Synthesis, characterization, and in vivo anti-cancer activity of new metal complexes derived from isatin-N(4)antipyrinethiosemicarbazone ligand against ehrlich ascites carcinoma cells

Article abstract of DOI:10.3390/molecules24183313

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)antipyrinethiosemicarbazone 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.

Full text of DOI:10.3390/molecules24183313

Article
Synthesis, Characterization, and In Vivo Anti-Cancer
Activity of New Metal Complexes Derived from
Isatin-N(4)antipyrinethiosemicarbazone Ligand
Against Ehrlich Ascites Carcinoma Cells
1
2,3,
4
1
5,6
Fathy El-Saied , Bishoy El-Aarag *, Tarek Salem , Ghada Said , Shaden A. M. Khalifa
1,7,8,9,
and Hesham R. El-Seedi
*
1
Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom 32512, Egypt;
elsayedfathy139@yahoo.com (F.E.-S.); ghadasaid_2007@yahoo.com (G.S.)
Biochemistry Division, Chemistry Department, Faculty of Science, Menoufia University,
Shebin El-Koom 32512, Egypt
Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology,
Okayama University, Okayama 7008530, Japan
Department of Molecular Biology, Genetic Engineering & Biotechnology Institute, University of Sadat City,
Sadat City 32958, Egypt; salem_tarek@yahoo.com
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University,
SE 106 91 Stockholm, Sweden; shaden.khalifa@su.se
Department of Experimental Cancer Medicine (ECM); Novum, 14157 Huddinge, Stockholm, Sweden
Pharmacognosy Group, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre,
Box 574, SE-751 23 Uppsala, Sweden
International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China
Al-Rayan Research and Innovation Center, Al-Rayan Colleges, Medina 42541, Saudi Arabia
2
3
4
5
6
7
8
9
* Correspondence: bishoy.yousef@gmail.com (B.E.-A.); hesham.el-seedi@ilk.uu.se (H.R.E.-S.);
Tel.: +20-1271732703 (B.E.-A.); +46-700434343 (H.R.E.-S.)
Received: 18 July 2019; Accepted: 9 September 2019; Published: 11 September 2019
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
1
metal complexes 2−12 were characterized using elemental, spectral ( H-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.
Molecules 2019, 24, 3313; doi:10.3390/molecules24183313
www.mdpi.com/journal/molecules

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Keywords: metal complexes; isatin-N(4)antipyrinethiosemicarbazone; Ehrlich ascites carcinoma;
tumor volume; VEGF; 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 [3−6].
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. 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.

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Figure 1. Cont.
Figure 1. Chemical structures of metal complexes 2−12.

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Table 1. Analytical and physical data of the ligand 1 and its metal complexes 2–11.
Molar
Conductance
Analysis (%)/Found (Calcd)
No.
Molar ratio/Compound
Color
m.p. (°C)
F.W.
Yield%
C
H
N
S
1
2
3
4
5
6
7
8
9
HL C20H18N6O2S
HL+CuCl2(1:1),(2:1),(1:2) [CuLCl]·H2O
HL+CuBr2(1:1) [CuLBr]·3H2O
Yellow
Deep brown
Brown
Brown
Brown
Brown
Brown
Brown
Brown
210–212
238–240
250
406.64
522.6
602.5
548.5
563.5
604.5
1036
614.5
1014.9
558.9
912.2
993.7
94
79
89
80
74
91
66
75
84
70
69
78
59.14(59)
46.0(46)
5.15(4.43)
4.1(3.64)
3.54(3.82)
3.29(3.5)
4.82(4.30)
3.75(3.48)
4.17(3.7)
5.3(5.04)
4.94(4.33)
4.19(4.3)
3.7(4.2)
20.91(20.66)
16.3(16.10)
15.02(13.94)
18(17.9)
15.13 (14.9)
14.67(13.91)
17(16.21)
7.85(7.87)
6.88(6.13)
5.03(5.31)
6(5.8)
-
10.0
37.9
34.3
11.9
70.32
47.9
4.6
13.5
2.6
1.4
38.81(39.83)
43.4(43.5)
46.12(46.90)
38.92(39.7)
47(46.33)
42.96(41.9)
46.84(47.3)
47.76(47.24)
51.9(52.6)
47.62(48.3)
HL+Cu(NO3)2.3H2O (1:1)[Cu(NO3) L]·H2O
HL+Cu(OAc)2 (1:1) [Cu(OAc) L]·2H2O
HL+Cu(ClO4)2(1:1) [Cu(ClO4)L]·2H2O
HL+CoCl2 (1:1) [CoClL]2·2H2O
HL+Co(OAc)2 (1:1) [Co(OAc) L(H2O)]·4H2O
Hl+NiCl2 (1:1) [NiCl2(HL)2]·4H2O
HL+Ni(OAc)2 (1:1) [Ni(OAc) L]·2H2O
HL+Zn(OAc)2 (1:1) [ZnL2·H2O
247
212–216
250–252
258-260
Over 300
254–255
Over 300
Over 300
210–212
5.1(5.7)
5.18(5.29)
6.4(6.18)
4.87(5.2)
5.89(6.3)
6.71(5.78)
6.64 (7)
14.2(13.7)
16.95(16.55)
15. 6(15.03)
18.8 (18.42)
16.95(16.91)
10
11
12
Brown
Deep yellow
Deep green
HL+FeCl3 (1:1) [FeCl2(HL)2]Cl·H2O
3.9(3.8)
5.6(6.44)
54.09

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1
2.1. H-NMR Spectra
1
The H-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
1
H-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
−3
The molar conductance values of the metal complexes in DMF (10 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
−1
2
−1
Ω cm mol 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

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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; 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
−1
groups [19,20]. The spectrum also displays bands at 1738, 1645, 1688, 1623 and 1595 cm . 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)
−1
−1
(thioamide IV) observed at 881 cm [15] and a weak band observed at 2650 cm , 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–
−1
848 cm ) compared to that of the free ligand, indicating that the C-S group takes part in the coordination.
−1
The appearance of a band at 1622–1560 cm , 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
1
coordination rather than the thion form, as suggested by the H-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
−1
spectra of the complexes 9 and 12, each exhibits four bands at 1735–1737, 1700–1660, 1645 and 1620 cm ,
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
−1
ν(C=N) of the isatin moiety at 1595 and 1520 cm , 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
−1
cm , assigned to υ(C=O) and υ(C-O), respectively. The difference between these two bands is
−1
approximately 287−243 cm , 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 (Td→C3v). 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
−1
showed that both the υ3 band and υ4 band are split into two bands, at 1118 and 1085 cm and at 694 and 631
−1
cm , respectively, indicating that the perchlorate coordinates to the metal ion as a monodentate ligand.
−1
The infrared spectrum of complex 4, [Cu(NO3)L]·H2O showed two bands at 1462 and 1382 cm ,
attributable to a monodentate nitrato ligand [25]. The infrared spectra of the hydrated complexes display a
−1
broad band at 3491–3388 cm from ν(OH) of the water molecule [26]. The IR spectra of all the complexes

Molecules 2019, 24, 3313
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−1
display a new band at 591–498 cm , assigned to ν(M-N) [27−29], and the spectra of all the metal complexes
−1
except 7 and 8 show a new band at 693–589 cm , assigned to υ(M-O) [19−29].

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Table 2. IR spectral bands (cm−1) for ligand 1 and its metal complexes 2−12 and their assignments.
4
No.
Ligand/Complexes
Ligand C20H18N6O2S
[CuClL].2H2O]
ν(H2O)
-
ν(N H)
3442,3348,3287
,3251,3141
ν(C=O)
ν(C=N)
1688 ,1623,
1595
1616, 1539
ν(C=S)
881
808
-
ν(M-O)
ν(M-N)
ν(OAc/ClO4/NO3
a
a
b
b
1
2
3
1738 , 1645
-
-
-
-
-
a
3395
3446
3161, 3100
1702 , 1645
632
614
498
504
a
1695 ,
[CuBrL].3H2O
3106
1622, 1578
b
1643(sh)
a
b
4
5
6
7
[Cu (NO3) L].H2O
[Cu(OAc)L].2H2O
[Cu(ClO4)L].2H2O
[CoClL]2.2H2O
[Co (OAc)
3425
3417
3428
3388
3166
3237
3325, 3170
3150
1703 ,1645
1620,1539
1560, 1525
1568, 1516
1600,1515
808
847
838
840
638
595
631
629
586
503
505
585
1382–1462
1615, 1333
1085,1049
-
a
b
b
1680 , 1645
a
1628 , 1645
a
b
1734 ,1645
a
b
8
3491,3422
3196
1734 , 1645
1590,1545
841
630
540
1619,1372
L(H2O)].4H2O
a
1735 ,1660
9
[NiCl2(HL)2].4H2O
3441
3280
a,1645 b.1610
1688, 1620
877
648
591
-
b
a
1664
10
11
12
[Ni(OAc) L].2H2O
[Zn L2].2H2O
3445
3413
3310, 3200
3223
1580, 1540
1609
829
848
881
590
612
589
503
580
512
1620,1342
b
(sh),1640
a
b
1700 , 1643
1737 , 1700
1645 , 1610
-
-
a
a
,
[FeCl2(HL)2]Cl. H2O
3429(s,br)
3280
1685, 1620
b
a
a indigo moiety, b antipyrine moeity, -: indicates no value available.

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2.5. Magnetic and Electronic Spectra
The room-temperature magnetic moments (μeff BM per metal atom) and electronic spectral bands for
the metal complexes in the solid state are described in Table 3. 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) [38−40] 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
2
2
2
675−580 nm, assignable to the B1g(dx2-y2)→2A1g(dz2), B1g(dx2-y2)→2B2g(dxy) (υ2) and B1g(dx2-y2)→2Eg(dxz,dyz)
(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 [43−45].
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
4
approximately 675 nm, which is attributable to the A2→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,
3
3
3
assignable to the A2g(F)→3T2g(F)(υ1), A2g(F)→3T1g(F)(υ2) and A2g(F)→3T1g(P) transitions, respectively, in an
octahedral environment [50−52]. The υ2/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
d configuration. The electronic spectrum of complex 12 showed strong bands at 391, 450, 530 and 584 nm.
5
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;
6
6
6
4
examples of such transitions include the A1g→4T1g (G), A1g→4T2g (G) and A1g→4Eg (G) or Eg(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.

Molecules 2019, 24, 3313
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Table 3. The electronic absorption spectral bands (nm) and magnetic moments of ligand 1 and its metal
complexes 2−12.
No
1
2
3
4
5
6
7
8
Compound
HL C20H18N6O2S
[CuLCl]·2H2O
π→π* (nm)
268
n→π*
280, 369
310, 395
325, 445
310, 455
310
363, 400
318, 345
320
310, 396
305
319, 396
300, 391
Charge transfer
d→d bands
µeff (β.M)
-
291
295
270
270
270
260
290
290
600, 672, 770
580, 631
675, 720
560, 671
500(br), 770(br)
600, 675, 700
600, 675, 780
500
1.03
1.1
1
1.11
1.6
0.42
3.7
-
[CuLBr]·3H2O
[Cu(NO3) L]·H2O
[Cu(OAc) L]·2H2O
[Cu(ClO4)L]·2H2O
[CoClL]2·2H2O
520, 574
[Co(OAc) L(H2O)]·4H2O
[NiCl2(HL)2]·4H2O
[Ni (OAc) L]·2H2O
[ZnL2]·2H2O
9
420
10
11
12
291
280
270
567, 950
-
584
2.26
-
4.67
480, 520(sh)
450
[FeCl2(HL)2]Cl·H2O
-: indicates no value available.
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. The EPR spectra of the
Cu(II) complexes have low g1 and gav 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
2
indicative of a d(x2 − y2) ground state with axial symmetry, resulting in a B1g ground state that is known for
2+
2+
Cu complexes [58,59]. These g values are consistent with Cu -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
2+
exchange interactions between the Cu(II) centres. Kivelson and Neiman reported that the g||-values of Cu
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⊥)
2
2
components were calculated according to the following equations: k|| = (g|| − 2.0023)∆2/8λ0, k⊥ = (g⊥
−
2
2
2
−1
2.0023) ∆1/2 λ0, k = (K|| + 2⊥ )/3 where λo is the spin orbit coupling (−828 cm ) of the free Cu(II) ion, and Δ1
and Δ2 are the electronic transitions B1g→2B2g (dx2 − y2→dxy) and B1g→2Eg (dx2 − y2→dxzdyz), respectively. The
2
2
2
2
2
calculated k|| , k⊥ and k 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 B1g is the ground state for these complexes.
2
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

Molecules 2019, 24, 3313
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displayed hyperfine structure, confirming the presence of intermolecular interactions, but these
interactions were not strong enough to overcome the dipolar interactions [69].
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
g⊥ = 2.038
2.038
2.095
5.53
2.25
2.12, 2.027
2.074
2.132
3.38
g⊥ or g2, g3 2.121, 2.057 2.079, 1.989 2.121, 1.977
g⊥
gav
G
2.089
2.12
2.12
0.94
2.034
2.125
5.82
2.049
2.128
5.86
R
0.76
0.87
-
0.72
k||2/k||
k⊥2/k⊥
K2/k
0.419/0.647 0.468/0.684 0.637/0.798 0.424/0.651 0.554/0.744
0.722/0.850 0.29/0.539 0.391/0.625 0.248/0.498 0.137/0.370
0.621/0.788 0.349/0.591 0.473/0.688 0.307/0.554 0.276/0.525
-: indicates no value available.
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. 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.

Molecules 2019, 24, 3313
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Table 5. Thermal (TGA) data for metal complexes 2, 3, 5, and 7−12.
Weight loss
Found
(calc)%
Temp. Range
Residual’s
Formula
No
Complex
Assignment
(˚C)
30−120
213−898
26−75
191−763
30−109
109−800
45−145
195−470
625−660
4.10 (3.44)
83.90(84.30)
9.3(8.96)
79.54(80.6)
6.60(6.40)
81.55(82.40)
4.20(3.50)
69.23(70.30)
8.30(8.10)
Loss of water of hydration (1H2O)
Continuous decomposition of complex
Loss of water of hydration (3H2O)
Continuous decomposition of complex
Loss of water of hydration (2H2O)
Continuous decomposition of complex
Loss of water of hydration (2H2O)
Partial decomposition of complex
oxidation of carbon to CO2
[Cu(L)Cl]
CuO
[Cu(L)Br]
CuO
[Cu(L)OAc]
CuO
[Co(L)Cl]2
2CoS + 7C
2CoS
2
3
5
[Cu(L)Cl]·H2O]
[Cu(L)Br]·3H2O
[Cu(L)OAc]·2H2O
7
[Co(L)Cl]2·2H2O
Loss of water of hydration (4H2O)
Loss of coordinated water (1H2O)
Continuous decomposition of complex to form metal oxide
and carbon
27−80
80−175
195−550
600−898
11.30(11.73)
3.63(2.93)
66.00(66.51)
3.12(3.91)
[Co(L)OAc (H2O)].
CoO +3C
8 [Co(L)OAc(H2O)]·4H2O
CoO +1C
Partial oxidation of carbon
Loss of water of hydration (4H2O)
decomposition of complex and formation of cobalt oxide
and carbon
42−133
133−550
7.50(7.10)
77.73(78.85)
[Ni (HL)2Cl2].
NiO +7C
9
[Ni(HL)2Cl2]·4H2O
27−75
75−255
255−899
45−131
131−999
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
Loss of water of hydration (2H2O)
Decomposition of complex
[Ni2(L)2(OAc)2]
[Ni2(L)2(OAc)]
NiO
10 [Ni2(L)2(OAc)2]·4H2O
[Zn(L2)].
ZnS +7C
11
[Zn(L)2]·2H2O
3.98(3.95)
Loss of water of hydration (1H2O)
Decomposition of complex and formation of iron sulfide
and carbon
55−160
160−350
605−700
1.60 (1.80)
70.30(68.80)
8.97(8.45)
[FeCl2(HL)2]Cl
Fe2S3 +7C
Fe2S3
12 [Fe(HL)2Cl2]Cl·H2O
Oxidation of carbon to CO2
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, 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.
Table 6. Effect of ligand 1 and its metal complexes 2, 7 and 9 on the volume of solid tumor.
Groups
Tumor Volume (mm3)
334.8 ± 17.5
Inhibition %
Tumor-bearing mice + DMSO
Tumor-bearing mice + ligand 1
Tumor-bearing mice + complex 2
Tumor-bearing mice + complex 7
Tumor-bearing mice + complex 9
-
212.2 ± 22 *
112.2 ± 52 **
99.2 ± 17.9 **
82.6 ± 3.9 **
36.6
66.5
70.37
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.
2.8.2. Effect of Ligand 1 and Its Metal Complexes 2, 7 and 9 on the Biochemical Analysis of Serum

Molecules 2019, 24, 3313
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As shown in Table 7, 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.
Table 7. Effect of ligand 1 and its metal complexes 2, 7 and 9 on serum biochemical parameters of mice
bearing solid tumor.
Albumin
(g/dL)
Glucose
(mg/dL)
Groups
ALT (U/L)
AST (U/L)
Normal control
39 ± 3.1
77.8 ± 11.5#
43.2 ± 9*
194 ± 35.1
438.6 ± 66.4 #
387 ± 45.6 *
275.6 ± 60.9 *
206.6 ± 54.3 **
208 ± 51.2 **
3.3 ± 0.3
2.5 ± 0.2#
2.3 ± 0.3
2.4 ± 0.2
2.5 ± 0.1
2.7 ± 0.4
128 ± 8.8
Solid tumor + DMSO
Solid tumor + ligand 1
Solid tumor + complex 2
Solid tumor + complex 7
Solid tumor + complex 9
80.2 ± 9.5#
86.4 ± 11.2
122.2 ± 11.6 **
126 ± 9.6 ***
127.4 ± 13.5 **
42.2 ± 5*
39.2 ± 7.9*
36.6 ± 3.9**
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).
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 2 A and 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.

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Figure 2. Effect of ligand 1 and its metal complexes 2, 7 and 9 on vascular endothelial growth factor (VEGF)
expression in tumor tissues (A) and in liver tissues (B) of Ehrlich ascites carcinoma (EAC)-bearing mice.
VEGF expression was determined by immunohostochemistry assay. VEGF expression was expressed as
labeling index percentage. Data are expressed as mean ± SEM. Significantly (## p < 0.01) different from the
normal mice and significantly (** p < 0.01) different from mice bearing solid tumor treated with vehicle
(DMSO).
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 Figurea 3 A and 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.
Figure 3. Effect of ligand 1 and its metal complexes 2, 7 and 9 on cysteine aspartyl-specific protease-7
(caspase-7) expression in tumor tissues (A) and in liver tissues (B) of Ehrlich ascites carcinoma
(EAC)-bearing mice. Caspase-7 expression was determined by immunohistochemistry assay. Caspase-7
expression was expressed as labeling index percentage. Data are expressed as mean ± SEM. Significantly (##

Molecules 2019, 24, 3313
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p < 0.01) different from the normal mice and significantly (** p < 0.01 and *** p < 0.001) different from mice
bearing solid tumor treated with vehicle (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
3
mL), and the mixture was maintained below 5 °C using ice. Refrigerator-cooled CS2 (11.36 cm , 0.2 mol)
was then added dropwise to the stirred solution over l h while the solution was maintained below 10 °C.
3
To the resulting yellow solution, MeI (11.68 cm , 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.
CS2
CH3I
S- methylantipyrine carbodithioate
Scheme 1. Preparation of S-methylantipyrine carbodithioate.
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).
S- methylantipyrine carbodithioate
N(4)-antipyrinylthiosemicarbazide

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Scheme 2. Preparation of N(4)-antipyrinylthiosemicarbazide.
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 H
2
SO
4
at reflux (80 °C) for 2 h as shown in Scheme 3.
Scheme 3. Preparation of Isatin N(4)-antipyrine-thiosemicarbazide 1 (ligand)
Ligand 1: Yield (87%), m.p. = 210−212 °C; color: yellow, Elemental analysis for C20
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 ), 3442, 3348,
H
18
N
6
O
2
S, (F.W. = 406.):
−1
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.
1
(Nujol mulls) (nm) 268, 280, 369 nm, H-NMR (MHz, DMSO-d
6
, 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-CH ), 2.135 (s, 3 H, CH ). GC–MS: m/z (relative abundance); 406.27, 363.47 (27.15), 291 (100),
3
3
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 (CuCl
2
·2H
2
O, CuBr
2
, Cu(NO
3
)2
·3H
2
O, Cu(OAc)
2
·H
2
O, Cu(ClO
4
)2
, CoCl
2
·6H
2
O, Co(OAc)
2
·4H O,
2
NiCl ·6H O, Ni(OAc)
2
2
2
·4H O, ZnCl
2
2
and FeCl
3
·6H
2
O) 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 CaCl . Due to the poor
2
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.
−1
2
−1
[Cu(L)Cl]·H
analysis for C20
%S 6.88( 6.13): IR (KBr, cm ), 3395 ν(H
2
O (2): Yield (79%), m.p. = 238−240 °C; color: dark brown, Λ
M
: 10.0 ohm cm mol , Elemental
H
19
N6
O
3
SCuCl., (F.W. = 522.6): Found (calcd) %C 46.0( 46.0), %H 4.1(3.64), %N 16.3( 16.10 ),
−1
2
O), 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, μeff (B.M) = 1.83.
−1
2
−1
[Cu(L)Br]·3H
for, C20
5.03(5.31) IR (KBr, cm ), 3446 ν(H
2
O (3): Yield (89%), m.p. = 250 °C; color: brown, Λ
5
M
: 37.9 ohm cm mol , Elemental analysis
H23
N
6
O
SCuBr, (F.W. = 602.5): Found (calcd) %C 38.81( 39.83), %H 3.54(3.82), %N 15.02(13.94), %S
−1
2
O), 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, μeff (B.M) = 1.81.
−1
2
−1
[Cu(L)(NO
for, C20
IR (KBr, cm ), 3425ν(H
3
)]·H
2
O (4): Yield (80%), m.p. = 247 °C; color: brown, Λ
SCu, (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);
O), 3166ν(NH), 1703 ν(C=O)isatin, 1645 ν(C=O)antipyrine , 1620, 1539 ν(C=N), 1382 ,1462
M
: 34.3 ohm cm mol , Elemental analysis
H
19
N
7
O6
−1
2

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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, μeff (B.M) = 1.78.
−1
2
−1
[Cu(L)OAc]·2H2O (5): Yield (74%), m.p. = 212−216 °C; color: brown, ΛM: 11.9 ohm cm mol , 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),
−1
%S 5.1(5.7); IR (KBr, cm ), 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, μeff (B.M) = 1.79.
−1
2
−1
[Cu(L)ClO4]·2H2O (6): Yield (91%), m.p. = 250−252 °C; color: brown, ΛM: 45.50 ohm cm mol ; Elemental
analysis for, C20H21N6O8SCuCl, (F.W. = 604.5): Found (calcd) %C 38.92(39.70), %H 3.75(3.48), %N
−1
14.67(13.91), %S 5.18( 5.29 ); IR (KBr, cm ), 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, μeff (B.M) = 1.76.
−1
2
−1
[Co(L)Cl]2·2H2O (7): Yield (66%), m.p. = 258−260 °C; color: brown, ΛM : 17.9 ohm cm mol , Elemental
analysis for C40H38N12O6S2Co2Cl2, (F.W. = 1034.8): Found (calcd) %C 47.0(46.38), %H 4.17(3.7), %N
−1
17.0(16.21), %S .4(6.18), IR (KBr, cm ), 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.
−1
2
−1
[Co(L)OAc(H2O)]·4H2O (8): Yield (75%), m.p. ˃ 300 °C; color: brown, ΛM: 4.6 ohm cm mol , 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),
−1
%S 4.87(5.22); IR (KBr, cm ), 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, μeff (B.M) = 3.7
−1
2
−1
[Ni(HL)2Cl2]·4H2O (9): Yield (84%), m.p. = 254−255 °C; color: brown, ΛM: 13.5 ohm cm mol , Elemental
analysis for C40H44N12O8S2 NiCl2., (F.W. = 1014.9): Found (calcd) %C 46.84(47.3), %H 4.94(4.33), %N
−1
16.95(16.55), %S 5.89(6.30); IR (KBr, cm ), 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, μeff (B.M) = 2.98.
−1
2
−1
[Ni(L)OAc]2·4H2O (10): Yield (70%), m.p. ˃ 300 °C; color: brown, ΛM: 2.60 ohm cm mol , Elemental
analysis for C44H48N12O12S2 Ni2, (F.W. = 1017.4): Found (calcd) %C 47.76(47.24), %H 4.19(4.3), %N, 15.
−1
60(15.03), %S 6.71(5.78); IR (KBr, cm ), 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, μeff (B.M) = 3.06.
−1
2
−1
[Zn(L)2]·2H2O (11): Yield (69%), m.p˃300 °C; color: dark yellow, ΛM: 1.40 ohm cm mol , 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),
−1
%S 6.64 (7.0); IR (KBr, cm 3413 ν(H2O), 3223ν(NH), 1700 ν(C=O)isatin, 1643 ν(C=O)antipyrine, 1609, 1570
1
ν(C=N), 815 ν(C=S), 612 ν(Zn-O), 580 ν(Zn-N): H-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).
−1
2
−1
[Fe(HL)2Cl2]Cl·H2O (12): Yield (78%), m.p. = 210−212 °C; color: dark green, ΛM: 74.50 ohm cm mol ,
Elemental analysis for C40H38N12O5S2FeCl3, (F.W. = 992.50): Found (calcd) %C 47.62(48.3), %H 3.9(3.8), %N
−1
16.95 (16.91), %S 5.6 (6.44), IR (KBr, cm ), 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, μeff (B.M) = 5.95

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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,
1
Glasgow, UK). The H-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
−3
conductance of a 1 × 10 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:
ꢀꢁꢂꢂ = 2.84 ꢄꢆꢇꢈꢈ ⋅ ꢉ
ꢃ
ꢅ
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
6
by serial intraperitoneal transplantation of 2.5 × 10 cells weekly.
3.2.4. Induction of Solid Tumor and Animals Grouping
−6
The solid tumor was induced by subcutaneously (SC) injecting 1 × 10 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.

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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
3
2
removed. The volume of the tumors was assessed [86] using the following formula: TV (mm ) = 4π (A/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. Conclusion
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

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anti-cancer agents those exert their activities via the induction of apoptosis and the inhibition of
angiogenesis, leading to reduction of the tumor size.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1; 1HNMR, Mass, IR, EPR,
TGA, and UV-Visible spectra.
Author Contributions: Conceptualization, F.E.-S., B.E.-A. and T.S.; Formal analysis, B.E.-A. and F.E.-S.; Funding
acquisition, S.A.M.K. and H.R.E.-S.; Methodology, B.E.-A., and G.S.; Visualization, B.E.-A. and F.E.-S.; Writing –
original draft, B.E.-A., F.E.-S., and G.S.; Writing – review & editing, B.E.-A., G.S., S.A.M.K., and H.R.E.-S. All authors
have read and approved the manuscript.
Funding: We are very grateful to the Swedish Research links grant 2016-05885 (VR for the years 2017 - 2019) for
generious financial support.
Acknowledgments: H.R.E.-S. is very graful to Jiangsu University, China for fellowship.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2019. CA 2019, 69, 7–34.
Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1995, 1, 27–30.
Thapa, D.; Lee, J.S.; Heo, S.W.; Lee, Y.R.; Kang, K.W.; Kwak, M.K. Novel hexahydrocannabinol analogs as
potential anti-cancer agents inhibit cell proliferation and tumor angiogenesis. Eur. J. Pharmacol. 2011, 650, 61–71.
El-Aarag, B.Y.; Kasai, T.; Zahran, M.A.; Zakhary, N.I.; Shigehiro, T.; Sekhar, S.C.; Agwa, H.S.; Mizutani, A.;
Murakami, H.; Kakuta, H.; et al. In vitro anti-proliferative and anti-angiogenic activities of thalidomide
dithiocarbamate analogs. Int. Immunopharmacol. 2014, 21, 283–292.
El-Aarag, B.; Kasai, T.; Masuda, J.; Zahran, M.; Agwa, H.; Seno, M. Anticancer effects of novel thalidomide
analogs in A549 cells through inhibition of vascular endothelial growth factor and matrix metalloproteinase-2.
Biomed. Pharmacother. 2017, 85, 549–555.
Zahran, M.; Agwa, H.; Osman, A.; Hammad, S.; El-Aarag, B.; Ismail, N.; Salem, T.; Gamal-Eldeen, A. Synthesis
and biological evaluation of phthalimide dithiocarbamate and dithioate derivatives as anti-proliferative and
anti-angiogenic agents-I. Eur. J. Chem. 2017, 8, 391–399.
Kamal, A.; Kumar, G.B.; Nayak, V.L.; Reddy, V.S.; Shaik, A.B.; Reddy, M.K. Design, synthesis and biological
evaluation of imidazopyridine/imidazopyrimidine-benzimidazole conjugates as potential anticancer agents.
Med. Chem. Commun. 2015, 6, 606–612.
4.
5.
6.
7.
8.
9.
Yang, L.L.; Lee, C.Y.; Yen, K.Y. Induction of apoptosis by hydrolyzable tannins from Eugenia jambos L. on
human leukemia cells. Cancer Lett. 2000, 157, 65–75.
Rosu, T.; Negoiu, M.; Pasculescu, S.; Pahontu, E.; Poirier, D.; Gulea, A. Metal-based biologically active agents:
Synthesis, characterization, antibacterial and antileukemia activity evaluation of Cu(II), V(IV) and Ni(II)
complexes with antipyrine-derived compounds. Eur. J. Med. Chem. 2009, 45(2): 774-781.
10. Leovac, V.M.; Bogdanovic, G.A.; Jovanovic, L.S.; Joksovic, L.C.; Markovic, I.; Joksovic, M.D. Synthesis,
characterization and antitumor activity of polymeric copper(II) complexes with thiosemicarbazones of
3-methyl-5-oxo-1-phenyl-3-pyrazolin-4-carboxaldehyde and 5-oxo-3-phenyl-3-pyrazolin-4-carboxaldehyde. J.
Inorg. Biochem. 2011, 105, 1413-1421.
11. Refata, M.S. N.M. El-Metwaly, Spectral, thermal and biological studies of Mn(II) and Cu(II) complexes with two
thiosemicarbazide derivatives. Spectrochim. Acta Part A 2012, 92, 336−346.
12. Mendes, I.C.; Moreira, J.P.; Mangrich, A.S.; Balena, S.P.; Rodrigues, B.L.; Beraldo, H. Coordination to copper (II)
strongly enhances the in vitro antimicrobial activity of pyridine-derived N(4)-tolyl thiosemicarbazones.
Polyhedron 2007, 26, 3263−3270.
13. Vinuelas-Zahinos, E.; Maldonado-Rogado, M.A.; Luna-Giles, F.; Barros-Garcia, F.J. Coordination behaviour of
Schiff base 2-acetyl-2-thiazoline hydrazone (ATH) towards cobalt(II), nickel(II) and copper(II). Polyhedron 2008,
27, 879−886.
14. Bharti, N.; Shailendra, Y.; Sharma, S.; Naqvi, F.; Azam, A. New palladium(II) complexes of
5-nitrothiophene-2-carboxaldehyde thiosemicarbazones. synthesis, spectral studies and in vitro anti-amoebic
activity.Bioorg. Med. Chem. 2003, 11, 2923–2929.

Molecules 2019, 24, 3313
22 of 25
15. El-Sawaf, A.K.; West, D.X.; El-Saied, F.A.; El-Bahnasawy, R.M. Synthesis, magnetic and spectral studies of
iron(III), cobalt(II,III), nickel(II), copper(II) and zinc(II) complexes of 4–formylantipyrine
N(4)-antipyrinylthiosemicarbazone. Transit. Met. Chem. 1998, 23, 649–655.
16. El-Sawaf, A.K.; West, D.X.; El-Saied, F.A.; El-Bahnasawy, R.M. Copper(II) complexes of 4-formylantipyrine
N(4)-substitutedthiosemicarbazones. Transit. Met. Chem. 1998, 22, 360−365.
17. Ali, A.Q.; Teoh, S.G.; Salhin, A.; Eltayeb, N.E.; Ahamed, M.B.K.; Majid, A.A. Synthesis of isatin
thiosemicarbazones derivatives: In vitro anti-cancer, DNA binding and cleavage activities. Spectrochim. Acta A
2014, 125, 440−448.
18. El-Saied, F.A.; El-Enein, S.A.A.; Emam, S.M.; El-Shater, H.A. Synthesis and characterization of Cu(II), Ni(II),
Co(II), Mn(II), Zn(II), Ru(III), Hf(IV) and ZrO(II) complexes of 2-thiophenylidene-N-4-methoxy
anilinoacetohydrazone. Polish J. Chem. 2009, 83, 1871−1883.
19. Detrov, I.; Grupce, O.; Stafilov, T. The N-H stretching region of some imides and thioimides. Mol. Struct. 1986,
142, 275−278.
20. Castineiras, A.; Gomez, M.C.; Sevillano, P. 2-(2,3-Dihydro-1,3-benzothiazol-2-yl)phenyl(diphenyl) phosphine
oxide, Synthesis and characterization by IR and NMR spectroscopy and X-ray diffractometry. Mol. Struct. 2000,
554, 301−306.
21. Prasad, R.N.; Mathur, M.; Upadhayay, A. Synthesis and spectroscopic studies of Cr (III), Fe (III) and Co (II)
complexes of hexaazamacrocycles. Indian Chem. Soc. 2007, 84, 1202−1204.
22. Husain, K.; Bhat, A.; Azam, A. New Pd(II) complexes of the synthesized 1-N-substituted thiosemicarbazones of
3-indole carboxaldehyde: Characterization and antiamoebic assessment against E. histolytica. J. Med. Chem. 2008,
43, 2016−2028.
23. Mikuriya, M.; Okawa, H.; Kida, S. Binuclear Metal Complexes. XXXV. Synthesis and Magnetism of Binuclear
Copper(II) Complexes with 3-[2-(Dialkylamino)ethylthio]-1-propanols. Bull. Chem. Soc. 1980, 53, 3717−3718.
24. Rreti, C.; Tosr, G. Complexes of Co alt (II), Nickel (II) and Copper Acetates, Perchlorates, and Tetrafluoroborates with
Heterocyclic Ligands Containing VA and VIA group Donor Atoms. Can. J. chem. 1975, 53, 177−182.
25. Jain, S.K.; Garg, B.S.; Bhoon, Y.K. Iron(III) complexes of 2-acetylpyridine-4-phenyl-3-thiosemicarbazones:
Magnetic, E.s.r. and spectral studies. Transit. Met. Chem. 1986, 11, 89−95.
26. Maurya, R.C.; Rajput, S. Neutral dioxovanadium(V) complexes of biomimetic hydrazones ONO donor ligands of
bioinorganic and medicinal relevance: Synthesis via air oxidation of bis(acetylaceto-nato)oxovanadium(IV),
characterization, biological activity and 3D molecular modeling. J. Mol. Struct. 2007, 833, 133−144.
27. Sallam, S.A.; Orabi, A.S.; El-Shetary, B.A.; Lentz, A. Copper, nickel and cobalt complexes of Schiff-bases derived
from β-diketones. Transit. Met. Chem. 2002, 27, 447−453.
28. Tossidis, A.; Bolos, C.A. Monohalogenobenzoylhydrazones II. Synthesis and Study of Ti(IV) Complexes with
Monochlorobenzoylhydrazones
of
2-Furaldehyde,
2-Thiophenaldehyde,
2-Pyrrolaldehyde
and
Di-2-pyridylketone as Ligands. Inorg. Chim. Acta 1986, 112, 93−97.
29. Ainscough, E.W.; Brodie, A.M.; Dobbs, A.J.; Ranford, J.D.; Waters, J.M. Antitumour copper(II) salicylaldehyde
benzoylhydrazone (H2sb) complexes: Physicochemical properties and the single-crystal X-ray structures of
[{Cu(H2sb)(CCl3CO2)2}2] and [{Cu(Hsb) (ClO4) (C2H5OH)}2] and the related salicylaldehyde acetylhydrazone
(H2sa) complex, [Cu(Hsa)Cl(H2O)]·H2O. Inorg. Chim. Acta 1998, 267, 27−38.
30. El-Sawaf, A.K.; Nassar, A.A.; El-Samanody, E. Synthesis, magnetic, spectral and biological studies of copper (II)
complexes of 4-benzoyl-3-methyl-1-phenyl-2-pyrazolin-5-one N(4)-substituted thiosemicarbazones. Sci. J. Chem.
2014, 2, 17−26.
31. Nair, M.K.M.; Radhakrishnan, P.K. Iodide Complexes of Yttrium and Lanthanides with
4-N-(4′-Antipyrylmethylidene) aminoantipyrine. Synth. React. Inorg. Met. Org. Chem. 1996, 26, 529−541.
32. Afrasiabi, Z.; Sinn, E.; Padhye, S.; Dutta, S.; Padhye, S.; Newton, C.; Anson, C.E.; Powell, A.K. Transition metal
complexes of phenanthrenequinone thiosemicarbazone as potential anticancer agents: Synthesis, structure,
spectroscopy, electrochemistry and in vitro anticancer activity against human breast cancer cellline, T47D. J.
Inorg. Biochem. 2003, 95, 306−314.
33. Fouda, M.F.R.; Abd-Elzaher, M.M.; Shakdofa, M.M.; El-Saied, F.A.; Ayad, M.I.; el Tabl, A.S. Synthesis and
Characterization of a Hydrazone Ligand Containing Antipyrine and its Transition Metal Complexes. J. Coord.
Chem. 2008, 61, 1983−1996.

Molecules 2019, 24, 3313
23 of 25
34. Lever, A.B.P. Electronic Spectra of Some Transition Metal Complexes: Derivation of Dq and B. J. Chem. Educ.
1968, 45, 711−712.
35. Shebl, M.; El-ghamry, M.A.; Khalil, S.M.; kishk, M.A. Mono- and binuclear copper(II) complexes of new
hydrazone ligands derived from 4,6-diacetylresorcinol: Synthesis, spectral studies and antimicrobial activity.
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 21, 232−241.
36. Downes, J.M.; Whelan, J.; Bosnich, B. Biological analogs, Spectroscopic characteristics of mercapto- and
disulfide-copper(II) coordination in relation to type I proteins. Inorg. Chem. 1981, 20, 1081−1086.
37. Okawa, H.; Nakamoto, M.; Lzumitani, T.; Kida, S. Syntheses and Configurations of Copper(II) and Nickel(II)
Complexes of trans-1,2-Dicyano-1,2-cyclohexanediamine. Bull. Chem. Soc. 1982, 55, 2671−2672.
38. Suzuki, M.; Kanatomi, H.; Koyama, H.; Murase, I. Copper(I) and Copper(II) Complexes of Tripod Ligands,
Tris(2-alkylthioethyl)amine. Bull. Chem. Soc. 1980, 53, 1961−1964.
39. Kovala-Dermertzi, D.; Tsangaris, J.M.; Hadjiliadis, N. Complexes of aminobenzylamines. Part I. Complexes of
O-aminobenzylamine with copper(II), cobalt(II) and nickel(II). Transit. Met. Chem. 1983, 8, 140−146.
40. Suzuki, M.; Demura, H.K.Y.; Murase, I. Syntheses and Properties of Dinuclear Copper(II) Complexes with
2,6-Bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol. Bull. Chem. Soc. 1984, 57, 1003−1007.
41. El-Tabl, A.S.; El-Bahnasawy, R.M.; Hamdy, A.E.D. Synthesis, magnetic, spectral and antimicrobial studies on
metal complexes of 4- methylphenylaminoacetoisatin hydrazine. J. Chem. Res. 2009, 11, 659−664.
42. Chohan, Z.H.; Pervez, H.; Khan, K.M.; Supuran, C.T. Metal binding and antibacterial activity of ciprofloxacin
complexes. J. Enzym. Inhib. Med. Chem. 2005, 20, 303−307.
43. Lever, A.B.P. Inorganic Electronic Spectroscopy, Studies in Physical and Theoretical Chemistry; Elsevier Science Ltd.:
Amsterdam, The Netherlands, 1984.
44. Matovic, Z.D.; Miletic, V.D.; Samardzic, G.; Pelosi, G.; Ianelli, S.; Trifunovic, S. Square-planar copper(II)
complexes with a tetradentate amido-carboxylate ligands. Crystal structure of Na2[Cu(obap)]2⋅2H2O. Strain
analysis and spectral assignments of complexes. Inorg. Chim. Acta 2005, 358, 3135−3144.
45. Mangalam, N.A.; Kurup, M.R.P. Versatile binding properties of a di-2-pyridyl ketone nicotinoylhydrazone
ligand: Crystal structure of a Cu(II) complex. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2011, 78, 926−934.
46. Aly, M.M.; Imam, S.M. Characterization of Copper(II), nickel(II), cobalt(II) and palladium(II) complexes of
vicinal oxime-imine ligands; induced chelate isomerism in the same molecule of the nickel(II) complex. Mon.
Chem. Chem. Mon. 1995, 126, 173−185.
47. el Saied, F.A.; Shakdofa, M.M.E.; el Tabl, A.S.; Elzaher, M.M.A. Coordination behaviour of N′1,N′4-bis((1,
5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)methylene) succinohydrazide toward transition metal
ions and their antimicrobial activities. Main Group Chem. 2014, 13, 87−99.
48. Pahonțu, E.; Ilieș, D.C.; Shova, S.; Oprean, C.; Păunescu, V.; Olaru, O.T.; et al. Synthesis, Characterization,
Antimicrobial and Antiproliferative Activity Evaluation of Cu(II), Co(II), Zn(II), Ni(II) and Pt(II) Complexes with
Isoniazid-Derived Compound. Molecules. 2017, 22(4):650.
49. Earnshaw, A. Introduction to Magneto-Chemistry; Academic Press: New York, NY, USA, 1968.
50. Chandra, S.; Sharma, K.K. Cobalt(II) and nickel(II) complexes of some nitrogen-oxygen and nitrogen-sulphur
ligands. Transit. Met. Chem. 1984, 9, 1−3.
51. El-Tabl, A.S.; El-Saied, F.A.; Al-Hakimi, A.N. Synthesis, spectroscopic investigation and biological activity of
metal complexes with ONO trifunctionalalized hydrazone ligand. Transit. Met. Chem. 2007, 32, 689−701.
52. Fouda, M.F.R.; Abd-El-zaher, M.M.; Shakdofa, M.M.; El-Saied, F.A.; Ayad, M.I.; El-Tabl, A.S. Synthesis and
characterization
of
transition
metal
complexes
of
N′-[(1,
5-dimethyl-3-oxo-2-phenyl-2,
3-dihydro-1H-pyrazol-4-yl) methylene] thiophene-2-carbohydrazide. Transit. Met. Chem. 2008, 33, 219−228.
53. Al-Tabl, A.S.; AbouEl-Enein, S.A. Reactivity of the new potentially binucleating ligand,
2-(acetichydrazido-N-methylidene-α-naphthol)-benzothiazol, towards manganese (II), nickel (II), cobalt (II),
copper (II) and zinc (II) salts. J. Coord. Chem. 2004, 57, 281−294.
54. El-Reash,
G.M.A.;
Ibrahim,
K.M.;
Bekheit,
M.M.
Structural
studies
of
4(phenyl
or
benzamidos)-1-cyclohexa-none-3- thiosemicarbazone complexes. Transit. Met. Chem. 1990, 15, 148−151.
55. El-Saied, F.A.; El-Asmy, A.A.; Kaminsky, W.; West, D.X. Spectral and structural studies of cobalt(II,III),
nickel(II), and copper(II) complexes of dehydroacetic acid N4-dialkyl- and 3-azacyclothiosemicarbazones.
Transit. Met. Chem. 2003, 28, 954−960.

Molecules 2019, 24, 3313
24 of 25
56. El-Tabl, A.S.; Shakdofa, M.M.E.; El-Seidy, A.M.A.; Al-Hakimi, A.N. Synthesis, characterization and antifungal
activity of metal complexes of 2-(5-((2-chlorophenyl) diazenyl)-2-hydroxybenzylidene)
hydrazinecarbothioamide. Phosphorus Sulfur Silicon Relat. Elem. 2012, 187, 1312−1323.
57. Kasumov, V.T.; Köksal, F. Synthesis, ESR, UV-Visible and reactivity studies of new
bis(N-dimethoxyaniline-3,5-(t)Bu2-salicylaldiminato)copper(II) complexes. Spectrochim. Acta A Mol. Biomol.
Spectrosc. 2012, 98, 207−214.
58. Hathaway, B.J.; Billing, D.E. The electronic properties and stereochemistry of mono-nuclear complexes of the
copper(II) ion. Coord. Chem. Rev. 1970, 5, 1949.
59. Zaky, R.R.; Ibrahim, K.M.; Gabr, I.M. Bivalent transition metal complexes of o-hydroxyacetophenone
[N-(3-hydroxy-2-naphthoyl)] hydrazone: Spectroscopic, antibacterial, antifungal activity and thermogravimetric
studies. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2011, 81, 28−34.
60. Brown, D.R.; West, D.X. An ESR study of addition species formed between bis(2-thiopyridine N-oxide)Cu(II)
and heterocyclic and aliphatic amines. J. Inorg. Nucl. Chem. 1981, 43, 1017−1021.
61. Nagashri, K.; Joseph, J.; Dhanaraj, C.J. Copper (II) complexes of hydroxyflavone derivatives as potential
bioactive molecule to combat antioxidants: Synthesis, characterization and pharmacological activities. Appl.
Organomet. Chem. 2011, 25, 704−717.
62. Proctor, I.M.; Hathaway, B.J.; Nicholis, P. The electronic properties and stereochemistry of the copper(II) ion.
Part I. Bis(ethylenediamine)copper(II) complexes. J. Chem. Soc. A 1968, 1678−1684.
63. Kivelson, D.; Neiman, R. ESR Studies on the Bonding in Copper Complexes. J. Chem. Phys. 2004, 35, 149.
64. Shauib, N.M.; Elassar, A.Z.A.; El-Dissouky, A. Synthesis and spectroscopic characterization of copper(II)
complexes with the polydentate chelating ligand 4,4’-[1,4-phenylenedi(nitrilo)dipente-2-one. Spectrochim. Acta
Part A Mol. Biomol. Spectrosc. 2006, 63, 714−722.
65. Ray, R.K.; Kauffmann, G.B. An EPR study of copper(II)-substituted biguanide complexes. Part III. Inorg. Chim.
Acta 1990, 174, 237−244.
66. Hathaway, B.J. A new look at the stereochemistry and electronic properties of complexes of the copper(II) ion.
Complex Chem. 1984, 57, 55-118.
67. Sakaguchi, U.; Adison, A.W. Spectroscopic and redox studies of some copper(II) complexes with biomimetic
donor atoms: Implications for protein copper centres. J. Chem. Soc. Dalton Trans. 1979, 4, 600−608.
68. El-Tabl, A.S. Synthesis and spectral studies on mononuclear copper (II) complexes of isonitrosoacetylacetone
ligand. Polish J. Chem. 1997, 71, 1213−1222.
69. Mahajian, M.; Saxena, K.N.; Saxen, P.C. ESR study of Cu(II) complexes. Inorg. Nucl.Chem. 1981, 43, 2148−2149.
70. Oscar, C. Biopharmacological studies on certain new anticancer drugs. J. Biochem. 1988, 110, 701–707.
71. Tagmizyan, W.A. Metabolism of impaired glucose in patients with neoplasia. Am. Biol. 1990, 2, 53–58.
72. Silverstein, H.; Dervot, K.; Oscar, D. Studies on carbohydrate metabolism and different types of tumors bearing
animals. Lancet 1988, 22, 40–45.
73. Kind, P.R.N.; Gordon, M.; Laverick, M.; Nias, A.H.; Slavin, B.M. The effect of C3H mouse mammary tumor on
the levels of serum and urine analytes in vivo. Br. J. Cancer 1985, 52, 607–612.
74. Parveen, A.; Subedi, L.; Kim, H.W.; Khan, Z.; Zahra, Z.; Farooqi, M.Q.; Kim, S.Y. Phytochemicals Targeting
VEGF and VEGF-Related Multifactors as Anticancer Therapy. J. Clin. Med. 2019, 8, 350.
75. Abd-Alhaseeb, M.M.; Zaitone, S.A.; Abou-El-Ela, S.H.; Moustafa, Y.M. Olmesartan Potentiates the
Anti-Angiogenic Effect of Sorafenib in Mice Bearing Ehrlich’s Ascites Carcinoma: Role of Angiotensin (1–7).
PLoS ONE 2014, 9, e85891.
76. Liang, Y.; Brekken, R.A.; Hyder, S.M. Vascular endothelial growth factor induces proliferation of breast cancer
cells and inhibits the anti-proliferative activity of anti-hormones. Endocr. Relat. Cancer 2006, 13, 905–919.
77. Kumar, S.; Cieplak, P. Effect of phosphorylation and single nucleotide polymorphisms on caspase substrates
processing. Apoptosis 2018, 23, 194−200.
78. Hassan, M.; Watari, H.; AbuAlmaaty, A.; Ohba, Y.; Sakuragi, N. Apoptosis and Molecular Targeting Therapy in
Cancer. BioMed Res. Int. 2014, 2014, 150845.
79. Cao, Z.; Yang, Q.; Yin, H.; Qi, Q.; Li, H.; Sun, G.; Wang, H.; Liu, W.; Li, J. Peroxynitrite induces apoptosis of
mouse cochlear hair cells via a Caspase-independent pathway in vitro. Apoptosis 2017, 22, 1419−1430.
80. Vance, N.R.; Gakhar, L.; Spies, M.A. Allosteric Tuning of Caspase-7: A Fragment-Based Drug Discovery
Approach. Angew. Chem. Int. Ed. Engl. 2017, 56, 14443−14447.

Molecules 2019, 24, 3313
25 of 25
81. Zahran, M.A.-H.; El-Aarag, B.; Mehany, A.B.M.; Belal, A.; Younes, A.S. Design, synthesis, biological evaluations,
molecular docking, and in vivo studies of novel phthalimide analogs. Arch. Pharm. Chem. Life Sci. 2018, 351, 1–12.
82. Salem, M.L.; Shoukry, N.M.; Teleb, W.K.; Abdel-Daim, M.M.; Abdel-Rahman, M.A. In vitro and in vivo
antitumor effects of the Egyptian scorpion Androctonus amoreuxi venom in an Ehrlich ascites tumor model.
SpringerPlus 2016, 5, 570.
83. Marcelli, M.; Cunningham, G.R.; Walkup, M.; He, Z.; Sturgis, L.; Kagan, C.; Mannucci, R.; Nicoletti, I.; Teng, B.;
Denner, L. Signaling Pathway Activated during Apoptosis of the Prostate Cancer Cell Line LNCaP:
Overexpression of Caspase-7 as a New Gene Therapy Strategy for Prostate Cancer. Cancer Res. 1999, 59, 382–390.
84. Guirgis, A.A.; Zahran, M.; Mohamed, A.S.; Talaat, R.M.; Abdou, B.Y.; Agwa, H.S. Effect of thalidomide
dithiocarbamate analogs on the intercellular adhesion molecule-1 expression. Int. Immunopharmacol. 2010, 10,
805–811.
85. Zahra, M.H.; Salem, T.A.; El-Aarag, B.; Yosri, N.; EL-Ghlban, S.; Zaki, K.; Marei, A.H.; Abd El-Wahed, A.; Saeed,
A.; Khatib, A.; et al. Alpinia zerumbet (Pers.): Food and Medicinal Plant with Potential In Vitro and In Vivo
Anti-Cancer Activities. Molecules 2019, 24, 2495.
86. Papadopoulos, D.; Kimler, B.F.; Estes, N.C.; Durham, F.J. Growth delay effect of combined interstitial
hyperthermia and brachytherapy in a rat solid tumor model. Anticancer Res. 1989, 9, 45–47.
87. El-Aarag, B.; Hussein, W.; Ibrahim, W.; Zahran, M. Thymoquinone Improves Anti-Diabetic Activity of
Metformin in Streptozotocin-Induced Diabetic Male Rats. J. Diabetes Metab. 2017, 8, 780.
88. Chen, J.; Stavro, P.M.; Thompson, L.U. Dietary Flaxseed Inhibits Human Breast Cancer Growth and Metastasis
and Downregulates Expression of Insulin-Like Growth Factor and Epidermal Growth Factor Receptor. Nutr.
Cancer 2002, 43, 187–192.
Sample Availability: Samples of the compounds are available from the authors.
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