Thiazole Functionalization of Thiosemicarbazone for Cu(II) Complexation: Moving toward Highly Efficient Anticancer Drugs with Promising Oral Bioavailability

In this work, we report the synthesis of a new thiosemicarbazone-based drug of N′-(di(pyridin-2-yl)methylene)-4-(thiazol-2-yl)piperazine-1-carbothiohydrazide (HL) featuring a thiazole spectator for efficient coordination with Cu(II) to give [CuCl(L)]2 (1) and [Cu(NO3)(L)]2 (2). Both 1 and 2 exhibit dimeric structures ascribed to the presence of di-2-pyridylketone moieties that demonstrate dual functions of chelation and intermolecular bridging. HL, 1, and 2 are highly toxic against hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7 with half maximal inhibitory concentration (IC50) values as low as 3.26 nmol/mL (HL), 2.18 nmol/mL (1), and 2.54 × 10−5 nmol/mL (2) for PLC/PRF/5. While the free ligand HL may elicit its anticancer effect via the sequestration of bio-relevant metal ions (i.e., Fe3+ and Cu2+), 1 and 2 are also capable of generating cytotoxic reactive oxygen species (ROS) to inhibit cancer cell proliferation. Our preliminary pharmacokinetic studies revealed that oral administration (per os, PO) of HL has a significantly longer half-life t1/2 of 21.61 ± 9.4 h, nearly doubled as compared with that of the intravenous (i.v.) administration of 11.88 ± 1.66 h, certifying HL as an effective chemotherapeutic drug via PO administration.


Introduction
Thiosemicarbazones (TSCs) are a class of Schiff base derivatives that exhibit diverse biologically beneficial activities, such as antibacterial, antiviral, and enzyme inhibitory activities [1,2].Most notably, TSCs have long been considered as a potential class of anticancer drug candidates for a broad spectrum of cancers through a wide range of action mechanisms, such as ribonuclease reductase and topoisomerase II inhibition [3][4][5][6].Other mechanisms include the quick sequestration of cell-proliferative-dependent ions (i.e., Fe 3+ and Cu 2+ ) to elicit the anticancer effect [4].The anticancer effect of TSCs is primarily linked to, and profoundly affected by, their structures that feature an S, N chelator [4,5].In this regard, the additional introduction of an N coordination site, such as α-pyridyl, serves as an advantage, as it forms a more stable N, N, S pincer-like chelation that results in more effective ion removal [7].In this sense, TSCs are particularly effective for treating iron overload diseases such as leukemia and neuroblastoma [8].Recently, findings have also suggested that, upon the capturing of metal ions by TSCs, a different anticancer mechanism based on reactive oxygen species (ROS) chemistry can be commenced by taking advantage of the unique characteristics of the tumor microenvironment (TME), such as the overexpression of H 2 O 2 and GSH [9][10][11][12].The ROS generation can subsequently strengthen the chemotherapeutic outcome of TSCs.

Results and Discussion
2.1.Synthesis and X-ray Structure Characterization of HL, [CuCl(L)] 2 (1), and [Cu(NO 3 )(L)] 2 (2) The synthesis of ligand HL was achieved by a five-step protocol to give an overall yield of 9.3% (Scheme 2).The purity of HL was confirmed by microanalysis, 1 H, and 13 C nuclear magnetic resonance (NMR) spectroscopy (Figure S1).The assumed connectivity of HL was also unambiguously determined by single-crystal X-ray diffraction studies (Figure 1a).Coordination complexes of 1 and 2 were prepared accordingly by reacting HL and the respective Cu(II) sources under appropriate conditions (Scheme 3).The single-crystal X-ray structure data for 1 (Figure 1b) and 2 (Figure 1c) indicated that both compounds form dimeric structures in the solid state, enabled by the head-to-tail complementary Cu−N coordination.In 1, each Cu 2+ shows a square pyramidal coordination geometry, fulfilled by an S, one azomethinic N, one α-pyridyl N atom from an L ligand, and one Cl − that defines the square plane, in addition to one pyridyl N atom from adjacent L ligand that occupies the apical position.A similar connectivity is also found in 2, except that one  Coordination complexes of 1 and 2 were prepared accordingly by reacting HL and the respective Cu(II) sources under appropriate conditions (Scheme 3).The single-crystal X-ray structure data for 1 (Figure 1b) and 2 (Figure 1c) indicated that both compounds form dimeric structures in the solid state, enabled by the head-to-tail complementary Cu−N coordination.In 1, each Cu 2+ shows a square pyramidal coordination geometry, fulfilled by an S, one azomethinic N, one α-pyridyl N atom from an L ligand, and one Cl − that defines the square plane, in addition to one pyridyl N atom from adjacent L ligand that occupies the apical position.A similar connectivity is also found in 2, except that one Coordination complexes of 1 and 2 were prepared accordingly by reacting HL and the respective Cu(II) sources under appropriate conditions (Scheme 3).The single-crystal X-ray structure data for 1 (Figure 1b) and 2 (Figure 1c) indicated that both compounds form dimeric structures in the solid state, enabled by the head-to-tail complementary Cu−N coordination.In 1, each Cu 2+ shows a square pyramidal coordination geometry, fulfilled by an S, one azomethinic N, one α-pyridyl N atom from an L ligand, and one Cl − that defines the square plane, in addition to one pyridyl N atom from adjacent L ligand that occupies the apical position.A similar connectivity is also found in 2, except that one Cl − is replaced by a chelating NO 3 − , yielding a distorted octahedral coordination geometry for Cu 2+ .
Notably, the ligands in both 1 and 2 undergo a change of conjugation with the loss of hydrazinic proton and the conversion of C=S into a C−S bond.Such a structure variation is seen in a variety of transition metal complexes of α-pyridyl thiosemicarbazone chelators, such as Triapine complexes of Fe 3+ /Ga 3+ [17,18], di-2-pyridyl ketone 4,4dimethyl-3-thiosemicarbazone (HDp44mT) complexes of Fe 3+ [19], as well as several Cu 2+ complexes.[20] Bormio Nunes et al. also reported that, for Fe 3+ /Cu 2+ complexes of COTI-2, the N−N single bond of the ligand is inherited in the final chelating complexes.[16] As a result of the C=S to C−S bond variation, the C=S bond distance in HL of 1.681(5) Å increased to 1.739(4) Å in 1 and 1.744(6) Å in 2 (Table S1).Nevertheless, the N−N distance in HL of 1.354(5) Å remained nearly unaltered as compared with the N=N distances in 1 (1.354(4)Å) and 2 (1.351(6) Å) due to the additional coordination of one N to Cu 2+ .Scheme 3. The synthetic procedure for 1 and 2 from HL.

Spectroscopic Characterizations of HL, 1, and 2
The energy-dispersive X-ray spectroscopy (EDS) reveal an atomic ratio of Cu:Cl:S of 2.0:2.2:4.2 in 1 and Cu:S of 2.0:4.6 in 2 (Figure S2), consistent with the derived ratio of Cu:Cl:S of 1.0:1.0:2.0 (1) and Cu:S of 1.0:2.0(2) from the single-crystal data, and the elements Cu, S, N, and C are evenly distributed in their elemental mapping diagrams (Figure S2).The Fourier transform infrared (FT-IR) spectra show that upon coordination with Cu 2+ , the C=N bond vibration peaks at 1571 cm −1 in HL and shifts to 1593 cm −1 for both 1 and 2 (Figure S3).Meanwhile, bands at 850 cm −1 (1) and 856 cm −1 (2), which are assignable as C=S bond vibrations, also shift as compared with that of HL (869 cm −1 ) upon bonding with Cu 2+ .[21,22] In the ultraviolet-visible (UV-Vis) spectroscopy (Figure 2), ligand-to-metal charge transfer (LMCT) bands in 1 and 2 are found at 426 nm as compared with HL. [23] Meanwhile, the ligand-centered UV-Vis absorption band of the HL undergoes a significant blue shift due to the conjugation change and charge transfer reactions.The LMCT leads to the partial reduction of Cu 2+ , which is favorable for Cu +induced reactions, such as the ROS generation via the Fenton-like process.[24] The Cu 2p X-ray photoelectron spectroscopy (XPS) of both 1 and 2 revealed two sets of binding energies (Figure 3).Peaks at 933.8/953.5 eV (1) and 933.8/953.8eV (2) are assignable as the spin-orbit splitting of Cu 2p3/2 and 2p1/2 of Cu 2+ .[25][26][27] Two satellite peaks at 943.3/963.3eV (1) and 943.4/963.0eV (2) are also attributed to Cu 2+ ions due to its d 9 electronic configuration, which is susceptible to photoreduction to give a more stable d 10 Scheme 3. The synthetic procedure for 1 and 2 from HL.
Notably, the ligands in both 1 and 2 undergo a change of conjugation with the loss of hydrazinic proton and the conversion of C=S into a C−S bond.Such a structure variation is seen in a variety of transition metal complexes of α-pyridyl thiosemicarbazone chelators, such as Triapine complexes of Fe 3+ /Ga 3+ [17,18], di-2-pyridyl ketone 4,4-dimethyl-3-thiosemicarbazone (HDp44mT) complexes of Fe 3+ [19], as well as several Cu 2+ complexes [20].Bormio Nunes et al. also reported that, for Fe 3+ /Cu 2+ complexes of COTI-2, the N−N single bond of the ligand is inherited in the final chelating complexes [16].As a result of the C=S to C−S bond variation, the C=S bond distance in HL of 1.681(5) Å increased to 1.739(4) Å in 1 and 1.744(6) Å in 2 (Table S1).Nevertheless, the N−N distance in HL of 1.354(5) Å remained nearly unaltered as compared with the N=N distances in 1 (1.354(4)Å) and 2 (1.351(6) Å) due to the additional coordination of one N to Cu 2+ .

Spectroscopic Characterizations of HL, 1, and 2
The energy-dispersive X-ray spectroscopy (EDS) reveal an atomic ratio of Cu:Cl:S of 2.0:2.2:4.2 in 1 and Cu:S of 2.0:4.6 in 2 (Figure S2), consistent with the derived ratio of Cu:Cl:S of 1.0:1.0:2.0 (1) and Cu:S of 1.0:2.0(2) from the single-crystal data, and the elements Cu, S, N, and C are evenly distributed in their elemental mapping diagrams (Figure S2).The Fourier transform infrared (FT-IR) spectra show that upon coordination with Cu 2+ , the C=N bond vibration peaks at 1571 cm −1 in HL and shifts to 1593 cm −1 for both 1 and 2 (Figure S3).Meanwhile, bands at 850 cm −1 (1) and 856 cm −1 (2), which are assignable as C=S bond vibrations, also shift as compared with that of HL (869 cm −1 ) upon bonding with Cu 2+ [21,22].In the ultraviolet-visible (UV-Vis) spectroscopy (Figure 2), ligand-to-metal charge transfer (LMCT) bands in 1 and 2 are found at 426 nm as compared with HL [23].Meanwhile, the ligand-centered UV-Vis absorption band of the HL undergoes a significant blue shift due to the conjugation change and charge transfer reactions.The LMCT leads to the partial reduction of Cu 2+ , which is favorable for Cu + -induced reactions, such as the ROS generation via the Fenton-like process [24].The Cu 2p X-ray photoelectron spectroscopy (XPS) of both 1 and 2 revealed two sets of binding energies (Figure 3).Peaks at 933.8/953.5 eV (1) and 933.8/953.8eV (2) are assignable as the spin-orbit splitting of Cu 2p 3/2 and 2p 1/2 of Cu 2+ [25][26][27].Two satellite peaks at 943.3/963.3eV (1) and 943.4/963.0eV (2) are also attributed to Cu 2+ ions due to its d 9 electronic configuration, which is susceptible to photoreduction to give a more stable d 10 configuration.The liquid chromatography-mass spectrometry (LC-MS) for both 1 (Figure S4a) and 2 (Figure S4b) show peaks at 470.9 m/z and 488.8 m/z, assignable as the

Hydroxyl Radical (•OH) Production
The generation of •OH from H2O2 by HL, 1, and 2 was assayed by 3,3′,5,5′tetramethylbenzidine (TMB).[12,28,29] The colorless TMB can be quickly oxidized by •OH to give a characteristic blue charge-transfer complex oxTMB with intense absorption at 652 nm.[30,31] Different concentrations of HL (5, 10, 20, 30 µg/mL) or 1 and 2 containing equivalent HL were treated with (TMB) solution containing 100 µM H2O2, which is assumed as approximately the concentration reported to be present in tumor cells, or five times that in normal cells.[32,33] After 20 min of incubation with HL, there is no oxTMB found, as suggested by both the UV-Vis results and the direct observation of solution colors (Figure S5).In sharp contrast, both 1 and 2 can induce the generation of •OH under otherwise identical conditions in a concentration-dependent manner, with 2 further outperforming 1 (Figures 4 and S5).In addition, the MeOH solutions of HL, 1, and 2 at

Hydroxyl Radical (•OH) Production
The generation of •OH from H2O2 by HL, 1, and 2 was assayed by 3,3′,5,5′tetramethylbenzidine (TMB).[12,28,29] The colorless TMB can be quickly oxidized by •OH to give a characteristic blue charge-transfer complex oxTMB with intense absorption at 652 nm.[30,31] Different concentrations of HL (5, 10, 20, 30 µg/mL) or 1 and 2 containing equivalent HL were treated with (TMB) solution containing 100 µM H2O2, which is assumed as approximately the concentration reported to be present in tumor cells, or five times that in normal cells.[32,33] After 20 min of incubation with HL, there is no oxTMB found, as suggested by both the UV-Vis results and the direct observation of solution colors (Figure S5).In sharp contrast, both 1 and 2 can induce the generation of •OH under otherwise identical conditions in a concentration-dependent manner, with 2 further

Hydroxyl Radical (•OH) Production
The generation of •OH from H 2 O 2 by HL, 1, and 2 was assayed by 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) [12,28,29].The colorless TMB can be quickly oxidized by •OH to give a characteristic blue charge-transfer complex oxTMB with intense absorption at 652 nm [30,31].Different concentrations of HL (5, 10, 20, 30 µg/mL) or 1 and 2 containing equivalent HL were treated with (TMB) solution containing 100 µM H 2 O 2 , which is assumed as approximately the concentration reported to be present in tumor cells, or five times that in normal cells [32,33].After 20 min of incubation with HL, there is no oxTMB found, as suggested by both the UV-Vis results and the direct observation of solution colors (Figure S5).In sharp contrast, both 1 and 2 can induce the generation of •OH under otherwise identical conditions in a concentration-dependent manner, with 2 further outperforming 1 (Figures 4 and S5).In addition, the MeOH solutions of HL, 1, and 2 at HL/L concentrations of 30 µg/mL remained stable upon keeping for 72 h (Figure S6), as evidenced by their unaltered UV-Vis absorption patterns throughout the experiments.

CCK-8 Assay for HL, 1, and 2
The cytotoxicity of HL, 1, and 2 against three hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7 were evaluated by the cell counting kit-8 (CCK-8; APExBIO Technology, Houston, TX, USA) assay.As shown in Figure 5, all three complexes demonstrate superior cytotoxicity originating from the ligand, particularly for the PLC/PRF/5 cell line.Notably, 1 and 2 demonstrated higher cytotoxicity as compared with HL, presumably due to the additional ROS generation induced by the overexpressed H2O2 in the cancer cells.The IC50 values for HL, 1, and 2 against PLC/PRF/5 were as low as 3.26 nmol/mL, 2.18 nmol/mL, and 2.54 × 10 −5 nmol/mL, which is favorably lower than those against the Hep-G2 (78.83 nmol/mL for HL, 38.11 nmol/mL for 1, and 16.86 nmol/mL for 2) and HuH-7 (192.20 nmol/mL for HL, 176.60 nmol/mL for 1, and 87.53 nmol/mL for 2) cell lines (Table S2).

Intracellular ROS Generation by HL, 1, and 2
Given the efficient •OH production in water induced by 1 and 2, we next investigated in vitro ROS generation by 1 and 2 in the representative HuH-7 cell line using 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe and compared it with that of HL. [34,35] The non-emissive DCFH-DA can be readily uptaken by the cell, where it is hydrolyzed into 2,7-dichlorodihydrofluorescein (DCFH) by cellular enzymes.Upon oxidation by ROS, DCFH can convert to 2,7-dichlorofluorescein (DCF), which gives bright green fluorescence.[29,36] As shown in Figure 6, HL alone produced negligible

CCK-8 Assay for HL, 1, and 2
The cytotoxicity of HL, 1, and 2 against three hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7 were evaluated by the cell counting kit-8 (CCK-8; APExBIO Technology, Houston, TX, USA) assay.As shown in Figure 5, all three complexes demonstrate superior cytotoxicity originating from the ligand, particularly for the PLC/PRF/5 cell line.Notably, 1 and 2 demonstrated higher cytotoxicity as compared with HL, presumably due to the additional ROS generation induced by the overexpressed H 2 O 2 in the cancer cells.The IC 50 values for HL, 1, and 2 against PLC/PRF/5 were as low as 3.26 nmol/mL, 2.18 nmol/mL, and 2.54 × 10 −5 nmol/mL, which is favorably lower than those against the Hep-G2 (78.83 nmol/mL for HL, 38.11 nmol/mL for 1, and 16.86 nmol/mL for 2) and HuH-7 (192.20 nmol/mL for HL, 176.60 nmol/mL for 1, and 87.53 nmol/mL for 2) cell lines (Table S2).

CCK-8 Assay for HL, 1, and 2
The cytotoxicity of HL, 1, and 2 against three hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7 were evaluated by the cell counting kit-8 (CCK-8; APExBIO Technology, Houston, TX, USA) assay.As shown in Figure 5, all three complexes demonstrate superior cytotoxicity originating from the ligand, particularly for the PLC/PRF/5 cell line.Notably, 1 and 2 demonstrated higher cytotoxicity as compared with HL, presumably due to the additional ROS generation induced by the overexpressed H2O2 in the cancer cells.The IC50 values for HL, 1, and 2 against PLC/PRF/5 were as low as 3.26 nmol/mL, 2.18 nmol/mL, and 2.54 × 10 −5 nmol/mL, which is favorably lower than those against the Hep-G2 (78.83 nmol/mL for HL, 38.11 nmol/mL for 1, and 16.86 nmol/mL for 2) and HuH-7 (192.20 nmol/mL for HL, 176.60 nmol/mL for 1, and 87.53 nmol/mL for 2) cell lines (Table S2).

Intracellular ROS Generation by HL, 1, and 2
Given the efficient •OH production in water induced by 1 and 2, we next investigated in vitro ROS generation by 1 and 2 in the representative HuH-7 cell line using 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe and compared it with that of HL. [34,35] The non-emissive DCFH-DA can be readily uptaken by the cell, where it is hydrolyzed into 2,7-dichlorodihydrofluorescein (DCFH) by cellular enzymes.Upon oxidation by ROS, DCFH can convert to 2,7-dichlorofluorescein (DCF), which gives bright green fluorescence.[29,36] As shown in Figure 6, HL alone produced negligible

Intracellular ROS Generation by HL, 1, and 2
Given the efficient •OH production in water induced by 1 and 2, we next investigated in vitro ROS generation by 1 and 2 in the representative HuH-7 cell line using 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe and compared it with that of HL [34,35].The non-emissive DCFH-DA can be readily uptaken by the cell, where it is hydrolyzed into 2,7-dichlorodihydrofluorescein (DCFH) by cellular enzymes.Upon oxidation by ROS, DCFH can convert to 2,7-dichlorofluorescein (DCF), which gives bright green fluorescence [29,36].As shown in Figure 6, HL alone produced negligible ROS.In sharp contrast, 1 and 2 can induce the formation of strong fluorescence, characterizing the effective generation of ROS in the HuH-7 cells.

In Vivo Pharmacokinetic Study of HL
For pharmacokinetic studies, CD ® (SD) IGS rats (5−6 weeks old) were randomly divided into two groups (three for each group) for oral administration (per os, PO) or intravenous administration (i.v.) of HL with dosages of 30 mg kg −1 and 0.5 mg kg −1 , respectively (Tables S3 and S4).The blood was then collected from the orbital sinus with a heparinized syringe at different time intervals (0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for PO, and 0.03, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for i.v.).High-performance liquid chromatography (HPLC) provided mean area under the curve (AUC0-inf) values of 10409.01 ± 5313.4 ng h mL −1 for PO and 5701.36 ± 2647.15 ng h mL −1 for i.v.HL achieved the largest Cmax value of 425.93 ± 176.1 ng mL −1 at a Tmax of 1.0 h for PO, while for i.v., the Cmax value of 806.43 ± 46.66 ng mL −1 was immediately observed at Tmax of 0.03 h, which is expected for i.v.administration.Notably, the PO administration exhibited a significantly longer half-life t1/2 of 21.61 ± 9.4 h, nearly doubled as compared with that of the i.v.administration of 11.88 ± 1.66 h (Figure 7), certifying that HL has a favorable pharmacokinetic via PO administration.

In Vivo Pharmacokinetic Study of HL
For pharmacokinetic studies, CD ® (SD) IGS rats (5−6 weeks old) were randomly divided into two groups (three for each group) for oral administration (per os, PO) or intravenous administration (i.v.) of HL with dosages of 30 mg kg −1 and 0.5 mg kg −1 , respectively (Tables S3 and S4).The blood was then collected from the orbital sinus with a heparinized syringe at different time intervals (0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for PO, and 0.03, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for i.v.).High-performance liquid chromatography (HPLC) provided mean area under the curve (AUC 0-inf ) values of 10409.01 ± 5313.4 ng h mL −1 for PO and 5701.36 ± 2647.15 ng h mL −1 for i.v.HL achieved the largest C max value of 425.93 ± 176.1 ng mL −1 at a T max of 1.0 h for PO, while for i.v., the C max value of 806.43 ± 46.66 ng mL −1 was immediately observed at T max of 0.03 h, which is expected for i.v.administration.Notably, the PO administration exhibited a significantly longer half-life t 1/2 of 21.61 ± 9.4 h, nearly doubled as compared with that of the i.v.administration of 11.88 ± 1.66 h (Figure 7), certifying that HL has a favorable pharmacokinetic via PO administration.

Toxicological Studies of HL
For toxicological studies, CD ® (SD) IGS rats were randomly divided into six groups (three in each group; n = 3) for daily administration of HL (PO) with dosages of 5 mg kg −1 (12.2 µmol kg −1 , groups 3 and 4) and 10 mg kg −1 (24.4 µmol kg −1 , groups 5 and 6).Their weight and status were monitored and compared with the control group (groups 1 and 2) that was fed sterilizing solutions.As shown in Table S5, for groups 3 and 4, the rats remain alive throughout the experimental process with their weight constantly growing, similar to that found in groups 1 and 2 (Figure S7).In comparison, at a dosage of 10 mg kg −1 , part of the rats (four of six) showed abnormal behaviors such as decreasing food uptake, cachexia, and lethargy on day 9, and these rats either died or were euthanized after drug suspension.The remaining two rats showed normal and stable behaviors throughout the experiments, with their final weights being comparable with those in control groups 1 and 2. This highlights that a 5 mg kg −1 drug dosage of HL by PO administration is safe for in vivo studies.

Toxicological Studies of HL
For toxicological studies, CD ® (SD) IGS rats were randomly divided into six groups (three in each group; n = 3) for daily administration of HL (PO) with dosages of 5 mg kg −1 (12.2 µmol kg −1 , groups 3 and 4) and 10 mg kg −1 (24.4 µmol kg −1 , groups 5 and 6).Their weight and status were monitored and compared with the control group (groups 1 and 2) that was fed sterilizing solutions.As shown in Table S5, for groups 3 and 4, the rats remain alive throughout the experimental process with their weight constantly growing, similar to that found in groups 1 and 2 (Figure S7).In comparison, at a dosage of 10 mg kg −1 , part of the rats (four of six) showed abnormal behaviors such as decreasing food uptake, cachexia, and lethargy on day 9, and these rats either died or were euthanized after drug suspension.The remaining two rats showed normal and stable behaviors throughout the experiments, with their final weights being comparable with those in control groups 1 and 2. This highlights that a 5 mg kg −1 drug dosage of HL by PO administration is safe for in vivo studies.

Synthetic Steps for HL
Ligand HL can be synthesized via a five-step process as indicated below.

Single-Crystal X-ray Crystallography
Diffraction data for HL, 1, and 2 were acquired either on a Bruker APEX II CCD X-ray diffractometer (Bruker AXS GmbH, Germany) using Mo-Kα (λ = 0.71073 Å) (HL) or Ga-Kα (λ = 1.34138Å) irradiation (1 and 2).Refinement and reduction of the collected data were achieved using the program Bruker SAINT, and absorption corrections were performed using a multi-scan method [37].All crystal structures were solved by direct methods and refined on F 2 by full-matrix least-squares techniques with SHELXTL-2016 [38].Crystallographic data for HL, 1, and 2 have been deposited in the Cambridge Crystallographic Data Center (CCDC) as supplementary publication numbers 2,341,904 (HL), 2,341,905 (1), and 2,341,906 (2).These data can be obtained free of charge, either from the CCDC via www.ccdc.cam.ac.uk/data_request/cif (accessed on 21 March 2024) or from the Supplementary Materials.A summary of the key crystallographic data for HL, 1, and 2 is listed in Table 1.Selected bond distances and angles were listed in Table S1.
where n = number of reflections and p = total number of parameters refined.

In Vitro Cytotoxicity Evaluation by CCK-8 Assay
The HuH-7 cell line was cultured in DMEM + 10% FBS + 1% P/S and DMEM + 1% P/S.Cells grew as a monolayer and were detached upon confluence using trypsin (0.5% w/v in PBS).The cells were harvested from the cell culture medium by incubation in trypsin solution for 3 min, and they were then centrifuged with the supernatant and subsequently discarded.A 3 mL portion of serum-supplemented cell culture medium was added to neutralize any residual trypsin.The cells were re-suspended in serumsupplemented DMEM at a concentration of 5 × 10 4 cells/mL.Cells were cultured at 37 • C and 5% CO 2 for the CCK-8 studies.HuH-7 cells were seeded at a density of 2 × 10 4 cells per well in 90 µL of culture medium (DMEM + 10% FBS + 1% P/S) and cultured for 24 h at 37 • C and 5% CO 2 for attachment.The culture medium was then replaced by a serum-free medium (DMEM + 1% P/S) containing various concentrations of HL, 1, and 2 (pharmaceuticals solubilized with 2 parts per thousand DMSO).All experiments were carried out with three replicates (n = 3), and the untreated cells served as the 100% cell viability control, while the cell-free medium (DMEM + 10% FBS + 1% P/S + CCK-8) served as the blank.HuH-7 cells were directly incubated for a period of 72 h.After incubation, 100 µL of culture medium (DMEM + 10% FBS + 1% P/S) and 10 µL of CCK-8 was introduced and cultured for an additional 2 h before spectrophotometric measurement at 450 nm on a microplate reader.The relative cell viability (%) related to control cells was calculated using the equation: where V% is the percentage of cell viability, [A] experimental is the absorbance of the wells culturing the treated cells, [A] blank is the absorbance of the blank, and [A] control is the absorbance of the wells culturing untreated cells.The cytotoxicity assessment for Hep-G2 and PLC/PRF/5 resembled that of HuH-7, except that the culturing media was different, viz.MEM (NEAA) + 10% FBS + 1% P/S and MEM (NEAA) + 1% P/S for both Hep-G2 and PLC/PRF/5.

Detection of Intracellular Reactive Oxygen Species
HuH-7 cells were introduced into a 12-well plate (4 × 10 5 per well) in 1 mL of growing media (DMEM + 10% FBS + 1% P/S), and the cells were incubated at 37 • C under 5% CO 2 for 24 h for attachment.The growing media were removed, and the wells were replenished with HL, 1, and 2 in 1 mL of growing media (with 2 ppm of DMSO) with equivalent HL concentrations of 2 µg/mL.The cells were incubated for an additional 2 h, and then the culturing media was replaced with 10 µmol/mL DCFH-DA in DMEM + 1% P/S (1 mL), prepared by diluting (1:1000) a concentrated DCFH-DA following the supplied protocols.The cells were incubated for a further 20 min, and then the culturing media was removed.The cells were further rinsed with culturing media (DMEM + 1% P/S) three times to remove the residual DCFH-DA and were observed under a BD5000 inverted microscope to estimate and compare the ROS generation abilities of these materials.

Pharmacokinetics of HL
CD ® (SD) IGS rats (5-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).All animal experiments were conducted in accordance with the requirements of the Experimental Animal Welfare Ethics Committee of Transcenta Diagnostic Technology (Suzhou) Co., Ltd.(PZ-20211210001).For pharmacokinetic experiments, SD rats were randomly divided into two groups, with 3 for PO administration (30 mg kg −1 ) and i.v.administration (0.5 mg kg −1 ), respectively.Plasma was collected from the jugular vein with the presence of a heparinized syringe at different time intervals, viz.0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for PO and 0.03, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h for i.v.Plasma was first centrifuged at 3500 rpm for 10 min, and then 0.05 mL of the plasma was extracted; trifluoroacetic acid was used to acid precipitate protein, and NaOH was used to neutralize the solution.The mixture was diluted using MeCN: H 2 O (5:5 v/v).After 10 min of precipitating, supernatant fluids were collected by centrifugation at 10,000 rpm for 5 min and filtered with a syringe through a 0.22 µm hydrophilic membrane filter and measured using the HPLC method.For the HPLC assay, the analytical column was an Agilent ZORBAX SB C 18 column (4.6 mm × 150 mm, 5 µm).The mobile phase was MeCN:H 2 O (5:5 v/v), the flow rate of the mobile phase was 0.8 mL min −1 , and the UV detector was set at 245 nm.

Toxicological Studies
For toxicological studies, CD ® (SD) IGS rats (5−6 weeks old) were randomly divided into six groups (three for each group) for the PO administration of HL with daily dosages of 5 mg kg −1 (12.2 µmol kg −1 , groups 3 and 4) and 10 mg kg −1 (24.4 µmol kg −1 , groups 5 and 6), and they were compared with the control group that was fed sterilizing solutions (groups 1 and 2, Table S5).The weight of the rats was continuously monitored over 27 days, and they were fed on day 1, 4, 8, 13, 16, 19, and 23.For groups 5 and 6, the drug dosage was suspended when abnormal behaviors were observed, including decreasing food uptake, cachexia, and lethargy.

Conclusions
In this work, we report that the thiosemicarbazone derivative of HL and its Cu(II) complexes 1 and 2 demonstrated superior anticancer performances against hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7.For 1 and 2, the involvement of ROS as catalyzed by the Fenton-like process is also obvious.Intriguingly, the pharmacokinetic studies revealed that HL can be successfully absorbed via PO administration, with a favorable half-life of 21.61 ± 9.4 h, almost double that of via i.v.administration, rendering HL as having promising clinical potential.We believe that the association of such a type

Scheme 2 .
Scheme 2. The five-step synthetic procedure for HL.
[CuL] + (molecular weight of 471.0) and [CuL(H 2 O)] + (molecular weight of 489.0), indicating that the dimeric structure of 1 and 2 presumably remain as monomers in solutions.Molecules 2024, 29, x FOR PEER REVIEW 5 of 15 configuration.The liquid chromatography-mass spectrometry (LC-MS) for both 1 (Figure S4a) and 2 (Figure S4b) show peaks at 470.9 m/z and 488.8 m/z, assignable as the [CuL] + (molecular weight of 471.0) and [CuL(H2O)] + (molecular weight of 489.0), indicating that the dimeric structure of 1 and 2 presumably remain as monomers in solutions.
Molecules 2024, 29, x FOR PEER REVIEW 6 of 15 HL/L concentrations of 30 µg/mL remained stable upon keeping for 72 h (FigureS6), as evidenced by their unaltered UV-Vis absorption patterns throughout the experiments.

Figure 4 .
Figure 4. Change in UV-Vis absorption intensity in the range of 500-750 nm upon treatment with different concentrations of 1 in the presence of 100 µM H2O2 (a; inset: photographs showing color change upon introducing gradient concentrations of 1).The intensity change at 652 nm (oxTMB) upon treating gradient concentrations of HL, 1, and 2 with 100 µM H2O2 (b).

Figure 4 .
Figure 4. Change in UV-Vis absorption intensity in the range of 500-750 nm upon treatment with different concentrations of 1 in the presence of 100 µM H 2 O 2 (a; inset: photographs showing color change upon introducing gradient concentrations of 1).The intensity change at 652 nm (oxTMB) upon treating gradient concentrations of HL, 1, and 2 with 100 µM H 2 O 2 (b).

Molecules 2024 ,
29, x FOR PEER REVIEW 6 of 15HL/L concentrations of 30 µg/mL remained stable upon keeping for 72 h (FigureS6), as evidenced by their unaltered UV-Vis absorption patterns throughout the experiments.

Figure 4 .
Figure 4. Change in UV-Vis absorption intensity in the range of 500-750 nm upon treatment with different concentrations of 1 in the presence of 100 µM H2O2 (a; inset: photographs showing color change upon introducing gradient concentrations of 1).The intensity change at 652 nm (oxTMB) upon treating gradient concentrations of HL, 1, and 2 with 100 µM H2O2 (b).

Molecules 2024 ,
29, x FOR PEER REVIEW 7 of 15 ROS.In sharp contrast, 1 and 2 can induce the formation of strong fluorescence, characterizing the effective generation of ROS in the HuH-7 cells.

Figure 6 .
Figure 6.Comparison of the intracellular ROS generating ability of HL, 1, and 2 in HuH-7 using DCFH-DA as a fluorescence probe.

Figure 6 .
Figure 6.Comparison of the intracellular ROS generating ability of HL, 1, and 2 in HuH-7 using DCFH-DA as a fluorescence probe.
• C overnight.Upon cooling to r.t., the crude powder of HL was recrystallized with MeOH/MTBE (5:1, v/v) to give a pure yellow-green solid product of HL.The yield was 18.8 mg, 28%, based on S4.Single crystals of HL were grown by slow evaporation of a MeOH/DMSO (v/v = 1:1) solution of HL.Anal.calcd for C 19 H 19 N 7 S 2 : C 55.72, H 4.68, N 23.94; found: C 55.26; H 4.93; N, 23.49.Method 1: HL (100 mg, 0.244 mmol) was introduced in 20 mL of MeOH to give a yellow suspension, and CuCl 2 •2H 2 O (42 mg, 0.246 mmol) in 10 mL of MeOH was subsequently introduced dropwise.The formed mixture was stirred at 40 • C for 8 h.The solvent was removed, and the formed brown powder was recrystallized in MeOH and Et 2 O to obtain the crystal of 1. Method 2: CuO (21.4 mg, 0.269 mmol) and HCl (19.6 mg, 0.537 mmol) were mixed in 3 mL of MeOH, and the mixture was stirred at 40 • C. With the gradual dissolution of CuO, the solution turned pale green.After 10 min, HL (100 mg, 0.244 mmol) was introduced, and the solution became dark brown.The mixture was stirred for an additional 2 h and then filtered, and it was washed with a mixture of MeOH/MTBE (1:1, v/v; MTBE = methyl tert-butyl ether) to give a brown powder of [CuCl(L)] 2 , which was dried under vacuo.Yield (220 mg, 89% based on HL); anal.calcd for C 38 H 40 Cl 2 Cu 2 N 14 S 4 : C 44.79, H 3.96, N 19.24; found: C 44.18, H 3.37, N 18.96; IR (ATR, cm

Table 1 .
A summary of the cell parameters and refinement results for HL, 1, and 2.