Synthesis, Characterization, and In Vivo Anti-Cancer Activity of New Metal Complexes Derived from Isatin-N(4)antipyrinethiosemicarbazone Ligand Against Ehrlich Ascites Carcinoma Cells

Abstract

The current study aimed to synthesize new metal coordination complexes with potential biomedical applications. Metal complexes were prepared via the reaction of isatin-N(4)anti- pyrinethiosemicarbazone ligand 1 with Cu(II), Ni(II), Co(II), Zn(II), and Fe(III) ions. The obtained metal complexes 2–12 were characterized using elemental, spectral (¹H-NMR, EPR, Mass, IR, UV-Vis) and thermal (TGA) techniques, as well as magnetic moment and molar conductance measurements. In addition, their geometries were studied using EPR and UV–Vis spectroscopy. To evaluate the in vivo anti-cancer activities of these complexes, the ligand 1 and its metal complexes 2, 7 and 9 were tested against solid tumors. The solid tumors were induced by subcutaneous (SC) injection of Ehrlich ascites carcinoma (EAC) cells in mice. The impact of the selected complexes on the reduction of tumor volume was determined. Also, the expression levels of vascular endothelial growth factor (VEGF) and cysteine aspartyl-specific protease-7 (caspase-7) in tumor and liver tissues of mice bearing EAC tumor were determined. Moreover, their effects on alanine transaminase (ALT), aspartate transaminase (AST), albumin, and glucose levels were measured. The results revealed that the tested compounds, especially complex 9, reduced tumor volume, inhibited the expression of VEGF, and induced the expression of caspase-7. Additionally, they restored the levels of ALT, AST, albumin, and glucose close to their normal levels. Taken together, our newly synthesized metal complexes are promising anti-cancer agents against solid tumors induced by EAC cells as supported by the inhibition of VEGF and induction of caspase-7.

1. Introduction

Cancer is considered one of the most devastating life-threatening disease worldwide. It is characterized by uncontrolled cell growth and the rapid spread of abnormal cells [1]. Angiogenesis is a vital regulator of tumor growth, spread and metastasis [2]. Tumor angiogenesis, which is the development of new blood vessels, is regulated by the production of angiogenic stimulators such as vascular endothelial growth factor (VEGF). VEGF is a key regulatory factor in the prognosis of various cancers; therefore, the inhibition of VEGF production is an alternative therapeutic approach for cancer treatment [3,4,5,6].

Chemotherapy, along with surgery, radiation, hormone therapy, and immune therapy, is used to treat cancer [7]. Despite the success of controlling the tumor growth in several cases, the incidence and prevelance of cancer is still dramtically increasing and calling for new chemotherapeutics to be introduced to the pharmaceutical industry and the market [8]. Antipyrine (2,3-dimethyl-1-phenyl-5-pyrazolone) and its derivatives possess a diverse array of potentially useful biological properties [9]. Similarly, thiosemicarbazones have demonstrated significant biological applications, as antitumor [10], antifungal [11], antiviral [12], antibacterial [13] and antimalarial [14] agents. Previous investigations involving the synthesis, characterization and biological evaluation of iron(III), cobalt(II), cobalt(III), nickel(II), zinc(II), and copper(II) complexes of 4-formylantipyrine N(4)-substituted thiosemicarbazones have been reported [15,16].

Based on these results and as a continuation of our ongoing interest in compounds with antitumor activities, the present study was carried out to synthesize and characterize metal complexes of isatin-N(4)antipyrinethiosemicarbazone and to evaluate their anti-tumor, anti-angiogenic and apoptotic activities against solid tumors induced by Ehrlich ascites carcinoma (EAC) cells in mice.

2. Results and Discussion

All the prepared metal complexes, shown in Figure 1, are stable at room temperature, non-hygroscopic and insoluble in most organic solvents and water, but they are completely soluble in dimethylformamide (DMF) and dimethylsulfoxide (DMSO). The elemental analyses, physical data, are listed in

Table 1. Analytical and physical data of the ligand 1 and its metal complexes 2–11.

No.	Molar Ratio/Compound	Color	m.p. (°C)	F.W.	Yield%	Analysis (%)/Found (Calcd)				Molar Conductance
						C	H	N	S
1	HL C20H18N6O2S	Yellow	210–212	406.64	94	59.14(59)	5.15(4.43)	20.91(20.66)	7.85(7.87)	-
2	HL+CuCl2(1:1),(2:1),(1:2) [CuLCl]·H2O	Deep brown	238–240	522.6	79	46.0(46)	4.1(3.64)	16.3(16.10)	6.88(6.13)	10.0
3	HL+CuBr2(1:1) [CuLBr]·3H2O	Brown	250	602.5	89	38.81(39.83)	3.54(3.82)	15.02(13.94)	5.03(5.31)	37.9
4	HL+Cu(NO3)2·3H2O (1:1)[Cu(NO3) L]·H2O	Brown	247	548.5	80	43.4(43.5)	3.29(3.5)	18(17.9)	6(5.8)	34.3
5	HL+Cu(OAc)2 (1:1) [Cu(OAc) L]·2H2O	Brown	212–216	563.5	74	46.12(46.90)	4.82(4.30)	15.13 (14.9)	5.1(5.7)	11.9
6	HL+Cu(ClO4)2(1:1) [Cu(ClO4)L]·2H2O	Brown	250–252	604.5	91	38.92(39.7)	3.75(3.48)	14.67(13.91)	5.18(5.29)	70.32
7	HL+CoCl2 (1:1) [CoClL]2·2H2O	Brown	258-260	1036	66	47(46.33)	4.17(3.7)	17(16.21)	6.4(6.18)	47.9
8	HL+Co(OAc)2 (1:1) [Co(OAc) L(H2O)]·4H2O	Brown	Over 300	614.5	75	42.96(41.9)	5.3(5.04)	14.2(13.7)	4.87(5.2)	4.6
9	Hl+NiCl2 (1:1) [NiCl2(HL)2]·4H2O	Brown	254–255	1014.9	84	46.84(47.3)	4.94(4.33)	16.95(16.55)	5.89(6.3)	13.5
10	HL+Ni(OAc)2 (1:1) [Ni(OAc) L]·2H2O	Brown	Over 300	558.9	70	47.76(47.24)	4.19(4.3)	15. 6(15.03)	6.71(5.78)	2.6
11	HL+Zn(OAc)2 (1:1) [ZnL2·H2O	Deep yellow	Over 300	912.2	69	51.9(52.6)	3.7(4.2)	18.8 (18.42)	6.64 (7)	1.4
12	HL+FeCl3 (1:1) [FeCl2(HL)2]Cl·H2O	Deep green	210–212	993.7	78	47.62(48.3)	3.9(3.8)	16.95(16.91)	5.6(6.44)	54.09

. The elemental analyses confirmed that complexes 2–6, 8 and 10 are formed in a 1:1 molar ratio of the metal ion and ligand, whereas complexes 9, 11 and 12 are formed in a 1:2 molar ratio of the metal ion and the ligand. Complex 7 is a binuclear complex.

2.1. ¹H-NMR Spectra

The ¹H-NMR spectra of the ligand 1 and the zinc complex 11 are illustrated in the Supplementary Materials. The data shows three separate singlets at 12.819, 11.219 and 10.115 ppm, which can be attributed to the thiosemicarbazide N-N(2)H, the indole N-H moiety and the thiosemicabazide CS-N(4)-H, respectively [17]. Multiple signals appear in the 7.702–6.876 ppm region, and these can be attributed to the aromatic protons of the indole and phenyl rings of antipyrine. The other signals for the ligand at 3.068 (s, 3H) and 2.135 (s, 3H) correspond to the N-CH₃ and C-CH₃ of the antipyrine moiety, respectively [15]. The ¹H-NMR spectrum of complex 11 does not show a signal corresponding to N-N(2)H of thiosemicarbazide, suggesting the loss of the thiol proton, and the binding ligand in its thiol form. The spectrum displays signals attributable to the indole N-H moiety, the CS-N(4)-H of thiosemicabazide and all the other characteristic signals at the same positions as those of the free ligand.

2.2. Mass Spectra

The mass spectra of ligand 1 and complexes 7, 8, 11, and 12 are shown in the Supplementary Materials. The molecular ion peak at m/z 406.27 amu in the spectrum of the ligand 1 is consistent with the molecular weight of its proposed structure. Moreover, the fragmentation pattern shows ion peaks at m/z 363.47, 291.15, 276.68, 261.69, 223.71, 204.07, 203.30, 145.64, 130.57, 118.18 and 76.3 amu corresponding to C₁₉H₁₇N₅OS, C₁₃H₁₇N₅OS, C₁₂H₁₃N₅OS, C₁₂H₁₃N₄OS, C₉H₁₀N₄OS, C₉H₆N₃OS, C₁₁H₁₃N₃O, C₈H₅N₂O, C₈H₅NO, C₇H₅NO and C₆H₅ fragments, respectively.

The mas spectrum of complex 7 (F.W. = C₄₀H₃₈N₁₂O₆S₂Co₂Cl₂) shows a molecular ion peak at m/z 999.5 amu, consistent with a molecular weight of 998.8 g/mol, which is the molecular weight of the complex after the loss of one molecule of hydration water (F.W. = C₄₀H₃₄N₁₂O₄S₂Co₂Cl₂). The spectrophotometer at hand cannot detect molecular ions with molecular weights greater than 1000 g/mol. The fragment ion peaks at m/z 868.9 and 463.9 amu correspond to the formulas C₄₀H₃₄N₁₂O₄S₂Co and C₂₀H₁₇N₆O₂Co.

The mass spectrum of complex 8 (C₂₂H₃₀N₆O₉SCo, F.W. = 612.5) displays a molecular ion peak at m/z 612.85 (100%) amu, confirming the suggested structure of the complex. Moreover, the spectrum shows several peaks, including peaks at m/z 576.23 (94.07), 561.30 (61.93), 541.85 (6.86), and 523.22 (39.20), corresponding to the formula C₂₂H₂₆N₆O₇SCo, C₂₂H₂₄N₆O₆SCo, C₂₂H₂₂N₆O₅SCo, and C₂₂H₂₀N₆O₄SCo, respectively, due to the sequential loss of two molecules of water of hydration, another water of hydration, a final water of hydration, and one coordinated water molecule, respectively.

The mass spectrum of complex 11 (C₄₀H₃₈N₁₂O₆S₂Zn, F.W. = 911.50) displays a molecular ion peak at m/z 911.95 (1.19), consistent with the suggested structure.The mass spectrum of complex 12 (C₄₀H₃₈N₁₂O₅S₂FeCl₃, F.W. = 992.50) exhibits a molecular ion peak at m/z 992.50 amu, confirming the putative molecular weight of the complex. Moreover, the spectrum shows several ion peaks, including peaks at 973.50 (1.90), 938.30 (12.96), 869.80 (8.27), and 464.40 (2.48) corresponding to the formula C₄₀H₃₆N₁₂O₄S₂FeCl₃, C₄₀H₃₈N₁₂O₅S₂FeCl₃, C₄₀H₃₈N₁₂O₅S₂FeCl₂, C₄₀H₃₈N₁₂O₅S₂Fe and C₂₀H₁₈N₆O₂SFe, which are attributable to the losses of one water of hydration, an ionisable chloride, two coordinated chloro ligands and one ligand, respectively.

2.3. The Molar Conductance

The molar conductance values of the metal complexes in DMF (10⁻³ M) were illustrated in Table 1. The results showed that none of the complexes except 12 are electrolytes [16], indicating that the anions in all these complexes are directly bound to the metal ion. Complex 12 gave a molar conductance of 74.5 Ω⁻¹cm²mol⁻¹ attributable to a 1:1 electrolyte, and indicating the presence of a chloride ion in the outer coordination sphere [18]. The high value of molar conductance of complex 6 may be associated with the partial displacement of the perchlorato ligand by DMF [15].

2.4. Infrared (IR) Spectra

The IR spectra of the ligand 1 and its metal complexes 2–12 were shown in Supplementary Materials. The most diagnostic infrared spectral bands are illustrated in

Table 2. IR spectral bands (cm⁻¹) for ligand 1 and its metal complexes 2–12 and their assignments.

No.	Ligand/Complexes	ν(H2O)	ν(N4H)	ν(C=O)	ν(C=N)	ν(C=S)	ν(M-O)	ν(M-N)	ν(OAc/ClO4/NO3
1	Ligand C20H18N6O2S	-	3442, 3348, 3287, 3251, 3141	1738 a, 1645 b	1688 a, 1623, 1595	881	-	-	-
2	[CuClL]·2H2O]	3395	3161, 3100	1702 a, 1645 b	1616, 1539	808	632	498	-
3	[CuBrL]·3H2O	3446	3106	1695 a, 1643(sh) b	1622, 1578	-	614	504	-
4	[Cu (NO3) L]·H2O	3425	3166	1703 a, 1645 b	1620, 1539	808	638	586	1382–1462
5	[Cu(OAc)L]·2H2O	3417	3237	1680 a, 1645 b	1560, 1525	847	595	503	1615, 1333
6	[Cu(ClO4)L]·2H2O	3428	3325, 3170	1628 a, 1645 b	1568, 1516	838	631	505	1085, 1049
7	[CoClL]2·2H2O	3388	3150	1734 a, 1645 b	1600, 1515	840	629	585	-
8	[Co (OAc) L(H2O)]·4H2O	3491, 3422	3196	1734 a, 1645 b	1590, 1545	841	630	540	1619, 1372
9	[NiCl2(HL)2]·4H2O	3441	3280	1735 a, 1660 a, 1645 b. 1610 b	1688, 1620	877	648	591	-
10	[Ni(OAc) L]·2H2O	3445	3310, 3200	1664 a (sh), 1640 b	1580, 1540	829	590	503	1620, 1342
11	[Zn L2]·2H2O	3413	3223	1700 a, 1643 b	1609	848	612	580	-
12	[FeCl2(HL)2]Cl·H2O	3429(s,br)	3280	1737 a, 1700 a, 1645 b, 1610 a	1685, 1620	881	589	512	-

^a indigo moiety, ^b antipyrine moeity, -: indicates no value available.

; the infrared spectrum of the ligand 1 displays bands at 3442, 3348, 3287, 3251, and 3141 attributable to ν(N-H) absorptions characteristic of –NH groups [19,20]. The spectrum also displays bands at 1738, 1645, 1688, 1623 and 1595 cm⁻¹. The first band can be assigned to ν(C=O) of the isatin moiety [19,20], and the second band can be assigned to ν(C=O) of the antipyrine moiety. The latter three bands are associated with ν(C=N) [21]. The appearance of multiple bands characteristic of ν(C=N) can be explained by the formation of tautomers secondary to the presence of the isatin carbonyl group and C=S with the adjacent –NH groups. The absorption band of ν(C=S) (thioamide IV) observed at 881 cm⁻¹ [15] and a weak band observed at 2650 cm⁻¹, assigned to ν(C-SH), supported the thione-thiol tautomerization expected from the C=S group with an adjacent proton [22].

The binding mode of the ligand in each metal complex has been deduced by comparing the IR spectrum of the ligand with that of the corresponding metal complex. In the infrared spectra of all the complexes, the band characteristic of the ν(C=N) of the isatin moiety is shifted to lower wave numbers compared to that of the free ligand, indicating the coordination of C=N group with the metal via the nitrogen atom in all complexes. In the IR spectra of the metal complexes, except for those of 7 and 8, the bands corresponding to the C=O of the isatin moiety appeared at lower wavenumbers compared to that of the ligand as a result of the participation of the carbonyl oxygen atom of this group in the coordination. This band did not shift in the infrared spectra of complexes 7 and 8, indicating that C=O is not involved in the coordination.

In the infrared spectra of the complexes, except 9, 10 and 12, the absorption band is attributable to the ν(C=O) of the antipyrine moiety appeared at the same position as that of the ligand, indicating that this group only participates in coordination to the metal ion in complexes 9, 10 and 12. In the infrared spectra of all complexes, except 9 and 12, the band corresponding to ν(C=S) appeared at lower wavenumbers (808–848 cm⁻¹) compared to that of the free ligand, indicating that the C-S group takes part in the coordination. The appearance of a band at 1622–1560 cm⁻¹, which is assignable to υ(–N=C-S-), accompanies the shift in the ν(C-S) band. The appearance of the later band indicates the participation of the thiol form in coordination rather than the thion form, as suggested by the ¹H-NMR spectrum of complex 11. The ν(C=S) bands in the infrared spectra of complexes 9 and 12 were at the same position as that of the free ligand, indicating that the C=S moiety does not participate in coordination in complexes 9 and 12.

Complexes 9 and 12 are composed of two ligand molecules that bind to one metal ion, and their infrared spectra showed that the two ligand molecules have different binding modes. In the infrared spectra of the complexes 9 and 12, each exhibits four bands at 1735–1737, 1700–1660, 1645 and 1620 cm⁻¹, attributable to the uncoordinated ν(C=O) of the isatin moiety, the coordinated ν(C=O) of the isatin moiety, the uncoordinated ν(C=O) of the antipyrine moiety and the coordinated ν(C=O) of the antipyrine moiety, respectively. Moreover, each of the spectra of complexes 9 and 12 displays two bands corresponding to the ν(C=N) of the isatin moiety at 1595 and 1520 cm⁻¹, which are assignable to the free uncoordinated C=N and the coordinated C=N, respectively. The above results illustrate that one of the ligand molecules behaves as a neutral, bidentate ligand and coordinates via the C=N and C=O groups of the isatin moiety and that the second molecule also behaves as a neutral, bidentate ligand but coordinates via the C=O and N-H groups of the antipyrine moiety.

The spectra of acetato complexes 5, 8 and 10 show two absorption bands at 1620–1615 and 1372–1333 cm⁻¹, assigned to υ(C=O) and υ(C-O), respectively. The difference between these two bands is approximately 287−243 cm⁻¹, which indicates that the acetate in these complexes coordinates to the metal ion as a monodentate ligand [23]. The monodentate coordination of a perchlorate ligand lowers the symmetry of the system (T_d→C_3v). The modes of the free perchlorate that were initially degenerated (υ₃ and υ₄) are both split into two bands [24]. The infrared spectrum of the perchlorate [Cu(ClO₄)L]·2H₂O complex showed that both the υ₃ band and υ₄ band are split into two bands, at 1118 and 1085 cm⁻¹ and at 694 and 631 cm⁻¹, respectively, indicating that the perchlorate coordinates to the metal ion as a monodentate ligand. The infrared spectrum of complex 4, [Cu(NO₃)L]·H₂O showed two bands at 1462 and 1382 cm⁻¹, attributable to a monodentate nitrato ligand [25]. The infrared spectra of the hydrated complexes display a broad band at 3491–3388 cm⁻¹ from ν(OH) of the water molecule [26]. The IR spectra of all the complexes display a new band at 591–498 cm⁻¹, assigned to ν(M-N) [27,28,29], and the spectra of all the metal complexes except 7 and 8 show a new band at 693–589 cm⁻¹, assigned to υ(M-O) [19,20,21,22,23,24,25,26,27,28,29].

2.5. Magnetic and Electronic Spectra

The room-temperature magnetic moments (μ_eff BM per metal atom) and electronic spectral bands for the metal complexes in the solid state are described in

Table 3. The electronic absorption spectral bands (nm) and magnetic moments of ligand 1 and its metal complexes 2–12.

No.	Compound	π→π* (nm)	n→π*	Charge Transfer	d→d Bands	µeff (β.M)
1	HL C20H18N6O2S	268	280, 369			-
2	[CuLCl]·2H2O	291	310, 395		600, 672, 770	1.03
3	[CuLBr]·3H2O	295	325, 445		580, 631	1.1
4	[Cu(NO3) L]·H2O	270	310, 455	520, 574	675, 720	1
5	[Cu(OAc) L]·2H2O	270	310		560, 671	1.11
6	[Cu(ClO4)L]·2H2O	270	363, 400		500(br), 770(br)	1.6
7	[CoClL]2·2H2O	260	318, 345		600, 675, 700	0.42
8	[Co(OAc) L(H2O)]·4H2O	290	320		600, 675, 780	3.7
9	[NiCl2(HL)2]·4H2O	290	310, 396	420	500	-
10	[Ni (OAc) L]·2H2O	291	305		567, 950	2.26
11	[ZnL2]·2H2O	280	319, 396	480, 520(sh)	-	-
12	[FeCl2(HL)2]Cl·H2O	270	300, 391	450	584	4.67

-: indicates no value available.

. The data showed that the electronic absorption spectrum of ligand 1 exhibits bands at 268 and 280 nm, attributable to intra-ligand π→π* electronic transitions [15,30]. These bands do not change significantly upon complex formation [31].

The somewhat broad band at 369 nm is assignable to the intra-ligand n→π* electronic transitions associated with the combination of the azomethine function of the thiosemicabazone, the thione function of the thiosemicabazone and the heterocyclic moiety [15,32]. In the spectra of the metal complexes, this band generally shifts to a higher energy as a result of the participation of the azomethine and/or thiol groups in coordination [33,34]. In the spectra of the metal complexes, new bands were observed in the 357–530 nm region due to S(π)→metal(II,III) charge-transfer processes [35,36], and the spectrum of bromo complex 3 showed a Br→metal(II) charge-transfer band [37]. Cl→metal(II,III) [38,39,40] and O→metal(II,III) [35] charge-transfer bands were generally observed at approximately 333 nm and were obscured by the stronger intra-ligand transfer bands. Copper(II) complexes 2–6 showed magnetic moments of 1.76–1.87 BM. These values are close to the spin-only value for one unpaired spin (~1.73 BM) and indicate that there is no molecular association between the copper(II) ions in the square planar environment [34,41,42]. The electronic absorption spectra of Cu(II) complexes 2–5 showed two or three bands at 870–660, 740–631 and 675–580 nm, assignable to the ²B₁g(d_x2−y2)→²A_1g(d_z2), ²B₁g(d_x2−y2)→²B_2g(d_xy) (υ₂) and ²B₁g(d_x2−y2)→²Eg(d_xz,d_yz) (υ₃) transitions, suggesting a square planar geometry around the copper(II) ion. Complex 6 displays a broad band at 790 nm that could be attributed to the abovementioned transitions in the ligands in a square planar geometry around the copper(II) ion [43,44,45].

Cobalt(II) complex 7 gave a magnetic moment of 3.1 BM, which is considerably less than what is expected for high-spin tetrahedral cobalt(II). This unexpected value could be explained by the occurrence of antiferromagnetic exchange through the chloro bridge linking the two cobalt(II) ions [46,47]. Complex 8 gave a magnetic moment of 3.7 BM per metal ion, which is consistent with high-spin tetrahedral cobalt(II) complexes [48]. The electronic spectra of complexes 7 and 8 displayed a broad band with several features at approximately 675 nm, which is attributable to the ⁴A₂→⁴T₁(P) (v₃) transition in a tetrahedral geometry around the Co(II) ion [15].

The nickel(II) complexes were found to be paramagnetic, which ruled out square planar configurations. The magnetic moments of nickel(II) complexes 9 and 10 are 2.98 and 3.06 BM, respectively, corresponding to two unpaired electrons [49], suggesting geometries other than square planar. The electronic spectra of Ni(II) complexes 9 and 10 showed bands at 950, 690-675 nm and 560−567 nm, assignable to the ³A_2g(F)→³T_2g(F)(υ₁), ³A_2g(F)→³T_1g(F)(υ₂) and ³A_2g(F)→³T_1g(P) transitions, respectively, in an octahedral environment [50,51,52]. The υ_2/υ₁ ratios for these complexes are 1.41 and 1.37, which are below the usual range of 1.5–1.75. These ratios suggest a tetragonally distorted octahedral geometry around the nickel(II) ions [51,53].

Iron(III) complex 12 gave a magnetic moment of 5.95 BM per metal ion, corresponding to a high-spin d⁵ configuration. The electronic spectrum of complex 12 showed strong bands at 391, 450, 530 and 584 nm. The first three bands can be assigned to ligand-to-metal charge-transfer (LMCT) processes. The last band at 584 nm is broad and of relatively high intensity. The relatively high intensity of this band can be attributed to overlap with a low-lying ligand charge-transfer band. This is because in the case of iron(III), the d-d transitions are considered forbidden because they take place between ions with different multiplicities; examples of such transitions include the ⁶A₁g→⁴T_1g (G), ⁶A₁g→⁴T_2g (G) and ⁶A₁g→⁴E_g (G) or ⁴E_g(D) transitions in tetragonally distorted octahedral Fe(III) environments [54]. The electronic spectrum of the zinc(II) complex showed bands at 396, 440, 491, and 520(sh), which can be attributed to ligand-to-metal (L→M) charge-transfer processes.

2.6. EPR Spectra of the Copper(II) Complexes

The EPR spectra of the Cu(II) complexes are illustrated in the Supplementary Materials. The EPR data of Cu(II) complexes 2–6 in the polycrystalline state at 298 K are listed in

Table 4. EPR spectral assignments and bonding parameters for Cu(II) complexes in polycrystalline state at 298 K.

Parameter	Complex 2	Complex 3	Complex 4	Complex 5	Complex 6
g‖ or g1	2.189	2.198	2.287	g‖ = 2.21	2.25
g⊥ or g2, g3	2.121, 2.057	2.079, 1.989	2.121, 1.977	g⊥ = 2.038	2.12, 2.027
g⊥	2.089	2.034	2.049	2.038	2.074
gav	2.12	2.125	2.128	2.095	2.132
G	2.12	5.82	5.86	5.53	3.38
R	0.94	0.76	0.87	-	0.72
k‖2/k‖	0.419/0.647	0.468/0.684	0.637/0.798	0.424/0.651	0.554/0.744
k⊥2/k⊥	0.722/0.850	0.29/0.539	0.391/0.625	0.248/0.498	0.137/0.370
K2/k	0.621/0.788	0.349/0.591	0.473/0.688	0.307/0.554	0.276/0.525

-: indicates no value available.

. The EPR spectra of the Cu(II) complexes have low g₁ and g_av values, indicating that the bonds have considerable covalent characteristics [18,55]. The EPR spectra of the Cu(II) complexes other than complex 5 ([Cu(L)(OAc)]·2H₂O) show rhombic-type distortion with three g values, g₁ = 2.189, 2.198, 2.287, and 2.25; g₂ = 2.121, 2.079, 2.121, and 2.12; and g₃ = 2.057, 1.989, 1.977, and 2.027, respectively. The rhombic symmetry of these complexes is attributable to the bulkiness of the ligand and to the unequal bond lengths of the four Cu-S, Cu-N, Cu-O, and Cu-X bonds (X=Cl, Br, NO₃ or ClO₄) [55]. The g tensors, which follow g₁ ˃ g₂ ˃ g₃ ˃ 2, are indicative of complexes with a d_(x2−y2) ground state. This result is supported by the value of R (R = (g₂ − g₁/(g₃ − g₂) < 1 (= 0.94 − 0.72)), confirming a d_(x2−y2) ground state with covalent bonding character [56,57].

The EPR spectral data of complex 5 demonstrate anisotropic signals with g_‖ > g_⊥ > 2.0023, which are indicative of a d_(x2−y2) ground state with axial symmetry, resulting in a ²B_1g ground state that is known for Cu²⁺ complexes [58,59]. These g values are consistent with Cu²⁺-complex 5 having a square planar geometry [60,61]. The geometric parameter G, which is a measure of the exchange interaction between the copper centres in the solid state, was calculated using the equation G = (g_‖ − 2.0023)/(g_⊥ − 2.0023) for axial and rhombic compounds, g_‖ = g₁ and g_⊥ = g₂ + g₃/2. If G ˃ 4, the exchange interaction is negligible, and if it is less than 4, considerable exchange interactions exist [58,62]. Complexes 3, 4, and 5 have G values ˃ 4, indicating that exchange interactions are negligible, while for complexes 2 and 6, G ˂ 4, suggesting considerable exchange interactions between the Cu(II) centres. Kivelson and Neiman reported that the g_‖-values of Cu²⁺ complexes can be used to determine the nature of the copper-ligand bonding. If the g_‖-value is smaller than 2.3, the environment is essentially covalent, while if the value is larger than 2.3, the environment is essentially ionic [63]. For all the synthesized complexes, g_‖ < 2.3, indicating that there is considerable covalent character in the copper-ligand bonds.

According to [45,64,65], the orbital reduction factor (k) and its parallel (k_‖) and perpendicular (k_⊥) components were calculated according to the following equations: k_‖² = (g_‖ − 2.0023)∆₂/8λ₀, k_⊥² = (g_⊥ − 2.0023) ∆₁/2 λ₀, k² = (K_‖² + 2_⊥²)/3 where λ_o is the spin orbit coupling (−828 cm⁻¹) of the free Cu(II) ion, and Δ₁ and Δ₂ are the electronic transitions ²B_1g→²B_2g (d_x2−y2→d_xy) and ²B_1g→²E_g (d_x2−y2→d_xzd_yz), respectively. The calculated k_‖², k_⊥² and k² values in Table 1 showed that for complexes 3–6, k_‖ ˃ k_⊥, indicating the presence of significant out-of-plane π-bonding [66] and confirming that ²B_1g is the ground state for these complexes. For complex 2, k_‖ ˂ k_⊥, indicating the presence of significant in-plane π-bonding [67]. Moreover, when the environment is ionic, k = 1, but if this value is less than 1, the environment is covalent. These compounds showed k values lower than one, which is strong evidence of covalent character, which is in agreement with the results obtained from the g_‖ values [65,68]. None of the solid-state EPR spectra of the complexes displayed hyperfine structure, confirming the presence of intermolecular interactions, but these interactions were not strong enough to overcome the dipolar interactions [69].

2.7. Thermal Analysis

The TGA spectra of metal complexes are shown in the Supplementary Materials. The TGA results of solid complexes 2, 3, 5 and 7–12 are listed in

Table 5. Thermal (TGA) data for metal complexes 2, 3, 5, and 7–12.

No.	Complex	Temp. Range (°C)	Weight Loss Found (calc)%	Assignment	Residual’s Formula
2	[Cu(L)Cl]·H2O]	30–120 213–898	4.10 (3.44) 83.90(84.30)	Loss of water of hydration (1H2O) Continuous decomposition of complex	[Cu(L)Cl] CuO
3	[Cu(L)Br]·3H2O	26–75 191–763	9.3(8.96) 79.54(80.6)	Loss of water of hydration (3H2O) Continuous decomposition of complex	[Cu(L)Br] CuO
5	[Cu(L)OAc]·2H2O	30–109 109–800	6.60(6.40) 81.55(82.40)	Loss of water of hydration (2H2O) Continuous decomposition of complex	[Cu(L)OAc] CuO
7	[Co(L)Cl]2·2H2O	45–145 195–470 625–660	4.20(3.50) 69.23(70.30) 8.30(8.10)	Loss of water of hydration (2H2O) Partial decomposition of complex oxidation of carbon to CO2	[Co(L)Cl]2 2CoS + 7C 2CoS
8	[Co(L)OAc(H2O)]·4H2O	27–80 80–175 195–550 600–898	11.30(11.73) 3.63(2.93) 66.00(66.51) 3.12(3.91)	Loss of water of hydration (4H2O) Loss of coordinated water (1H2O) Continuous decomposition of complex to form metal oxide and carbon Partial oxidation of carbon	[Co(L)OAc (H2O)]·CoO + 3C CoO + 1C
9	[Ni(HL)2Cl2]·4H2O	42–133 133–550	7.50(7.10) 77.73(78.85)	Loss of water of hydration (4H2O) decomposition of complex and formation of cobalt oxide and carbon	[Ni (HL)2Cl2]·NiO + 7C
10	[Ni2(L)2(OAc)2]·4H2O	27–75 75–255 255–899	6.00(6.43) 4.50(4.30) 78.30(77.80)	Loss of water of hydration (4H2O) Loss of one acetato ligand Decomposition of complex	[Ni2(L)2(OAc)2] [Ni2(L)2(OAc)] NiO
11	[Zn(L)2]·2H2O	45–131 131–999	3.98(3.95)	Loss of water of hydration (2H2O) Decomposition of complex	[Zn(L2)]·ZnS + 7C
12	[Fe(HL)2Cl2]Cl·H2O	55–160 160–350 605–700	1.60 (1.80) 70.30(68.80) 8.97(8.45)	Loss of water of hydration (1H2O) Decomposition of complex and formation of iron sulfide and carbon Oxidation of carbon to CO2	[FeCl2(HL)2]Cl Fe2S3 + 7C Fe2S3

. The results show an agreement with the formulae suggested by the analytical, spectral and magnetic data. A general decomposition pattern, in which the complexes decompose in two or three stages, was observed. The first stage is the loss of waters by hydration at 29–160 °C; the second stage of decomposition corresponds to the loss of coordinated water molecules at 80–175 °C; complex 8 showed a third decomposition stage that is a result of overlapping processes including the loss of Cl⁻, Br⁻ or OAc⁻ anions; the loss the ligands to form metal oxides or metal sulfides mixed with carbon residues; and the oxidation of carbon to carbon dioxide leaving metal oxides or sulfides.

2.8. Biological Activities

2.8.1. Effect of Ligand 1 and Its Metal Complexes 2, 7 and 9 on Solid Tumor Volume

Mice bearing solid tumors were treated SC with 0.2 mM of ligand 1 and its metal complexes 2, 7 and 9. The treatment was started seven days after EAC implementation in mice and was continued for 14 consecutive days. Tumor volume was measured after 24 h from the last dose of treatment. As shown in

Table 6. Effect of ligand 1 and its metal complexes 2, 7 and 9 on the volume of solid tumor.

Groups	Tumor Volume (mm3)	Inhibition %
Tumor-bearing mice + DMSO	334.8 ± 17.5	-
Tumor-bearing mice + ligand 1	212.2 ± 22 *	36.6
Tumor-bearing mice + complex 2	112.2 ± 52 **	66.5
Tumor-bearing mice + complex 7	99.2 ± 17.9 **	70.37
Tumor-bearing mice + complex 9	82.6 ± 3.9 **	75.53

Data are presented as mean ± SEM. Significantly (* p < 0.01 and ** p < 0.001) different from mice bearing solid tumor treated with vehicle (DMSO). -: indicates no value available.

, treatment of solid tumor-bearing mice with the ligand 1 resulted in a significant reduction in tumor volume with inhibition of (36.6%) as compared with tumor-bearing mice treated with DMSO. Metal complexes 2, 7 and 9 exhibited potent effects on reduction of the volume of solid tumor. The recorded inhibition percents were 66.5%, 70.37% and 75.53%, respectively, as compared with tumor-bearing mice treated with the vehicle.

2.8.2. Effect of Ligand 1 and Its Metal Complexes 2, 7 and 9 on the Biochemical Analysis of Serum

As shown in

Table 7. Effect of ligand 1 and its metal complexes 2, 7 and 9 on serum biochemical parameters of mice bearing solid tumor.

Groups	ALT (U/L)	AST (U/L)	Albumin (g/dL)	Glucose (mg/dL)
Normal control	39 ± 3.1	194 ± 35.1	3.3 ± 0.3	128 ± 8.8
Solid tumor + DMSO	77.8 ± 11.5 #	438.6 ± 66.4 #	2.5 ± 0.2 #	80.2 ± 9.5 #
Solid tumor + ligand 1	43.2 ± 9 *	387 ± 45.6 *	2.3 ± 0.3	86.4 ± 11.2
Solid tumor + complex 2	42.2 ± 5 *	275.6 ± 60.9 *	2.4 ± 0.2	122.2 ± 11.6 **
Solid tumor + complex 7	39.2 ± 7.9 *	206.6 ± 54.3 **	2.5 ± 0.1	126 ± 9.6 ***
Solid tumor + complex 9	36.6 ± 3.9 **	208 ± 51.2 **	2.7 ± 0.4	127.4 ± 13.5 **

Data are presented as mean ± SEM. ALT: alanine aminotransferase; AST: aspartate aminotransferase. Significantly (^# p < 0.01) different from the normal mice (untreated) and significantly (* p < 0.05, ** p < 0.01, *** p < 0.001) different from mice bearing solid tumor treated with vehicle (DMSO).

, EAC cells induced a significant reduction in albumin and glucose and a significant elevation in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (p < 0.001) in comparison with normal mice (normal control group). Conversly, ligand 1 and its metal complexes 2, 7 and 9 exhibited significant increase of albumin and glucose levels and decrease ALT and AST levels relative to tumor-bearing mice treated with DMSO (Table 7).

An increase in glucose catabolism and a decrease in glucose synthesis in the presence of cancer cells were reported [70,71]. Therefore, the decrease in serum glucose in mice bearing solid tumor may be due to the tumors exhibit high rate of glycolysis. This is a way of stimulating the body into a constant state of gluconeogenesis which is accompanied with the reduction in serum albumin [72,73]. Treatment of tumor-bearing mice with ligand 1 and its metal complexes, especially complex 7, restored serum glucose, albumim, ALT and AST levels towards the standard levels when compared with the tumor-bearing mice treated with the vehicle.

2.8.3. Effect of Ligand 1 and Its Metal Complexes 2, 7 and 9 on the Expression of Vascular Endothelial Growth Factor (VEGF) in Tumor and Liver Tissues of EAC Mice

VEGF is a significant angiogenic factor released during the tumor formation to stimulate the angiogenesis process. Usage of VEGF antagonists is one of the essential anti-angiogenic therapies for cancer treatment [74]. Therefore, the effect of ligand 1 and its metal complexes 2, 7 and 9 on VEGF expression was studied in tumor and liver tissues of EAC mice. Results as presented in Figure 2A,B illustrated that VEGF is highly expressed in tumor and liver tissues of mice bearing solid tumor compared to normal mice.

The increasing in the VEGF expression level in mice bearing solid tumors was related to the presence of EAC cells. This finding is consistent with previously reported results which concluded that EAC cells has been found to exhibit strong VEGF expression level [75]. Moreover, cancer usually enhances VEGF expression through tumor aggression, therefore, VEGF is involved in cancer pathology [76]. Conversely, the treatment with ligand 1 and its metal complexes 2, 7 and 9 downregulated the expression of VEGF in tumor (Figure 2A) and in liver tissues (Figure 2B) of mice bearing solid tumor in comparison to mice bearing solid tumor treated with DMSO. Complex 9 exhibited the most potent anti-VEGF activity among the tested compounds. These results revealed that the anti-tumor activities of the tested compounds might be due to their ability to inhibit VEGF.

2.8.4. Effect of Ligand 1 and Its Metal Complexes 2, 7 and 9 on the Expression of Cysteine Aspartyl-Specific Protease-7 (Caspase-7) in Tumor and Liver Tissues of EAC Mice

Apoptosis is a programmed cell death that is caused by caspases that target cysteine aspartyl residues in target proteins [77]. Defects in apoptosis mechanisms can induce tumor pathogenesis [78], for example, the activation of caspases by the signal transduction pathway can lead to irreversible apoptosis through protein degradation [79]. Therefore, it is important to design and develop new agents that act as caspases activators to treat cancers [80,81]. Consequently, the effect of ligand 1 and its metal complexe 2, 7 and 9 on the stimulation of caspase-7 was examined in tumor and liver tissues of EAC mice.

As shown in Figure 3A,B, the induction of solid tumors is associated with low expression of caspase-7 in tumor and liver tissues of mice bearing solid tumor compared to normal mice. Our finding was compatible with the reported data demonstrating the declined expression of caspase-3 in mice bearing EAC cells [82]. On the other hand, the treatment with ligand 1 and its metal complexes 2, 7 and 9 upregulated the expression of caspase-7 in tumor (Figure 3A) and in liver tissues (Figure 3B) of mice bearing EAC tumor in comparison to mice bearing solid tumor treated with DMSO.

Complex 9 followed by complex 7 exhibited the most potent apoptotic activity among the tested compounds. Caspase-7 is a critical mediator of apoptosis and the overexpression of it induced apoptosis of cancer cells [83]. Therefore, these results revealed that the anti-tumor activities of the tested compounds might be due to their ability to stimulate caspase-7.

3. Materials and Methods

3.1. Chemistry

3.1.1. Preparation of N(4)-antipyrinylthiosemicarbazide

The preparation of S-methylantipyrine carbodithioate was illustrated in Scheme 1. In details, 4-Aminoantipyrine (40.6 g, 0.2 mol) was added in small portions to an aqueous KOH solution (11.2 g, 100 mL), and the mixture was maintained below 5 °C using ice. Refrigerator-cooled CS₂ (11.36 cm³, 0.2 mol) was then added dropwise to the stirred solution over l h while the solution was maintained below 10 °C. To the resulting yellow solution, MeI (11.68 cm³, 0.2 mol) dropwise was added over 2 h while the temperature was maintained below 10 °C. During the addition of MeI, the yellow colour of the mixture gradually diminished, and a white solid powder was formed. The crystals of S-methyl-antipyrinecarbodithioate were collected by vacuum filtration and washed with cold H₂O. Because of the noxious odour of this solid (m.p. 193 ± 2 °C), it was used immediately without purification.

The preparation of N(4)-antipyrinylthiosemicarbazide was presented in Scheme 2. S-Methylantipyrinecarbodithioate (29.3 g, 0.1 mol) dissolved in EtOH (30 mL) was added to hydrazine monohydrate (3.2 mL, 0.1 mol). The solution was heated at reflux until the evolution of methyl mercaptan was almost complete. The release of methyl mercaptan was detected based on the yellow colour it imparts to paper moistened with Pb(OAc)₂ placed at the mouth of the condenser. The reaction time for this step was 8 h. The resulting white solid crystals, N(4)-antipyrinyl-thiosemicarbazide, were collected by vacuum filtration, washed with cold i-PrOH and dried (m.p. 203 ± 2 °C).

3.1.2. Preparation of Isatin N(4)-antipyrine-thiosemicarbazide 1 (ligand)

Isatin N(4)-antipyrine-thiosemicarbazide 1 (ligand) was prepared by mixing equimolar amounts of isatin and N(4)-antipyrine-thiosemicarbazide in anhydrous EtOH with a few drops of concentrated H₂SO₄ at reflux (80 °C) for 2 h as shown in Scheme 3.

Ligand 1: Yield (87%), m.p. = 210–212 °C; color: yellow, Elemental analysis for C₂₀H₁₈N₆O₂S, (F.W. = 406.): Found (calcd) %C 59.14 (59.00), %H 5.15(4.43), %N 20.91(20.66), %S 7.85(7.87)); IR (KBr, cm⁻¹), 3442, 3348, 3287, 3251, 3141ν(NH), 1738 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1688, 1623, 1595 ν(C=N), 881 ν(C=S); UV-Vis. (Nujol mulls) (nm) 268, 280, 369 nm, ¹H-NMR (MHz, DMSO-d₆, δ, ppm): 12. 819 (s, 1 H, thiosemicabazide N-NH), 11.219 (s, 1H, indole N-H), 10.115 (s, 1H, CS-N-H), 7.702–6.876. (m, multiple, H aromatic protons), 3.068 (s, 3 H, N-CH₃), 2.135 (s, 3 H, CH₃). GC–MS: m/z (relative abundance); 406.27, 363.47 (27.15), 291 (100), 276.68 (72.92), 261.69 (28.97), 223.71 (90.05), 204.07 (22.73), 203.3 (26.0), 145.64 (51.57), 130.57 (28.60), 118.18 (78.92), 67.30 (18.68).

3.1.3. Preparation of the Metal Complexes

The metal complexes of the ligand were prepared by mixing a hot ethanolic solution (30 mL) of the metal salt (CuCl₂·2H₂O, CuBr₂, Cu(NO₃)₂·3H₂O, Cu(OAc)₂·H₂O, Cu(ClO₄)₂, CoCl₂·6H₂O, Co(OAc)₂·4H₂O, NiCl₂·6H₂O, Ni(OAc)₂·4H₂O, ZnCl₂ and FeCl₃·6H₂O) with the appropriate amount of a hot (75 °C) ethanolic solution of the ligand to form a 1:1 L/M (ligand/metal) solution. The reaction mixture was then refluxed for approximately 2 h. The obtained precipitates were separated by filtration, washed several times with ethanol and then diethyl ether, and then dried under vacuum over anhydrous CaCl₂. Due to the poor solubility of these complexes in most organic solvents and water, all attempts to prepare a single crystal of the ligand or any of the metal complexes were unfortunately unsuccessful.

[Cu(L)Cl]·H₂O (2): Yield (79%), m.p. = 238–240 °C; color: dark brown, Λ_M: 10.0 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₂₀H₁₉N₆O₃SCuCl., (F.W. = 522.6): Found (calcd) %C 46.0(46.0), %H 4.1(3.64), %N 16.3(16.10), %S 6.88(6.13): IR (KBr, cm⁻¹), 3395 ν(H₂O), 3161, 3100 ν(NH), 1702 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1616, 1539 ν(C=N), 808 ν(C=S), 632 ν(Cu-O), 498 ν(Cu-N) UV-Vis. (Nujol mulls) (nm) 310, 325 n→π*, 395, 460, 525 Charge transfer, 600, 672, 725 d→d, µ_eff (B.M) = 1.83.

[Cu(L)Br]·3H₂O (3): Yield (89%), m.p. = 250 °C; color: brown, Λ_M: 37.9 ohm⁻¹ cm² mol⁻¹, Elemental analysis for, C₂₀H₂₃N₆O₅SCuBr, (F.W. = 602.5): Found (calcd) %C 38.81(39.83), %H 3.54(3.82), %N 15.02(13.94), %S 5.03(5.31) IR (KBr, cm⁻¹), 3446 ν(H₂O), 3106ν(NH), 1695 ν(C=O)_isatin, 1643 ν(C=O)_antipyrine, 1622, 1578 ν(C=N), 806 ν(C=S), 614 ν(Cu-O), 504 ν(Cu-N); UV-Vis. (Nujol mulls) (nm) 325 n→π*, 380, 445, 500 charge transfer, 580, 631, 660 d→d, µ_eff (B.M) = 1.81.

[Cu(L)(NO₃)]·H₂O (4): Yield (80%), m.p. = 247 °C; color: brown, Λ_M: 34.3 ohm⁻¹ cm² mol⁻¹, Elemental analysis for, C₂₀H₁₉N₇O₆SCu, (F.W. = 548.5): Found (calcd) %C 43.4(43.5), %H 3.29(3.50), %N 18.0(17.9), %S 6.0(5.8); IR (KBr, cm⁻¹), 3425ν(H₂O), 3166ν(NH), 1703 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1620, 1539 ν(C=N), 1382,1462 ν(NO3), 808 ν(C=S), 638 ν(Cu-O), 586 ν(Cu-N); UV-Vis. (Nujol mulls) (nm) 310 n→π*, 390, 455, 520, 574 charge transfer, 675, 720 d→d, µ_eff (B.M) = 1.78.

[Cu(L)OAc]·2H₂O (5): Yield (74%), m.p. = 212–216 °C; color: brown, Λ_M: 11.9 ohm⁻¹ cm² mol⁻¹, Elemental analysis for, C₂₂H₂₄N₆O₆SCu, (F.W. = 563.5): Found (calcd) %C 46.12(46.90), %H 4.82(4.30), %N 15.13 (14.9), %S 5.1(5.7); IR (KBr, cm⁻¹), 3417 ν(H₂O), 3237ν(NH), 1680 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1560, 1525 ν(C=N), 1615, 1333 ν(OAc), 847 ν(C=S), 595 ν(Cu-O), 503 ν(Cu-N); UV-Vis. (Nujol mulls) (nm) 310 n→π*, 395, 430, 460, 560 charge transfer, 671,740(br), 870 d→d, µ_eff (B.M) = 1.79.

[Cu(L)ClO₄]·2H₂O (6): Yield (91%), m.p. = 250–252 °C; color: brown, Λ_M: 45.50 ohm⁻¹ cm² mol⁻¹; Elemental analysis for, C₂₀H₂₁N₆O₈SCuCl, (F.W. = 604.5): Found (calcd) %C 38.92(39.70), %H 3.75(3.48), %N 14.67(13.91), %S 5.18(5.29); IR (KBr, cm⁻¹), 3428 ν(H₂O), 3325, 3170ν(NH), 1628 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1568, 1516ν(C=N), 1118,1085, 694,631 ν(ClO4), 838 ν(C=S), 693 ν(Cu-O), 505 ν(Cu-N); UV-Vis. (Nujol mulls) (nm) 310 n→π*, 357, 390, 480(br) charge transfer, 790(br) d→d, µ_eff (B.M) = 1.76.

[Co(L)Cl]₂·2H₂O (7): Yield (66%), m.p. = 258–260 °C; color: brown, Λ_M: 17.9 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₄₀H₃₈N₁₂O₆S₂Co₂Cl₂, (F.W. = 1034.8): Found (calcd) %C 47.0(46.38), %H 4.17(3.7), %N 17.0(16.21), %S .4(6.18), IR (KBr, cm⁻¹), 3388 ν(H₂O), 3150 ν(NH), 1734 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1600, 1515 ν(C=N), 841 ν(C=S), 585 ν(Co-N); GC–MS: m/z (relative abundance); 999.5 (22.46), 869.45 (15.13), 464.94 (4.00); UV-vis. (Nujol mulls) (nm) 318, 345 n→π*, 390, 450(br) charge transfer, 600, 675, 700 d→d, µeff (B.M) = 3.1.

[Co(L)OAc(H₂O)]·4H₂O (8): Yield (75%), m.p. ˃ 300 °C; color: brown, Λ_M: 4.6 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₂₂H₃₀N₆O₉SCo, (F.W. = 612.5): Found (calcd) %C 42.96(43.10), %H 5.3(4.89),%N 14.20(13.71), %S 4.87(5.22); IR (KBr, cm⁻¹), 3491, 3422 ν(H₂O), 3196ν(NH), 1734 ν(C=O)_isatin, 1645 ν(C=O)_antipyrine, 1590, 1545 ν(C=N), 1619, 1372 ν(OAc), 841 ν(C=S), 540 ν(Co-N); GC–MS: m/z (relative abundance); 612.85 (100), 576.23 (94.07, 561.30 (61.93), 541.85 (6.86), 523.22 (39.20); UV-Vis. (Nujol mulls) (nm) 320 n→π*, 390, 440(br), 530(br) charge transfer, 600, 675, 780d→d, µ_eff (B.M) = 3.7

[Ni(HL)₂Cl₂]·4H₂O (9): Yield (84%), m.p. = 254–255 °C; color: brown, Λ_M: 13.5 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₄₀H₄₄N₁₂O₈S₂ NiCl₂., (F.W. = 1014.9): Found (calcd) %C 46.84(47.3), %H 4.94(4.33), %N 16.95(16.55), %S 5.89(6.30); IR (KBr, cm⁻¹), 3441 ν(H₂O), 3280 ν(NH), 1735, 1660 ν(C=O)_isatin, 1645, 1620 ν(C=O)_antipyrine, 1595(sh), 1520(sh) ν(C=N), 880 ν(C=S), 648 ν(Ni-O), 591 ν(Ni-N); UV-VIS. (Nujol mulls) (nm) 320 n→π*, 396, 420, 530 charge transfer, 560(s), 690(w), 950(br) d→d, µ_eff (B.M) = 2.98.

[Ni(L)OAc]₂·4H₂O (10): Yield (70%), m.p. ˃ 300 °C; color: brown, Λ_M: 2.60 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₄₄H₄₈N₁₂O₁₂S₂ Ni₂, (F.W. = 1017.4): Found (calcd) %C 47.76(47.24), %H 4.19(4.3), %N, 15. 60(15.03), %S 6.71(5.78); IR (KBr, cm⁻¹), 3445 ν(H₂O), 3310, 3200 ν(NH), 1664 ν(C=O)_isatin, 1620 ν(C=O)_antipyrine, 1580, 1540 ν(C=N), 1620, 1342 ν(OAc), 829 ν(C=S), 590 ν(Ni-O), 503 ν(Ni-N); UV-Vis. (Nujol mulls) (nm) 305, 315 n→π, 395, 450, 530 charge transfer, 567(s), 670(sh). 950(br) d→d, µ_eff (B.M) = 3.06.

[Zn(L)₂]·2H₂O (11): Yield (69%), m.p. ˃ 300 °C; color: dark yellow, Λ_M: 1.40 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₄₀H₃₈N₁₂O₆S₂ Zn, (F.W. = 911.50): Found (calcd) %C 51.9(52.6), %H 3.7 (4.2), %N 18.8 (18.42), %S 6.64 (7.0); IR (KBr, cm⁻¹ 3413 ν(H₂O), 3223ν(NH), 1700 ν(C=O)_isatin, 1643 ν(C=O)_antipyrine, 1609, 1570 ν(C=N), 815 ν(C=S), 612 ν(Zn-O), 580 ν(Zn-N): ¹H-NMR (MHz, DMSO-d₆, δ, ppm): 11.22 (s, 1H, indole N-H), 11.008 (s,1H, CS-N-H), 7.704–6.879. (m, multiple, C-H aromatic), 3.111 (s, 3 H, N-CH3), 2.174 (s, 3 H, CH3); GC–MS: m/z (relative abundance); 911.5 (1.19) UV-Vis. (Nujol mulls) (nm) 319 n→π, 396, 440, 491, 520(sh).

[Fe(HL)₂Cl₂]Cl·H₂O (12): Yield (78%), m.p. = 210–212 °C; color: dark green, Λ_M: 74.50 ohm⁻¹ cm² mol⁻¹, Elemental analysis for C₄₀H₃₈N₁₂O₅S₂FeCl₃, (F.W. = 992.50): Found (calcd) %C 47.62(48.3), %H 3.9(3.8), %N 16.95 (16.91), %S 5.6 (6.44), IR (KBr, cm⁻¹), 3429(s,br) ν(H₂O), 3280 ν(NH), 1737, 1700 ν(C=O)_isatin, 1645, 1621 ν(C=O)_antipyrine, 1610, 1576 ν(C=N), 880 ν(C=S), 589 ν (Fe-O), 512 ν(Fe-N) GC–MS: m/z (relative abundance); 992.5 (11.39), 974.50 (1.90), 938.30 (12.96), 869.80 (8.27), 464.40 (2.48); UV-Vis. (Nujol mulls) (nm) 300, 340 n→π, 391, 450, 530(sh) charge transfer, 567(s), 584 d→d, µ_eff (B.M) = 5.95.

3.1.4. Instrumentation and Measurements

Elemental microanalyses [C, H, and N] were conducted in the Micro Analytical Unit. The IR spectra were acquired using KBr discs in a Spirit model FT-IR spectrophotometer (Shimadzu, Thermo Scientific, Glasgow, UK). The ¹H-NMR spectra of the ligand and zinc complex were recorded in DMSO-d₆ on a Gemini 200 NMR spectrophotometer (Varian, Palo Alto, CA, USA) at 300 MHz. Mass spectra were collected using a model ISQLT direct probe controller with a single quadrupole mass analyser (Thermo Scientific, Glasgow, UK) using Thermo X-calibur software (Xcalibur 4.1). Solid-state electronic spectra were recorded in Nujol mulls using a Shimadzu UV-Vis 1800 spectrophotometer (Thermo Scientific, Glasgow, UK). The molar conductance of a 1 × 10⁻³ M solution of the complexes in N,N-dimethylformamide (DMF) was measured at 25 °C with a Bibby MCl-type conductometer (Chelmsford, UK). The resistance was measured in ohms and the molar conductivities were calculated according to the following equation: ΛM = VxKxg/Mw* Ω where ΛM = molar conductivity (Ω-1cm2mol-1), V = volume of the complex solution (mL), K = cell constant (0.92 cm-1), Mw = molecular weight of the complex, g = weight of the complex (g), and Ω = resistance (Ω).

The solid-state electron paramagnetic resonance (EPR) spectra of the copper(II) complexes were recorded with an 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. Diphenyl picryl hydrazide (DPPH) was used as a g-marker to calibrate the spectra. The thermal analyses (TGA) were carried out in air using a Shimadzu DT-30 thermal analyser. Magnetic susceptibilities were measured at 27 °C using the modified Gouy method with a Johnson Matthey balance and were calculated using the following equation:

$$\mathit{Meff}=2.84\sqrt{x_m^{\mathit{corr}}·T}$$

3.2. Biological Evalution

3.2.1. Preparation of Solutions of the Complexes

Stock solutions (1 mM) of the newly synthesized complexes were prepared by dissolving each complex in dimethylsulfoxide (DMSO)/H₂O, and the solutions were stored at 4 °C. Only ligand 1 and complexes 2, 7 and 9 were successfully solubilized, allowing their antitumor activities to be determined.

3.2.2. Experimental Animals

Adult female Swiss albino mice (6–7 weeks old), weighing 20–22 g, were purchased and housed under standard ventilation and light cycle conditions. They were given food and water ad libitum. The current study protocol was approved by the Ethical Committee for Laboratory Animals of Science Faculty Menoufia University (No.: ECLA-SFMU-18015).

3.2.3. Tumor Cell Line

The Ehrlich ascites carcinoma (EAC) cell line was purchased from the National Cancer Institute (NCI, Cairo, Egypt). The EAC cell line was maintained in the peritoneal cavity of adult female Swiss albino mice by serial intraperitoneal transplantation of 2.5 × 10⁶ cells weekly.

3.2.4. Induction of Solid Tumor and Animals Grouping

The solid tumor was induced by subcutaneously (SC) injecting 1 × 10⁻⁶ EAC cells into the right thigh of the lower limb of mice [84,85]. Seven days after tumor inoculation, the tumor volume became palpable. The mice were randomly divided in separate cages into six groups:

• Group 1 (normal control): mice without any treatment.
• Group 2 (vehicle group): mice bearing solid tumors treated with DMSO/H₂O.
• Group 3: mice bearing solid tumor and treated subcutaneously with 0.2 mM of ligand 1.
• Group 4: mice bearing solid tumor and treated subcutaneously with 0.2 mM of complexe 2.
• Group 5: mice bearing solid tumor and treated subcutaneously with 0.2 mM of complexe 7.
• Group 6: mice bearing solid tumor and treated subcutaneously with 0.2 mM of complexe 9.

The doses were selected based on the LD₅₀ values of the complexes (data not shown). The treatment was started seven days after EAC implementation and was continued for 14 consecutive days.

3.2.5. Blood and Tissue Samples Collection

At the end of the experiment, mice were sacrificed. Blood samples were collected from jugular vein puncture into chilled non-heparinized tubes, which were centrifuged at 40× g for 10 min at 4 °C. The sera were frozen at −20 °C for future measurements. The livers and solid tumors were removed and cut into pieces, and the individual pieces were fixed in paraffin blocks for immunohistochemical assays.

3.2.6. Assessment of Tumor Volume (TV)

After 24 h from the last dose of treatment, mice were sacrificed and the solid tumors were carefully removed. The volume of the tumors was assessed [86] using the following formula: TV (mm³) = 4π (A/2)² × (B/2), where (A) is the minor tumor axis and (B) is the major tumor axis.

3.2.7. Biochemical Analysis of the Serum

To determine the effect of ligand 1 and its metal complexes 2, 7 and 9 on the serum biochemical profile. The levels of alanine transaminase (ALT), aspartate transaminase (AST), albumin and glucose were determined using a Beckman autoanalyzer apparatus, Brea, CA, USA).

3.2.8. Immunohistochemical Examination of VEGF and Caspase-7 in Solid Tumors and Liver Tissues

The effect of ligand 1 and its metal complexes 2, 7 and 9 on the expression of VEGF and caspase-7 in solid tumor tissues and in liver tissues of mice bearing solid tumors was deremined by immunohistochemical methods. In details, the sections of tumor and liver tissues in paraffin were dewaxed, hydrated, and immersed in antigen retrieval solution (EDTA solution, pH 8). The samples were treated with 0.3% hydrogen peroxide and protein block and then incubated with anti-VEGF (Santa Cruz, Dallas, TX, USA, 1:100 dilution) and anti-caspase-7 (Lab Vision, Thermo Fischer Scientific, Glasgow, UK; ready to use) at 4 °C overnight. The slides were rinsed three times with PBS and incubated with anti-mouse IgG secondary antibodies (EnVision + System HRP; Dako, SanJose, CA, USA) for 30 min at room temperature. The samples were visualized with diaminobenzidine commercial kits (Liquid DAB+Substrate Chromogen System; Dako) and finally counterstained with Mayer’s haematoxylin. In the negative control, the primary antibody was replaced with normal mouse serum. The positive cells were counted from seven high-power fields of 400×. The labelling index (%) was determined by dividing the number of positive cells by the total cells × 100 [87,88].

3.3. Statistical Analysis

Data are expressed as mean ± SEM and the statistical comparison between groups were analyzed using one-way ANOVA followed by Tukey post hoc test. A p of < 0.05 value was considered statistically significant.

4. Conclusions

In summary, new metal complexes derived from isatin-N-(4)antipyrinethiosemicarbazone ligand 1 were synthesized and characterized. Also, their potential anti-cancer activities against solid tumors induced by EAC cells in mice were investigated. The results revealed that the tested complexes significantly reduced the volume of solid tumors. This reduction was associated with an increase in the level of caspase-7 and a dramatic decrease in the VEGF level. The tested complexes are promising anti-cancer agents those exert their activities via the induction of apoptosis and the inhibition of angiogenesis, leading to reduction of the tumor size.