A Review on the Advancements in the Field of Metal Complexes with Schiff Bases as Antiproliferative Agents

: Metal complexes play an essential role in pharmaceutical sciences for their multiple and important activities. Schiff bases are versatile pharmacophores able to form chelating complexes with several metals in different oxidation states. Complexes with Schiff bases are widely described in the literature for their multiple actions and numerous advantages, such as low cost and easy synthesis. They show multiple biological activities, including antimicrobial, antioxidant, antimalarial, antinﬂammatory and antitumor. Schiff bases may also form complexes with lanthanides and actinides acting as catalysts (e.g., in various synthetic processes) and antitumor agents. This review intends to extend on our previous paper regarding Schiff bases as antitumorals, highlighting the importance, in the ﬁeld of the anticancer agents, of these tools as ligands of metal complexes.


Introduction
Schiff bases (R 1 R 2 C=NR 3 ) are interesting organic compounds containing an azomethine (-CH=N-) or an imine (-C=N-) group generally formed by the condensation of active carbonyl groups and amino compounds, in which the nitrogen atom is bonded to an aryl or alkyl group [1]. These compounds form highly stable complexes with transition metal ions [2]. Metal complexes in which the metal is coordinated to different ligands, able to stabilize the metal and modify its chemical and pharmaceutical properties, have gained considerable importance in medicinal chemistry [3] as antitumor agents [4][5][6][7]. Complexes containing the transition metals platinum, gold and silver have attracted much attention due to their antitumor effects. Schiff bases, as well as diarylureas [8][9][10], have been defined as "privileged" ligands in organic synthesis, thanks to their affordability, easy synthesis and different biological activities and ability to form complexes with almost all metals [11][12][13]. Actually, they may form complexes with transition metals, platinum group metals (PGM) [14], lanthanides [15,16], and actinides [17,18] and many of them have been extensively described in the literature [19,20]. Metal complexes bearing in the structure a Schiff base are known for their numerous applications and biological activities including antimicrobial [21], antioxidant [22], antitumor [23], antinflammatory [24], and ureases inhibitors [25], and occupy a central role in the development of coordination chemistry [26][27][28]. Recently, the study of these compounds has attracted interest particularly for the development of new anticancer drugs. Among them, a pyrazolone Schiff base copper complex has been suggested as a potential drug candidate for the treatment of liver cancer, being endowed with high antiproliferative activity against hepatocellular carcinoma (HCC) [29]. Moreover, the use of low frequency electromagnetic fields and Mn(II) complex of a Schiff base derived from the pyridoxal has been described for the treatment of breast cancer [30]. Finally, a recent study reported that the introduction of Schiff bases in the N-phenylcarbazole/triphenylamine modified half-sandwiched iridium(III) compound led to an enhancement of the antitumor activity of about thirteen times that of cisplatin [31]. Interestingly, Schiff base-Fe 2+ complexes have been incorporated into nanomicelles PAsc/Fe@Cy7QB, which represent chemodynamic theranostics able to specifically recognize and eradicate cancer cells in solid tumors [32]. Transition metal complexes with Schiff bases are also used as electrode modifiers in sensors to detect analytes of pharmaceutical, environmental and forensic interest, mainly to analyze illicit drugs [33]. Recently, the development of a coumarin Schiff base system has been described as a highly selective fluorescent/colorimetric probe for Cu 2+ and as a tumor biomarker for glutathione detection [34]. Herein, we want to focus the attention on the antitumor activity of complexes with Schiff bases, reviewing firstly the data regarding the antiproliferative activities of Schiff base complexes with transition metals; then, we will dwell on platinum group metals (PGM), such as Pt(II) and Au(III). Finally, we will deepen complexed with lanthanides and discuss their antiproliferative activities [35,36]. Particularly, we want to highlight the importance of the antiproliferative activity of the metal complex-based Schiff bases, as a significant starting point for the development of new anticancer drugs, giving an update of recent reviews regarding this relevant topic in medicinal chemistry [37][38][39][40][41][42].

Antiproliferative Activity of Schiff Bases Complexed with Transition Metals
Complexes of Schiff bases with transition metals are summarized in Table 1. Several complexes of Schiff bases with Cu(II), Co(II) and Ni(II) were studied as antitumor agents against colorectal adenocarcinoma (HT29) cells by Emam et al. (2017) [43]. The Co(II) complex 1 showed higher anticancer activity than its corresponding ligand, particularly being able to inhibit cancer cells growth of about the 83.22% after 72 h, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fetoh et al. (2019) [44] studied Schiff base complexes of Co(II) and Ni(II) ions with diethyl 3,3'-(ethane-1,2-diylbis(azaneylylidene))(3E,3 E)-dibutyrate (H 2 L 1 ) and (3E,3 E)-3,3'-(ethane-1,2-diylbis (azanylylidene))bis(N-(pyridin-2-yl)butanamide) (H 2 L 2 ) as ligands, and studied their antimicrobial and antiproliferative activity.  [45] studied five transition metal complexes TM 3 L 2 (OAc) 2 (TM = transition metal) with the N,N -bis[(2-hydroxy-1-naphthalenyl)methylene]-propane-1,3-diamine, which is a bis-Schiff base, and determined the in vitro anticancer activities against seven human cancer cell lines (namely, gastric cancer MGC80-3, HeLa, human bladder T-24, HepG2, MDA, A549, human hepatocellular carcinoma Bel-7402 and human lung Wi-38 cancer cell lines), by the MTT assay. The complexes with Cu, Ni and Fe (4)(5)(6) showed higher antitumor activities than cisplatin and the highest inhibitory effects were found for complex 4 (IC 50 lower than 0.5 µM for human bladder cancer cell line T-24) while showing nearly no toxicity to the normal cell HL-7702 as revealed by flow cytometry. The complex 4 induced tumor cell apoptosis by reducing the Bcl-2/Bax ratio, blocking the T-24 tumor cells at the G2/M phase, reducing the mitochondrial membrane potential, increasing the intracellular concentration of reactive oxygen species (ROS) and Ca 2+ , and changing protein expression. The apoptosis induced by complex 4 has been found to be triggered by Caspases 3/8 activation in T24 cells. Hassan et al. (2020) [46] synthesized the metal complexes of a tridentate Schiff base ligand (E)-1-(2-hydroxy-3-methoxybenzylidene)-3-phenylurea with Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Zr(IV) (7-9 are shown in Table 1). The ligand and its metal complexes were screened for their cytotoxic activity against two cell lines, i.e., human colon carcinoma (HCT-116) and breast carcinoma cells (MCF-7), using cisplatin as standard drug and obtaining IC 50 values of 7.22 µg/mL and 6.9 µg/mL against MCF-7 and HCT-116, respectively. All the complexes were more active than the free Schiff base ligand and, in particular, the higher cytotoxic activity was exerted by Cu, Co and Zn complexes (IC 50 [48] reported the synthesis and biological evaluation of new Schiff bases incorporating sulfisoxazole and sulfamethoxazole and their Cu(II) complexes. The anticancer activity was evaluated against three human cancer cell lines with the sulfonamide B test. Complexes of ligand S1M-S1 with Cu(II) named Cu(S2M-S1) 2 (11) and Cu(S1M-S1) 2 (12) ( Table 1) exhibited promising cytotoxic activity against breast (MCF7) cells (IC 50 = 40 µM versus 5-fluorouracil, 5-FU, IC 50 = 0.10 µM). Moreover, docking studies were carried out on these compounds, suggesting that hydrogen bonds seem to play a pivotal role in the reversible interaction of small molecules and proteins. In this case, the oxygen and nitrogen atoms on the sulfisoxazole ring, the oxygen atom of sulfonyl groups and oxygen and nitrogen atoms attached to the benzene ring are thought to be involved in the hydrogen bonds' formation. Ismail et al. (2021) [49] studied several complexes in which the N 1 ,N 2 -bis(furan-2-ylmethylene)-4-methylbenzene-1,2-diamine (L) is complexed with several metals, namely Ag(I), Cr(III), Fe(III), Co(II), Cu(II) and Cd(II) (13)(14)(15)(16)(17)(18), and studied their cytotoxicity against the HepG2 cell lines. The IC 50 values were 12.9 µg/mL, 14.8 µg/mL, 7.31 µg/mL, 8.53 µg/mL, 17.1 µg/mL and 1.95 µg/mL, respectively. The cadmium(II) complex showed the highest cytotoxic activity, being more potent than vinblastine used as standard drug (IC 50 = 2.93 µg/mL). Its activity was also higher with respect to that of the free ligand, suggesting an important role of the metal in the biological activity. Wongsuwan et al. (2021) [50] studied the in vitro anticancer activity of the Schiff base ligands (N-(8quinolyl)-X-salicylaldimine) and their complexes with Fe(II) and Fe(III) against the A549 human lung adenocarcinoma cell line. The Fe(III) complex (compound 19, Table 1) showed an anticancer activity against the A549 cells (IC 50 = 10 µM) higher than two well-known anticancer agents, i.e., etoposide and cisplatin (IC 50 = 19 and 16 µM, respectively). Naureen et al. (2021) [51] analyzed two Schiff base ligands (L1, L2) and their complexes with Fe(III) and Zn(II) (20,21), studying their antitumor activity by means of potato disc tumor induction assay utilizing the Agrobacterium tumefaciens strains. In this assay, the complexes of the two Schiff bases with Zn showed a better activity than the corresponding Fe compounds: ((L1) 2 Zn(Ac) 2 IC 50 = 6.81 µg/mL and (L2) 2 Zn(Ac) 2 IC 50 = 7.58 µg/mL versus (L1) 2 FeCl 3 IC 50 = 13.91 µg/mL and (L2) 2 FeCl 3 IC 50 = 70.89 µg/mL). Vincristine was used as positive control with an IC 50 value of 250 µg/mL. Additionally, the safety of these compounds was also evaluated, using the brine shrimp microwell cytotoxicity assay. The obtained results confirmed that Zn complexes showed a higher cytotoxicity than the corresponding Fe ones (IC 50 > 134 µg/mL); moreover, in both the assays the ligands were more pharmacologically active, as well. Alkiş Table 1 whereas the latter will be described below in Table 2. The compounds were studied for their in vitro anticancer activities against the human colon cancer cell line (Caco-2) by using the MTT assay and investigating the biocompatibility characteristics in the normal fibroblast cells line (L-929). Moreover, the effectiveness of electrochemotherapy (ECT) on cytotoxic activities of the compounds in Caco-2 cancer cell line was examined. Both the complexes showed interesting anticancer properties in the Caco-2 cells (IC 50 = 610 µM for Co complex) while showing a low cytotoxic effect on L-929. Moreover, the authors demonstrated that the combined application of electroporation (EP)+complexes was much more effective than the application of complexes alone in the treatment of Caco-2 colon cancer cells. The Co(II) complex increased its cytotoxicity levels by 2.07 times in its combined applications with EP.   [53] (28). The cytotoxicity was evaluated against HeLa, human colon cancer cells (LoVo), A549 and the cisplatin-resistant cell line (A549/cis) by MTT assays using cisplatin as a positive reference. The cytotoxicity against the normal cell lines LO2 was also evaluated. Complex M3 effectively inhibited the growth of all the tested cancer cells in a dose-dependent manner during a 48 h treatment. The IC 50 against HeLa was close to that of cisplatin (IC 50 = 12.40 µM versus 9.74 <µM). The activity of the complex against the cisplatin-resistant cell line A549/cis was higher than the reference (IC 50 = 18.03 µM versus 44.79 µM). Moreover, the complex 28 seems to be less toxic than cisplatin (IC 50 = 6.27 µM versus 2.61 µM) and with a better selectivity index (SI). Furthermore, anticancer mechanistic studies showed that the complex 28 inhibited cell proliferation by blocking DNA synthesis and acting on nuclear division of HeLa cells over time.             Cu(S1M-S1) 2 (12) IC 50

Antiproliferative Activity of Schiff Bases Complexed with Platinum Group Metals (PGM)
Platinum group metals are represented by Ru, Rh, Pd, Os, Ir and Pt and can be coordinated by Schiff base ligands as shown in Table 2

Antiproliferative Activity of Schiff Bases Complexed with Platinum Group Metals (PGM)
Platinum group metals are represented by Ru, Rh, Pd, Os, Ir and Pt and can be coordinated by Schiff base ligands as shown in Table 2

Antiproliferative Activity of Schiff Bases Complexed with Platinum Group Metals (PGM)
Platinum group metals are represented by Ru, Rh, Pd, Os, Ir and Pt and can be coordinated by Schiff base ligands as shown in Table 2. Mbugua et al. (2017) [56] reported a study on palladium(II) and platinum(II) complexes based on pyrrole Schiff bases and their anticancer activity against various human cancerous (Caco-2, HeLa, HepG2, MCF-7, and PC-3) and noncancerous (MCF-12A) cell lines using MTT and apoptosis assays, respectively. The 29 complex showed an enhanced and selective cytotoxicity particularly against the HepG2 (IC 50 = 0.3 µg/mL) and Caco-2 (IC 50 = 16.63 µg/mL) cells. It reduced the viability of the five cancerous cell lines, which included one breast cancer cell line, by more than 60%.  [52] has been already described above with regard to the Co(II) complex. The other complex, with Ru(II) [RuCl(p-cymene)L] (42), showed an in vitro higher activity than the Co(II) analogue complex (22), showing an IC 50 value of 510 µM against the human colon cancer cell line (Caco-2) and low cytotoxic effects on L-929, as determined by MTT assay. Furthermore, as well this time, Ru(II) complexes increased the cytotoxicity levels by 2.12 times in its combined applications with EP.

Antiproliferative Activity of Schiff Base Complexed with Lanthanides
Lanthanide-based complexes with Schiff bases are depicted in Table 3. Andiappan et al. (2018) [61] studied a series of lanthanides (i.e., praseodymium (Pr), erbium (Er), and ytterbium (Yb)) based with a Schiff base ligand (SBL), the N 2 ,N 3 -bis(anthracen-9-ylmethylene) pyridine-2,3-diamine and studied their cytotoxicity activity against Vero, human breast cancer (MCF7), and cervical (HeLa) anticancer cell lines by MTT assay. The SBLPr (43) and SBLEr (44) complexes exhibited anticancer activity against the above-mentioned cancer cell lines, reducing their viability by about 50% at a concentration of 25 µg/mL). Both the complexes were shown to induce the apoptosis of MCF7 and HeLa cells as observed by the acridine orange (AO)/ethidium bromide (EB) staining assay. a further confirmation was obtained by the propidium iodide (PI) staining assay that showed that both the complexes induced DNA fragmentation. Sathiyanarayanan et al. (2020) [62] described a series of lanthanum(III) complexes with a Schiff base, particularly praseodymium (Pr), samarium (Sm), and ytterbium (Yb) (45, 46 and 47, respectively). In vitro antiproliferative activity studies by MTT assay showed that the compounds were active against HeLa tumor cells, the compound 46 being the most active with an IC 50 equal to 34 µg/mL. Hoechst 33,258 staining and AO/EB dual staining suggested that the cells underwent the apoptosis mechanism in a dose-dependent manner and the flow cytometric analysis evidenced a block at the G1 phase, where there was no cell replication. Moreover, the docking studies suggested that the anti-cancer effect of complex 46 may occur via the caspase 3-specific substrate activation (polyADP-ribose polymerase, PARP, cleavage). The complex 46 binds DNA via intercalation producing an arrest at the S-phase of HeLa cells. Hua et al. (2021) [63] studied a hexanuclear Nd(III) complex {[Nd 6 (HL) 2 L 2×2 (NO 3 ) 8 (EtOH) 6 ](EtOH)(H 2 O) 3 } (48) (given the complexity of the structure it has not been reported herein and only the H 2 L and H 2 X ligands are depicted in Table 3) and studied its antitumor activity against the human hepatocellular carcinoma SMMC-7721 cells by MTT assay. It showed a higher activity than the reference compound (IC 50 = 4.42 µM versus 10.11 µM of gemcitabine).

Summary
The complexes of Schiff base are attracting much attention for their biological activities, including antibacterial, antifungal, anti-inflammatory, antioxidant and anticancer. The main aim of this review was to explore and summarize the available recent research studies regarding the Schiff bases' metal complexes as antitumorals. We considered complexes with transition metals, with PGM and lanthanides, focusing on the latest studies conduced by different authors in recent years. Some of them showed interesting antiproliferative activity against a large panel of cancer cell lines (MCF7, HeLa, HepG2, HCT-116, and so on) even higher than the adopted reference drugs. Most of their anticancer activities have been investigated using well-known and validated viability assays, revealing IC50 values equal or even lower than the clinical used drugs, used as references, principally the cisplatin. In many cases, a better cytotoxic profile has been recorded, the viability of the normal cells being not or little affected. In all cases, the authors prove that these complexes are able to induce cancer cell death by apoptosis, regulating the principal actors playing a specific role in initiating/sustaining the apoptotic mechanism. Moreover, in the majority of cases DNA represents the main target, as demonstrated by in vitro studies using the CTDNA as simple, but effective, assay to determine DNA intercalation properties. Some of these studies have been supported by in silico proofs, in which the ability of the specific groups of these complexes to bind DNA has been supposed. Finally, interesting evidence about a role in regulating the cell cytoskeleton targeting the actin have been reported. This makes us confident that many Schiff bases complexed with metals could represent promising molecules to develop new and suitable therapies for the treatment of numerous kinds of cancers, some of which with high incidence rates.

Summary
The complexes of Schiff base are attracting much attention for their biological activities, including antibacterial, antifungal, anti-inflammatory, antioxidant and anticancer. The main aim of this review was to explore and summarize the available recent research studies regarding the Schiff bases' metal complexes as antitumorals. We considered complexes with transition metals, with PGM and lanthanides, focusing on the latest studies conduced by different authors in recent years. Some of them showed interesting antiproliferative activity against a large panel of cancer cell lines (MCF7, HeLa, HepG2, HCT-116, and so on) even higher than the adopted reference drugs. Most of their anticancer activities have been investigated using well-known and validated viability assays, revealing IC50 values equal or even lower than the clinical used drugs, used as references, principally the cisplatin. In many cases, a better cytotoxic profile has been recorded, the viability of the normal cells being not or little affected. In all cases, the authors prove that these complexes are able to induce cancer cell death by apoptosis, regulating the principal actors playing a specific role in initiating/sustaining the apoptotic mechanism. Moreover, in the majority of cases DNA represents the main target, as demonstrated by in vitro studies using the CTDNA as simple, but effective, assay to determine DNA intercalation properties. Some of these studies have been supported by in silico proofs, in which the ability of the specific groups of these complexes to bind DNA has been supposed. Finally, interesting evidence about a role in regulating the cell cytoskeleton targeting the actin have been reported. This makes us confident that many Schiff bases complexed with metals could represent promising molecules to develop new and suitable therapies for the treatment of numerous kinds of cancers, some of which with high incidence rates.