 and 11 showed three bands at 582–602, 622–650 and 700–780 nm, owing to the transitions of 2B1g(dx2-y2) → 2A1g(dz2), 2B1g(dx2-y2) → 2B2g(dxy)(υ2) and 2B1g(dx2-y2) → 2Eg(dxz,dyz)(υ3), respectively deducing a square planar geometry around the copper(II) ion (Gupta et al., 2007, Ahmed and Lal, 2017).

3.6

3.6 EPR investigation

Copper(II) complexes generally exhibit two different types of EPR spectra, isotropic and anisotropic (Hathaway and Billing, 1970). The latter may be of axial symmetry (when the main component of g-tensor is as following: g1 = g2 = g⊥≠g3 = gιι) or orthorhombic (when g1 ≠ g2 ≠ g3). Most copper(II) complexes have anisotropic spectra with a ground state of dx2-y2 where gιι˃ g ⊥=2.0.

Cu(II) complexes 8, 9 and 11 and their EPR data are presented in (Table 4). Cu(II) complexes EPR spectra possess small g values proposing a considerable covalency in the binding (El-Saied et al., 2003). The ESR spectral data of complexes 8, 9 and 11 demonstrated anisotropic signals with gιι > g⊥>2.0023. This suggests an axial symmetry type of a d(x2-y2) ground state and resulting in a 2B1g ground state that is identified for Cu2+ complexes (Hathaway and Billing, 1970, Zaky et al., 2011). The square-planar geometry was supported with the obtained g-values of Cu(II) complexes (Brown and West, 1981, Nagashri et al., 2011). The geometric G parameter is measured using the following equation G= (gιι −2.0023)/(g⊥2.0023) for axial. When G˃4, the exchange interaction is insignificant, while, if G < 4 the significant effect of exchange interactions is indicated (Proctor et al., 1968). The value of G˃4 for complexes 8, 9 and 11 demonstrated that the exchange interaction between Cu(II) ions is insignificant. According to Kivelson and Neiman, 2004, the g||-values for Cu2+-complexes illustrated the bonding nature of copper-ligand. If the gιι < 2.3 the nature is essentially covalent, while if gιι˃2.3 the environment is mainly ionic. The g ιι < 2.3 of the complexes indicated the presence of significant covalent character for copper-ligand bonding.

Table 4 Assigned EPR spectra and bonding parameters for Cu(II) complexes 8, 9 and 11.
Complex no. gιι g⊥ gav G k‖2/ k‖ k⊥2/ k⊥ K2/k
8 2.121 2.02 2.054 6.7 – – –
9 2.48 2.078 2.212 6.3 – – –
11 2.12 2.026 2.057 4.97 0.29/0.54 0.25/0.5 0.26/0.51

(-): no value available.

Based on Hathaway, 1973, the values of the calculated k||2, k⊥2 and k2 for complex 11 are showed in Table 4. The results revealed k||˃ k⊥ suggesting a strong out-of-plane π-bonding and supporting the notion that 2B1g is the ground state for the complex. Also, in the case of ionic environment, k value equal 1. However, if K < 1 the environment is covalent. The covalent nature was established when k value was less than one which is well-matched with the obtained results from the gιι values (Ray and Kauffmann, 1990).

3.7

3.7 Thermal analysis

In the present work, thermal analysis was performed to obtain valuable information about the thermal stability of the synthesized complexes and to investigate the nature of solvent molecules to be outside or inside the inner coordination sphere of the metal (Shebl et al., 2016, El-Shafiy and Shebl, 2018, El-Shafiy and shebl, 2019). Table 5 in Supplementary Materials showed the TGA results of the solid complexes 1, 4–6, 8, 10 and 11 as well as TGA spectra. The complexes were thermally decomposed according to a general pattern that involved two or three steps. First step: molecules of hydrated water were lost at 26–170 °C, second step: the coordinated or ionic anions were lost at 80–170 °C and third step included the loss of anions, decomposition of complex to loss the ligands leading to formation of metal or metal sulfide. The suggested formulas were well-matched with the obtained results.

Table 5 TGA of complexes 1, 4, 5, 6, 8, 10 and 11.
No Complex Temp. range (˚C) Weight loss
Found (calc)%		 Assignment Residual's formula
1

 [Fe2(HL)2Cl4].2H2O 27–170

170–226
227–900		 3.8(3.6)

7.8(7.0)
72.8(71.9)		 Two moles of hydrated water were lost.
Two coordinated Cl- ions were lost.
Complete decomposition and gave ferrous sulfide.		 [Fe2(HL)2Cl4]
[Fe2(HL)2Cl2]2+
2 FeS
4 [Co2(HL)2].(NO3)2·H2O 31–65

65–175 175–550		 2.3(2.1)

7.06(6.6)
81.8(79.5)		 One mole of hydrated water was lost.
One nitrate ion was lost.
Complete delegation and cobaltous sulfide were formed.		 [Co2(HL)2].(NO3)2
[Co2(HL)2]+.NO3–
2CoS
5

 [Ni(HL)Cl].2H2O 31–90

90–210
225–780
780		 6.8(7.4)
zero
75.3(74.1)
zero		 Hydrated water was lost.
Stable

Delegation and metallic residue was formed.
 [Ni(HL)Cl]
[Ni(HL)Cl]
Ni
Ni
6

 [Ni(HL)(OAc)].H2O 31–100
100–252

252–890

890		 4.1(3.63)
Zero 77.7(78.0)

Zero		 Hydrated water was lost.
Stable
Decomposition of complex and formation of nickel (II) sulfide.
Nickel sulfide		 [Ni(HL)(OAc)]
[Ni(HL)(OAc)]
NiS

NiS
8

 [Cu(HL)Cl] 27–189

189–899		 zero
86.3(86.1)

 Stable up to 189˚C.
Decomposition of complex in overlapped steps and formation of copper metal.		 [Cu(HL)Cl] Cu
10

 [Cu(HL)(OAc)].2H2O 25–135
135–899

 7.3(7.0)
79.9(80.8)
 Hydrated water was lost.
Continuous loss of weight in overlapped steps leaving copper residue.		 [Cu(HL)(OAc)]
Cu
11
 [Cu(HL)(NO3)] 27–165
165–900

 zero
76.2(78.1)		 Stable up to 165 ˚C.

Decomposition of complex in continuous overlapped steps.		 [Cu(HL)(NO3)]
CuS

3.8

3.8 Biological results

3.8.1

3.8.1 Tumor volume

The effect of the ligand and metal complexes 2, 5 and 8 on the volume of the solid tumors induced in mice were illustrated in Table 6. Results revealed that compared to DMSO group, the treatment with the ligand and complexes at a dose of 0.36 mg/kg/day for two consecutive weeks resulted in the reduction of the solid tumors volume. The reduction percentages were evaluated to 9.7, 25.8, 98.1 and 71 for ligand, complexes 2, 5 and 8, respectively. Our result was compatible with the inhibition activity of complexes derived of acetoacetanilide N(4)-methyl(phenyl)thiosemicarbazone ligand towards the volume of Ehrlich solid tumor induced in mice (Priya et al., 2015).

Table 6 EAC tumors volumes of mice treated with DMSO, ligand and complexes 2, 5 and 8.
Mice Groups Tumor volume (mm3) Inhibition %
EAC + DMSO 310 ± 53.5 –
EAC + ligand 280 ± 10.8 9.7
EAC + complex 2 230 ± 40.6* 25.8
EAC + complex 5 5 ± 0.6** 98.4
EAC + complex 8 89.8 ± 4.7** 71

EAC: Ehrlich ascites carcinoma; DMSO: dimethyl sulfoxide; Values are showed as mean ± SD. Significant difference from EAC + DMSO group at * p < 0.01 and ** p < 0.001.

3.8.2

3.8.2 Hepatic functions

The influence of ligand and complexes 2, 5 and 8 on the activities of ALT and AST and albumin concentration was measured. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes are commonly used to differentiate hepatic and non-hepatic status. Results revealed that ALT and AST activities were significantly (p < 0.001) elevated while the albumin concentration declined significantly (p < 0.01) in mice bearing Ehrlich solid tumours compared to normal mice as demonstrated in Table 7. Mice treated with ligand and complexes 2, 5, 8 revealed significant reduction of ALT and AST activities and concomitant elevation in albumin concentration, relative to DMSO group, and especially, after the treatment with complexes 5 and 8. These findings were well-matched with the metal complexes derived from isatin-N(4)antipyrinethiosemicarbazone that restored the elevated hepatic enzymes functions in Ehrlich solid tumor bearing mice (El-Saied et al., 2019). Taken together, the results indicated the ameliorative effects of the new synthesized complexes towards hepatic functions.

Table 7 Hepatic functions of normal mice and mice bearing EAC tumor treated with DMSO, ligand and complexes 2, 5 and 8.
Mice Groups ALT
(U/L)		 AST
(U/L)		 Albumin
(g/dL)
Normal 43.7 ± 6 147.8 ± 16 3.4 ± 0.22
EAC tumor + DMSO 363.3 ± 125## 395.3 ± 83## 2.5 ± 0.09#
EAC tumor + ligand 139.2 ± 25* 206.6 ± 48 2.4 ± 0.11
EAC tumor + complex 2 92.6 ± 30** 187 ± 22* 2.5 ± 0.18
EAC tumor + complex 5 154.6 ± 12* 194 ± 35** 3.3 ± 0.07**
EAC tumor + complex 8 95.2 ± 15** 97 ± 26** 3 ± 0.25*

ALT: alanine aminotransferase; AST: aspartate aminotransferase; DMSO: dimethyl sulfoxide. Results are showed as mean ± SD. Significant difference from the normal group at # p < 0.01 and ## p < 0.001. Significant difference from EAC tumor + DMSO group at * p < 0.05 and ** p < 0.01.

3.8.3

3.8.3 Ki67 expression in EAC tumors and liver tissues

EAC cells are characterized by their speedy proliferation, therefore, testing the anti-proliferative capability of newly synthesized compounds was performed using this feature. Furthermore, the effects of ligand, complexes 2, 5 and 8 on the expression of proliferation marker Ki67 were investigated in both the tissues of solid tumors and liver tissues. Our results showed that compared to normal mice group, Ki67 is significantly increased in liver tissues of mice bearing EAC tumors (Fig. 1B). In comparison to DMSO group, the treatment with ligand, complexes 2, 5 and 8 reduced Ki67 expression in tissues of solid tumors as well, the effect was evidential with complex 5, (Fig. 1A). Complexes derived of hydroxyquinoline-thiosemicarbazone ligand revealed significant effects towards lung and breast cancer cell proliferation (Rogolino et al., 2017). Consequently, the antitumor activities of the complexes against EAC related to their ability to suppress the proliferation marker Ki67 expression.

Ki67 expression in the tissues of solid tumors (A) and in liver tissues (B) of mice bearing EAC solid tumors. Ki67 expression was measured through immunohistochemistry assay and presented as labeling index percentage. Results are showed as mean ± SD. Significant difference from the normal group at ## p < 0.01. Significant difference from EAC + DMSO group at * p < 0.05, ** p < 0.01 and *** p < 0.001.
Fig. 1
Ki67 expression in the tissues of solid tumors (A) and in liver tissues (B) of mice bearing EAC solid tumors. Ki67 expression was measured through immunohistochemistry assay and presented as labeling index percentage. Results are showed as mean ± SD. Significant difference from the normal group at ## p < 0.01. Significant difference from EAC + DMSO group at * p < 0.05, ** p < 0.01 and *** p < 0.001.

3.8.4

3.8.4 p53 expression in EAC tumors and liver tissues

P53, tumor suppressor protein, regulates the transcription of numerous genes involved in the apoptosis process. In addition, P53 has crucial roles in cancer prevention (EL Ablack et al., 2020). Accordingly, the effects of ligand, complexes 2, 5 and 8 on the expression level of p53 were determined in both EAC-tumor tissues and liver tissues of mice bearing EAC tumors as shown in Fig. 2. In the liver tissues, there is an increase in the expression levels of p53 due to the action of solid tumor induced in mice, compared to liver tissues of normal control mice group. However, the increased p53 expressions were declined after the treatment with ligand, complexes 2, 5 and 8, in comparison to liver tissues of tumor control group (Fig. 2 B).

p53 expression in the tissues of solid tumors (A) and in liver tissues (B) of mice bearing solid EAC tumors. p53 expression level was measured through immunohistochemistry assay and presented as labeling index percentage. Results are showed as mean ± SD. Significant difference from the normal group at ### p < 0.001. Significant difference from EAC + DMSO group at ** p < 0.01 and *** p < 0.001.
Fig. 2
p53 expression in the tissues of solid tumors (A) and in liver tissues (B) of mice bearing solid EAC tumors. p53 expression level was measured through immunohistochemistry assay and presented as labeling index percentage. Results are showed as mean ± SD. Significant difference from the normal group at ### p < 0.001. Significant difference from EAC + DMSO group at ** p < 0.01 and *** p < 0.001.

In the solid tumor tissues, the p53 expression was increased after mice treated with ligand, complexes 2, 5 and 8, in comparison to mice bearing solid tumor (Fig. 2A). The sequence of the complex’s potency towards the induction of p53 was 5 > 2 > 8 > ligand. The ability of chemical compounds to induce the apoptotic related proteins in cancer cells represented an innovative approach for cancer treatment (Mikuriya et al., 1980, Zahran et al., 2018). Trans-thiosemicarbazone schiff base palladium (II) complex induces the apoptosis in tumor xenograft model through the regulation of apoptosis related proteins (Zhang et al., 2017). Also, thiosemicarbazone with Ga(III) complex initiates a p53-dependent and -independent programmed cell death (Mendes et al., 2009). Therefore, the anti-tumor actions of the examined complexes were linked with their potent abilities to induce p53 in EAC-solid tumor tissues.

4

4 Conclusion

New metal DAMATSC complexes were synthesized and their molecular structures were characterized. In vivo antitumor activities were evaluated using EAC tumors model induced in mice. The tested complexes significantly reduced the volume of the solid tumors. In addition, they augmented the expression level of p53 while, declined the expression level of ki67 in both the hepatic and tumor tissues. Inhibition of the tumor proliferation and induction of the apoptosis were claimed to be the two mechanisms of action beyond the effect of these complexes administration paving the way to these complexes and ligands to be introduced to the anti-cancer drug industry.

Acknowledgements

We are very grateful to the Swedish Research links grant VR (Stockholm, Sweden), Grant number 2016-05908 for financial support for publication. Dr. S.A.M. Khalifa thanks the Department of Molecular Biosciences, Wenner-Grens Institute, Stockholm University, Sweden.

Funding

This research did not receive any specific grant from any funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Design the experiments, B.E.‐A. and F.E.-S.; Experimental work, B.E‐A. and N.K.; Supervision, F.E.-S., B.E.‐A. and T.S.; Writing the manuscript draft, B.E.‐A., F.E.-S. and N.K.; Review & editing, B.E.-A.; F.E.-S, S.A.M.K, and H.R.E.-S. All authors have read and approved the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.102993.

Appendix A

Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1


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