Effect of mono- and dinuclear thiosemicarbazone platinacycles in the proliferation of a colorectal carcinoma cell line

Dalton Transactions PAPER Cite this: DOI: 10.1039/d4dt01490a Received 21st May 2024, Accepted 20th August 2024 DOI: 10.1039/d4dt01490a rsc.li/dalton Effect of mono- and dinuclear thiosemicarbazone platinacycles in the proliferation of a colorectal carcinoma cell line† Francisco Reigosa-Chamorro,‡ a Sandra Cordeiro,‡ b,c M. Teresa Pereira, a Beatriz Filipe, b,c Pedro V. Baptista, b,c Alexandra R. Fernandes * b,c and José M. Vila * a Herein, we describe the synthesis and characterization of a series of thiosemicarbazone platinacycles. Their activity towards HCT116 and A2780 cancer cell lines as well as normal fibroblasts was explored and conclusions about the influence of their structures were drawn based on the results. Ligands L1–3, tetranuclear compounds [Pt(L1–3)] 4 ,[Pt(L1–3)(PPh 3 )], and [Pt(L1–L3) 2 {Ph 2 P(CH 2 ) 4 PPh 2 }], and phosphine derivatives, were deemed unpromising owing to their lack of activity. However, mono-coordinated diphosphine complexes [Pt(L1–L3)(Ph 2 PCH 2 PPh 2 -P)] showed high selectivity and low IC 50 values, and their antiproliferative activity was further studied. The three studied derivatives 3a,3b and 3c showed a fast internalization of HCT116 colorectal cancer cells with similar IC 50 values, which induced a depolarization of mitochondrial membrane potential, with the subsequent triggering of apoptosis and autophagy in the case of 3c. In the case of compounds 3a and 3b, cell death mechanisms (extrinsic and intrinsic apoptosis, respectively) were triggered via the induction of reactive oxygen species (ROS). The three compounds were not toxic to a chicken embryo in vivo (after 48 h), and, importantly, showed an anti-angiogenic potential after exposure to the IC 50 of compounds 3a,3b and 3c. Introduction Since the seminal paper by Cope and Siekman 1 describing cyclometallated compounds, i.e., metallacycles, achieving the activation of aromatic C–H bonds using transition metals was published, the chemistry of such species has attracted much attention. Although many transition metals can potentially be applied to achieve corresponding metallacycles, the primary ones include palladium and platinum. This is a result of the numerous applications of these two transition metals in reactivity, 2–4 metallomesogens, 5,6 and synthetic chemistry, 7,8 where they are used in the functionalization of aromatic carbons through insertion reactions. 9,10 Palladium and platinum have also found application in catalysis after the discovery of phosphine palladacycles by Herrmann et al. 11–13 Furthermore, being of particular note are the Suzuki 14–19 and Mizoroki–Heck cross-coupling reactions. 19,20 One application that has expanded the most for obvious reasons is their usage as antineoplastic substances, where the functionality of metallacycles is mainly marked by palladium or platinum. 21–26 The choice of the ligand in the synthesis of new compounds is of great importance, especially when bioactive applications are to be considered, since their properties will depend on the final structure of the compound. Thiosemicarbazone ligands are typical ligands in cyclometallation chemistry owing to the ease of their preparation via a simple condensation reaction, which is also compatible with a number of functional groups. Their selection is enhanced because they are bioactive on their own 27 as well as when they are combined with the metals of coordination compounds and cyclometallated species. 28 Their effects as antiplasmodic agents were studied for years, but research interests and new findings have shifted their application to other diseases such as cancer. 29 We have reported that in thiosemicarbazone metallacycles, the organic ligand is able to bind to a metal centre as tridentate [C,N,S] in a tetranuclear 30 structure through M–S chelating and M–S bridging bonds. †Electronic supplementary information (ESI) available. CCDC 2309646 (1b) and 2309120 (2c). For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4dt01490a ‡Both authors contributed equally. a Departamento de Química Inorgánica, Universidade de Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela, Spain. E-mail: [email protected] b Associate Laboratory i4HB –Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University Lisbon, 2819-516 Caparica, Portugal. E-mail: [email protected]t c UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal This journal is © The Royal Society of Chemistry 2024 Dalton Trans. Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal This favours the addition of an appropriate leaving group on the metal in the resulting mononuclear compound, holding fast the thiosemicarbazone/metal moiety. This safeguards metallacycle structural integrity during the transport in biological fluids until it reaches cancer cells, securing its biological/drug activity. This also allows to modify the characteristics of the compounds with diverse ancillary ligands, which has proved to be vital in the proficiency of the parent compound in catalytic reactions, spectroscopic and emissive chemiluminescence and antiproliferative effect. In this sense, phosphine ligands can be a great way to add variability to the resulting structures by using different ligands and changing the reaction conditions. Our previous experience with cyclometallated compounds tested the potential of mononuclear and dinuclear species as anticancer agents. The results allowed us to conclude that the inclusion of a second metal via palladacycle metallo-ligands bearing a monocoordinated diphosphine did not produce a noticeable improvement, and that the combination of the boronic acid function and dppm showed great potential. Nevertheless, the enhanced effect could not be attributed to one factor alone. Furthermore, it was unclear if all types of boronic acid derivatives, regardless of the ancillary ligands, could be as effective. 26 The results presented here confirm the effectiveness of the platinum analogues, and point to the dppm ligand as a key structural moiety in the biological effect of these species. In the present work, we aimed to expand on the knowledge of the family of thiosemicarbazone platinacycles by adjustment of the imine groups and the ancillary ligands in a series of compounds derived from acetylphenylboronic acid to determine the effect of this tuning in their bioactivity as anticancer drugs. Experimental section General procedures All solvents were used without any previous purification. All chemicals were of reagent grade. The phosphines PPh 3 (triphenylphosphine), PPh 2 (CH 2 )PPh 2 [bis(diphenylphosphino) methane, dppm] and PPh 2 (CH 2 ) 4 PPh 2 [bis(diphenylphosphino)-butane, dppb] were purchased from Sigma-Aldrich. Elemental analyses were performed in a THERMO FINNIGAN, model FLASH 1112. IR spectra were acquired with a JASCO FT/ IR-4600 spectrometer equipped with an ATR, model ATR-PRO ONE. The NMR spectra were acquired on Varian INOVA 400 or Bruker DPX-250 spectrometers, using the solvent signal (CDCl 3 ,δ 1 H = 7.26, DMSO-d 6 ,δ 1 H = 2.50; acetone-d 6 ,δ 1 H= 2.05), or external H 3 PO 4 (85%), as appropriate. Coupling constants are reported in Hz. Synthesis of the ligands L1 (a) 4-Acetylphenylboronic acid (500 mg, 3.05 mmol) was added to 3-thiosemicarbazide (277.92 mg, 3.05 mmol), hydrochloric acid (35%, 0.65 cm 3 ) and water (40 cm 3 ), resulting in a clear solution that was stirred at room temperature (RT) for 3 h. The formed white solid powder was filtered off, washed with cold water, and dried in vacuo. White solid. Yield: 650.7 mg, 90%. Anal. Found: 45.8; H, 5.2; N, 17.7; S, 13.5%; C 9 H 12 BN 3 O 2 S (237.08 g mol −1 ) requires C, 45.6; H, 5.1; N, 17.7; S, 13.5%. IR cm −1 ν(O–H) 3458; ν(N–H) 3272, 3330; ν(CvN) 1596; ν(B–O) 1352; ν(CvS) 813. 1 H NMR (400 MHz, DMSO-d 6 , δ): 10.24 (s, 1H, NNH), 8.31 (s, 1H, NH 2 ), 8.14 (s, 2H, B(OH) 2 ), 7.96 (s, 1H, NH 2 ), 7.89 (vd, N= 7.8 Hz, 2H, H2/H6), 7.78 (v, N= 7.8 Hz, 2H, H3/H5), 2.29 (s, 3H, MeCvN). L2 (b) was prepared similarly from 3.05 mmol of acetylphenylboronic acid and methylthiosemicarbazone. White solid. Yield: 719.8 mg, 94%. Anal. Found: C, 47.6; H, 5.5; N, 16.8; S, 12.8%; C 10 H 14 BN 3 O 2 S (251.11 g mol −1 ) requires C, 47.8; H, 5.6; N, 16.7; S, 12.8%. IR cm −1 ν(O–H) 3500; ν(N–H) 3314, 3341; ν(CvN) 1595; ν(B–O) 1363; ν(CvS) 831. 1 H NMR (400 MHz, DMSO-d 6 ,δ): 10.26 (s, 1H, NNH), 8.49 (br, 1H, NHMe), 8.17 (s, 2H, B(OH) 2 ), 7.90 (d, N= 7.6 Hz, 2H, H2/H6), 7.80 (vd, N= 7.8 Hz, 2H, H3/H5), 3.03 (d, 3 J= 4.0 Hz, 3H, NHMe), 2.29 (s, 3H, MeCvN). L3 (c) was made from 3.05 mmol of acetylphenylboronic acid and methylthiosemicarbazone following the same procedure. White solid. Yield: 768.1 mg, 95%. Anal. Found: C, 50.0; H, 6.1; N, 16.0; S, 12.0%; C 11 H 16 BN 3 O 2 S (265.14 g mol −1 ) requires C, 49.8; H, 6.1; N, 15.9; S, 12.1%. IR cm −1 ν(O–H) 3450; ν(N–H) 3317, 3321; ν(CvN) 1592; ν(B–O) 1357; ν(CvS) 819. 1 H NMR (400 MHz, DMSO-d 6 ,δ): 10.16 (s, 1H, NNH), 8.55 (br, 1H, NHEt), 8.13 (s, 2H, B(OH) 2 ), 7.88 (vd, N= 7.8 Hz, 2H, H2/H6), 7.80 (vd, N= 7.8 Hz, 2H, H3/H5), 3.62 (dq, 3 J= 7.0 Hz, 2H, CH 2 ), 2.28 (s, 3H, MeCvN), 1.14 (t, 3 J= 7.0 Hz, 3H, Me). [PtL1] 4 (1a).L1 (50 mg, 0.21 mmol) was added to a suspension of potassium tetrachloroplatinate (0.18 mmol, 1 eq.) in a mixture of ethanol (20 mL) and water (0.5 mL). The mixture was stirred at 55 °C for 48 h. Ethanol was removed under reduced pressure and the resulting orange solid was washed with water, centrifuged, and dried in vacuo. Yield: 84.9 mg, 92%. Anal. Found: C, 24.8; H, 2.2; N, 9.6; S, 7.6%; (C 9 H 10 BN 3 O 2 PtS) 4 (1720.6 g mol −1 ) requires C, 25.1; H, 2.3; N, 9.8; S, 7.5%. IR cm −1 ν(O–H) 3448; ν(N–H) 3289, 3162; ν(CvN) 1577; ν(B–O) 1317. 1 H NMR (400 MHz, DMSO-d 6 ,δ): 7.83 (s, 1H, H5), 7.16 (d, 3 J= 7.6 Hz, 1H, H3), 6.60 (s, 2H, NH 2 ), 6.41 (d, 3 J= 7.6 Hz, 1H, H2), 1.88 (s, 3H, MeCvN). [PtL2] 4 (1b) was prepared from L2 (50 mg, 0.20 mmol) and potassium tetrachloroplatinate (75 mg, 0.18 mmol) following the same procedure. Yield: 72.5 mg, 82%. Anal. Found: C, 27.0; H, 2.5; N, 9.3; S, 7.5%; (C 10 H 12 BN 3 O 2 PtS) 4 (1776.72 g mol −1 ) requires C, 27.1; H, 2.7; N, 9.5; S, 7.2%. IR cm −1 ν(O–H) 3472; ν(N–H) 3305, 3210; ν(CvN) 1582; ν(B–O) 1351. 1 H NMR (400 MHz, DMSO-d 6 ,δ): 7.93 (s, 1H, H5), 7.58 (s, 2H, B(OH) 2 ), 7.36 (d, 3 J= 7.6 Hz, 1H, H3), 6.70 (d, 3 J= 7.6 Hz, 1H, H2), 6.49 (s, 1H, NHMe), 2.93 (d, 3 J= 4.7 Hz, 3H, NHMe), 1.51 (s, 3H, MeCvN). [PtL3] 4 (1c) was prepared employing L3 (53 mg, 0.20 mmol) and potassium tetrachloroplatinate (75 mg, 0.18 mmol) as starting materials. Yield: 69.1 mg, 80%. Anal. Found: C, 28.9; H, 3.1; N, 9.4; S, 7.2%; (C 11 H 14 BN 3 O 2 PtS) 4 (1832.84 g mol −1 ) requires C, 28.8; H, 3.1; N, 9.2; S, 7.0%. IR cm −1 ν(O–H) 3419; ν(N–H) 3244, 2972; ν(CvN) 1575; ν(B–O) 1320. 1 H NMR Paper Dalton Transactions Dalton Trans. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online (400 MHz, DMSO-d 6 ,δ): 7.92 (s, 1H, H5), 7.35 (d, 3 J= 7.6 Hz, 1H, H3), 6.68 (d, 3 J= 7.6 Hz, 1H, H2), 6.29 (s, 1H, NHEt), 1.54 (s, 3H, MeCvN), 1.11 (t, 3 J= 7.2 Hz, 3H, Me). [PtL1(PPh 3 )] (2a). Compound 1a (50 mg, 0.029 mmol) and triphenylphosphine (30.5 mg, 0.11 mmol, 1 : 4) were added in a carrousel reaction flask fitted with a stirring rod, and vacuum/argon cycles were performed; then, deoxygenated acetone was added through a syringe. This mixture was stirred for 6 h to yield a clear solution. After solvent removal, the resulting solid was triturated with dichloromethane/n-hexane and centrifuged. The coral solid was then dried under vacuum. Yield: 63.6 mg, 79%. Anal. Found: 46.6; H, 3.6; N, 6.2; S, 4.8%; C 27 H 25 BN 3 O 2 PPtS (692.44 g mol −1 ) requires C, 46.8; H, 3.7; N, 6.1; S, 4.6%. IR cm −1 ν(O–H) 3452; ν(N–H) 3049; ν(CvN) 1574; ν(B–O) 1328. 1 H NMR (400 MHz, acetone-d 6 ,δ): δ7.71–7.64 (m, 6H, o-PPh 3 ), 7.49–7.39 (m, 11H, PPh 3 ), 7.31 (dd, J= 7.5, 1.2 Hz, 1H, H3), 6.98 (d, J= 7.6 Hz, 1H, H2), 6.96 (q, J= 1.8 Hz, 1H, H5), 6.05 (s, 2H, NH 2 ), 2.43 (s, 3H, MeCvN). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ23.91, 1 J(PtP) = 3892.86 Hz. The remaining phosphine and/or diphosphine derivatives were prepared similarly. [PtL2(PPh 3 )] (2b) was prepared from 0.1 mmol of triphenylphosphine and 0.025 mmol of 1b. Yield: 56.5 mg, 71%. Anal. Found: C, 47.4; H, 3.8; N, 6.0; S, 4.4%; C 28 H 27 BN 3 O 2 PPtS (706.47 g mol −1 ) requires C, 47.6; H, 3.8; N, 5.9; S, 4.5%. IR cm −1 ν(O–H) 3412; ν(N–H) 3324; ν(CvN) 1574; ν(B–O) 1335. 1 H NMR (400 MHz, acetone-d 6 ,δ): 7.75–7.63 (m, 6H, PPh 3 ), 7.47–7.35 (m, 9H, PPh 3 ), 7.30 (d, 3 J= 7.5 Hz, 1H, H3), 6.98 (d, 3 J= 8.0 Hz, 1H, H2), 6.94 (d, 4 J= 1.6 Hz, 1H, H5), 6.04 (s, 1H, NHMe), 2.96 (d, 3 J= 4.5 Hz, 3H, NHMe), 2.42 (s, 3H, Me). 31 P– { 1 H} NMR (400 MHz, acetone-d 6 )δ23.91 1 J(PtP) = 3888 Hz. [PtL3(PPh 3 )] (2c) was prepared from 0.1 mmol of triphenylphosphine and 0.025 mmol of 1c. Yield: 62.9 mg, 80%. Anal. Found: C, 48.1; H, 4.0; N, 5.9, S, 4.6%; C 29 H 29 BN 3 O 2 PPtS (720.50 g mol −1 ) requires C, 48.3; H, 4.1; N, 5.8, S, 4.5%. IR cm −1 ν(O–H) 3434; ν(N–H) 3052; ν(CvN) 1574; ν(B–O) 1330. 1 H NMR (400 MHz, acetone-d 6 ,δ): 7.67 (dd, 3 J(PH) = 11.3 Hz, 3 J= 7.7 Hz, 6H, o-PPh 3 ), 7.45 (dd, J= 8.2, 5.2 Hz, 9H), 7.31 (d, 3 J= 7.6 Hz, 1H, H3), 6.99 (d, 3 J= 8.3 Hz, 1H, H2), 6.96 (s, 1H, H5), 6.11 (s, 1H, NHEt), 3.40 (p, 3 J= 6.9 Hz, 2H, CH 2 ), 2.44 (s, 3H, Me), 1.17 (td, 3 J= 7.1, 2.3 Hz, 3H, Me). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ23.83 1 J(PtP) = 3912.3 Hz. [PtL1(dppm-P)] (3a) was prepared from 0.2 mmol of dppm and 0.05 mmol of 1a. Yield: 101.3 mg, 57% Anal. Found: C, 49.8; H, 3.7; N, 5.1; S, 3.8%; C 34 H 32 BN 3 O 2 P 2 PtS (814.55 g mol −1 ) requires C, 50.1; H, 3.9; N, 5.2; S, 3.9. IR cm −1 ν(O–H) 3444; ν(N–H) 3051; ν(N–H) 1575; ν(B–O) 1330. 1 H NMR (400 MHz, acetone-d 6 ,δ): 7.90–7.82 (m, 4H, PPh 2 ), 7.42–7.33 (m, 6H, PPh 2 ), 7.33–7.27 (m, 4H, PPh 2 ), 7.21–7.13 (m, 5H, PPh 2 ), 7.03 (d, J= 1.6 Hz, 1H, H5), 6.89 (d, 3 J= 7.6 Hz, 1H, H2), 6.28 (s, 2H, B(OH) 2 ), 6.08 (s, 2H, NH 2 ), 3.72–3.51 (m, 2H, PCH 2 P), 2.37 (s, 3H, MeCvN). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ12.54 (d, 2 J(PP) = 78.9 Hz, 3 J(PtP) = 3863.70 Hz), −23.51 (d, 2 J(PP) = 79.0 Hz, 3 J(PtP) = 77.76 Hz). [PtL2(dppm-P)] (3b) was prepared from 0.2 mmol of dppm and 0.05 mmol of 1b. Yield: 88.8 mg, 62%. Anal. Found: C, 50.6; H, 4.1; N, 5.0; S, 3.8%; C 35 H 34 BN 3 O 2 P 2 PtS (828.58 g mol −1 ) requires C, 50.7; H, 4.1; N, 5.1; S, 3.9%. IR cm −1 ν(O–H) 3391; ν(N–H) 3051; ν(CvN) 1574; ν(B–O) 1331. 1 H NMR (400 MHz, acetone-d 6 ,δ): 7.86 (t, J= 9.7 Hz, 4H, PPh 2 ), 7.33 (q, J= 7.3 Hz, 10H, PPh 2 ), 7.18 (p, J= 7.7 Hz, 6H, PPh 2 ), 7.03 (s, 1H, H5), 6.90 (d, 3 J= 7.6 Hz, 1H, H2), 6.32 (s, 2H, B(OH) 2 ), 6.12 (s, 1H, NHMe), 3.72–3.55 (m, 2H, PCH 2 P), 2.99 (t, 3 J= 3.5 Hz, 3H, NHMe), 2.37 (s, 3H, MeCvN). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ12.55 (d, 2 J(P–P) = 79.5 Hz, 1 J(Pt–P) = 3860.46 Hz), −23.59 (d, 2 J(PP) = 79.0 Hz, 3 J(PtP) = 38.6 Hz). [PtL3(dppm-P)] (3c) was prepared from 0.2 mmol of dppm and 0.05 mmol of 1c. Yield: 89.4 mg, 63%. Anal. Found: C, 51.1; H, 4.2; N, 5.1; S, 3.9%; C 36 H 36 BN 3 O 2 P 2 PtS (842.60 g mol −1 ) requires C, 51.3; H, 4.3; N, 5.0; S, 3.8%. IR cm −1 ν(O–H) 3391; ν(N–H) 3054; ν(CvN) 1574; ν(B–O) 1334. 1 H NMR (400 MHz, acetone-d 6 ,δ): 7.86 (t, J= 9.8 Hz, 4H), 7.49 (s, 1H), 7.33 (q, J= 7.5 Hz, 10H), 7.18 (t, J= 8,4 Hz, 6H), 7.03 (s, 1H, H5), 6.89 (dd, 3 J= 7.6, 2.5 Hz, 1H, H 2 ), 6.31 (d, J= 2.5 Hz, 2H, B(OH) 2 ), 6.12 (s, 1H, NHEt), 3.62 (dd, J= 17.9, 9.8 Hz, 2H, PCH 2 P), 3.43 (p, 3 J= 7.2 Hz, 2H, PCH 2 P), 2.36 (d, J= 2.5 Hz, 3H, MeCvN), 1.20 (td, 3 J= 7.2, 2.3 Hz, 3H, Me). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ12.55 (d, 2 J(P–P) = 78.9 Hz, 1 J(Pt–P) = 3859.6 Hz), −23.53 (d, 2 J(P–P) = 78.4 Hz, 3 J(Pt–P) = 38.7 Hz). [(PtL1) 2 (μ-dppb)] (4a) was prepared from 0.05 mmol of dppb and 0.025 mmol of 1a. Yield: 53.8 mg, 72%. Anal. Found: C, 42.6; H, 3.7; N, 6.7; S, 4.7%; C 46 H 48 B 2 N 6 O 4 P 2 Pt 2 S 2 (1286.78 g mol −1 ) requires C, 43.0; H, 3.8; N, 6.5; S, 5.0%. IR cm −1 ν(O–H) 3389; ν(N–H) 3050; ν(CvN) 1574; ν(B–O) 1331. 1 H NMR (400 MHz, acetone-d 6 )δ7.84–7.71 (m, 8H, PPh 2 ), 7.50 (q, J= 9.4, 8.1 Hz, 6H, PPh 2 ), 7.40 (td, J= 5.2, 2.7 Hz, 6H, PPh 2 ), 7.37–7.30 (m, 3H, PPh 2 ), 7.05 (d, 3 J= 7.5 Hz, 2H, H2), 6.94 (t, J= 6.6 Hz, 2H, H5), 6.43 (s, 4H, B(OH) 2 ), 6.17 (s, 4H, NH 2 ), 2.35 (d, J= 5.9 Hz, 6H, MeCvN), 2.30–2.16 (m, 4H, PCH 2 ), 1.92–1.85 (m, 4H, CH 2 ). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ16.04, 15.06, 1 J(PtP) = 3818.23 Hz. [(PtL2) 2 (μ-dppb)] (4b) was prepared from 0.05 mmol of dppb and 0.025 mmol of 1b. Yield: 50.3 mg, 68%. Anal. Found: C, 44.3; H, 4.1; N, 6.2; S, 4.7%; C 48 H 52 B 2 N 6 O 4 P 2 Pt 2 S 2 (1314.84 g mol −1 ) requires C, 43.9; H, 4.0; N, 6.4; S, 4.9%. IR cm −1 ν(O–H) 3394; ν(N–H) 3054; ν(CvN) 1576; ν(B–O) 1333. 1 H NMR (400 MHz, acetone-d 6 )δ7.82–7.75 (m, 6H, PPh 2 ), 7.55–7.46 (m, 6H, PPh 2 ), 7.39 (dq, J= 6.1, 2.5 Hz, 4H, PPh 2 ), 7.36–7.32 (m, 4H, PPh 2 ), 7.07 (t, J= 3.8 Hz, 2H, H5), 6.96 (d, 3 J = 7.5 Hz, 2H, H2), 6.33 (s, 4H, B(OH) 2 ), 6.10 (s, 2H, NHMe), 2.98 (dd, J= 8.6, 4.6 Hz, 6H, NHMe), 2.40 (d, J= 7.8 Hz, 6H, MeCvN), 2.31–2.17 (m, 4H, PCH 2 ), 1.92–1.76 (m, 4H, CH 2 ). 31 P–{ 1 H} NMR (400 MHz, acetone-d 6 )δ16.04, 15.06, 1 J(PtP) = 3818.23 Hz. [(PtL3) 2 (μ-dppb)] (4c) was prepared from 0.05 mmol of dppb and 0.025 mmol of 1c. Yield: 57.1 mg, 78%. Anal. Found: C, 44.9; H, 4.3; N, 6.1; S, 4.6%; C 50 H 56 B 2 N 6 O 4 P 2 Pt 2 S 2 (1342.89 g mol −1 ) requires C, 44.7; H, 4.2; N, 6.3; S, 4.8%. IR cm −1 ν(O–H) 3399; ν(N–H) 3046; ν(CvN) 1572; ν(B–O) 1330. 1 H NMR (400 MHz, acetone-d 6 )δ7.82–7.75 (m, 6H, PPh 2 ), 7.55–7.46 (m, 6H, PPh 2 ), 7.39 (dt, J= 6.1, 2.1 Hz, 4H, PPh 2 ), 7.36–7.32 (m, 4H, PPh 2 ), 7.09–7.05 (m, 2H, H5), 6.96 (d, J= 7.5 Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2024 Dalton Trans. Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Hz, 2H, H2), 6.31 (s, 4H, B(OH) 2 ), 6.17 (s, 2H, NHEt), 3.43 (ddd, J= 9.2, 7.1, 5.1 Hz, 4H, CH 2 ), 2.40 (d, J= 7.1 Hz, 6H, MeCvN), 2.34–2.17 (m, 4H, PCH 2 ), 1.94–1.73 (m, 4H, CH 2 ), 1.19 (q, 3 J= 7.1 Hz, 6H, Me). 31 P–{ 1 H} NMR (400 MHz, acetoned 6 )δ16.06, 15.01, 1 J(PtP) = 3821.58 Hz. The compound synthesis reactions are shown in Scheme 1. Cell culture The HCT116 colorectal cancer cell line and the primary human dermal fibroblasts were obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA) and cultured in Dulbecco’smodifiedEagle’s medium (DMEM), while the A2780 carcinoma cell line was acquired from Sigma-Aldrich (Madrid, Spain) and cultured in Roswell Park Memorial Institute (RPMI) medium. Media were supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) of a penicillin/streptomycin solution. The media and supplements were obtained from Thermo Fischer Scientific (Waltham, Massachusetts, USA). Cells were cultured in 25 cm 2 and/or 75 cm 2 T-flasks in a CO 2 incubator with a humidified atmosphere at 37 °C and 5% (v/v) CO 2 (SANYO CO 2 Incubator, Electric Biomedical Co., Osaka, Japan). Cell viability assays Cells were initially seeded in 96-well plates at a cell density of 0.75 × 10 5 cells per mL and incubated (37 °C, 5% (v/v) CO 2 ) for 24 h. After this period, the media were replaced with fresh media containing the desired compounds (concentrations ranging from 0.1 µM to 50 µM), 0.1% (v/v) of DMSO or 0.4 µM doxorubicin (Dox). After 48 h of incubation under the same conditions, the CellTiter 96® aqueous one solution cell proliferation assay kit (Promega, Madison, USA) was used to evaluate the cell viability by measuring the absorbance at 490 nm in a Tecan Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). Data were analysed with the GraphPad Prism 8 software, where viability-concentration response curves facilitated the calculation of the concentration of compounds that induced a 50% reduction in cell viability (IC 50 ). Scheme 1 Reaction sequence for the synthesis of the reported compounds. Paper Dalton Transactions Dalton Trans. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Complex stability in biological media The stability of the three platinum compounds (3a,3b and 3c) was analysed via UV-Visible spectroscopy (Shimadzu Scientific Instruments) with a quartz cuvette (1 cm path length) over a wavelength range of 220–700 nm for three different incubation times, namely 0, 3, 24 and 48 h. Compounds were diluted in RPMI medium without phenol red and FBS after being first dissolved in 100% (v/v) DMSO. Compounds 3a and 3c were analysed at a final concentration of 50 μM, while 3b was analysed at a final concentration of 150 μM. ICP-AES (inductively coupled plasma-atomic emission spectrometry) To evaluate the compound internalization in HCT116 cells, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was performed. HCT116 cells were seeded in 25 cm 2 T-flasks at a density of 5 × 10 5 cells/T-flask and incubated for 24 h. The culture medium was then replaced with fresh medium with 10× IC 50 concentrations of the studied compounds or 0.1% (v/v) DMSO, and cells were incubated for 3 h. After this period, the cell culture medium was recovered in a 15 mL Falcon tube and the cells were washed with PBS. After this washing step, the PBS washing solution was also recovered in the 15 mL Falcon tube. Cells were detached from the T-flask with 2 mL of TrypLE Express and centrifuged at 750g(Sigma 3-16K Sartorius, Germany) for 5 min. The supernatant was removed and added to the 15 mL Falcon tube, and the cell pellet was washed 2 more timeswithPBS.Aquaregia(3:1HCl/HNO 3 ) was prepared, and added to the 15 mL Falcon tubes containing the previous washing solutions and the supernatant (non-cellular fraction) or to the cell pellets (cellular fraction). Samples were incubated at room temperature (RT) for 24 h in a hood fume, and then delivered to Laboratório de Análises/LAQV to quantify the platinum levels by ICP-AES. Analysis of apoptosis induction by flow cytometry Apoptosis was evaluated using the Alexa Fluor® 488 Annexin V/ dead cell apoptosis kit (Invitrogen, Thermo Fisher Scientific, MA, USA). HCT116 cells were seeded in 6-well plates (at a density of 2 ×10 5 cellsperwell)for24h,andlaterincubatedfor48hperiod with the IC 50 concentrations of compounds 3a,3b and 3c. Cells treated with 0.1% (v/v) DMSO were used as the vehicle control, while cells treated with 0.4 μM Dox were used as positive controls. After 48 h of incubation, cells were washed with PBS and detached from the wells with TrypLE Express (Invitrogen), and then washed again with PBS. Lastly, cells were incubated with the Alexa® Fluor 488-Annexin V solution and 100 μgmL −1 of propidium iodide (PI) at RT for 15 min. Samples were evaluated by the Attune® acoustic focusing flow cytometer (Life Technologies, Carlsbad, USA), and the results were analysed with the Attune® Cytometric software. Quantification of BAX and BCL-2 protein levels by western blot HCT116 cells were cultivated, incubated and collected, as described previously. 32 After collection, cells were submitted to 5 ultrasound pulses on ice (2 min on ultrasound, followed by 30 s on ice; Elma D-78224; Singen/Htw, Germany) and centrifuged at 1000gfor 5 min. The total amount of protein in the supernatant extracts was quantified with Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). For SDS-PAGE, 20 μg of protein was loaded on 10% polyacrylamide gel and transferred to a 0.45 μm PVDF membrane (GE Healthcare Life Sciences, Germany) (BAX and BCL-2 proteins). The membrane was blocked for 2 h with 5% (w/v) non-fat milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20), and incubated at room temperature for 1 h under constant agitation with the respective primary antibody solution (anti-BAX, 1 : 5000, Abcam, United Kingdom; and anti BCL-2, 1 : 1000, Sigma, St Louis, MO, USA). After incubation, membranes were washed with TBST buffer three times, for a duration of 5 min each (procedure also repeated after the secondary antibody incubation) and exposed to the secondary antibody solution (1 : 3000, anti-mouse IgG, horseradish peroxidase HPR-linked antibody or 1 : 2000, anti-rabbit IgG, HPR- linked antibody; Cell Signalling Technology, USA). Membranes were treated with the WesternBright ECL substrate (Advansta, USA) for 5 min and later exposed to a film in a dark room. After that, to normalize the results, membranes were incubated two times with stripping buffer (0.1 M glycine, 20 mM magnesium acetate, 50 mM KCl, pH 2.0) for a duration of 10 and 20 min, respectively, and then incubated with anti-βactin (1 : 5000; Sigma, St Louis, USA). BAX and BCL-2 protein quantification (densitometry) was performed using the Image J software. BAX and BCL-2 protein levels were normalized to βactin levels. Mitochondrial membrane potential (ΔΨ m ) analysis by flow cytometry ΔΨ m was analysed using the JC-1 mitochondrial membrane potential assay kit (Abnova Corporation, Walnut, CA, USA). HCT116 cells were seeded in 6-well plates with a density of 2 × 10 5 cells per well, incubated for 24 h, and later incubated for another 48 h period with the IC 50 concentrations of compounds 3a,3b and 3c or with 0.1% (v/v) DMSO. As positive controls, 0.4 μM Dox and 5 μM Cis were used. After that, cells were washed with PBS, detached with TrypLE Express, and washed with DMEM medium. Cells were then resuspended in DMEM medium without phenol red + 5% (v/v) FBS, and stained with the JC-1 staining solution for 20 min at 37 °C. Lastly, cells were resuspended in DMEM medium without phenol red + 5% (v/v) FBS, and analysed in the Attune® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, CA, USA). Caspase-8 activity HCT116 cells were seeded in 25 cm 2 T-flasks at a density of 2 × 10 6 cells/T-flasks. After 24 h incubation, under the same conditions as described previously, the medium was replaced with fresh medium containing DMSO 0.1% (v/v), 0.4 µM Dox, 5 µM Cis or IC 50 concentrations of the compounds 3a,3b and 3c, and incubated for 48 h. After that, cells were detached using Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2024 Dalton Trans. Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online cold PBS and a cell scraper, and centrifuged at 500gfor 5 min. Then, the instructions provided in the caspase-8 assay kit (Abcam) procedure were followed. Caspase 8 activity was obtained by measuring the absorbance at 400 nm of each sample. Caspase 8 activities were normalized to the corresponding value in DMSO samples. Analysis of autophagy by flow cytometry The autophagic potential of HCT116 cells was evaluated using the autophagy assay kit (ab139484) (Abcam, Cambridge, United Kingdom). HCT116 cells were seeded in 6-well plates with a density of 2 × 10 5 cells per well and incubated for 24 h. After the replacement of the culture medium with fresh medium containing the IC 50 concentrations of compounds 3a, 3b and 3c, cells were incubated for another 48 h. 0.1% (v/v) DMSO, 0.4 μM Dox and 5 μM Cis were used as controls. In addition, 15 h before finishing the 48 h incubation time, 0.5 μM Rapamycin was added to the respective wells. After that, cells were washed with PBS, detached with TrypLE Express, washed with DMEM medium without phenol red + 5% (v/v) FBS, and then incubated with the green stain solution in DMEM medium without phenol red + 5% (v/v) FBS for 30 min at RT. After this period, cells were washed, resuspended in the assay buffer, and then analysed with the Attune® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, CA, USA). The results were analysed with the Attune® Cytometric software. Analysis of reactive oxygen species (ROS) production by flow cytometry HCT116 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and incubated for 24 h. The culture medium was replaced with fresh medium containing the IC 50 concentrations of compounds 3a,3b and 3c, 0.1% (v/v) DMSO, 0.4 μM Dox, 5 μM cisplatin or 30 μM TBHP (positive control as indicated by the manufacturer), and cells were incubated for another 48 h period. Cells were then washed with PBS, detached with TrypLE Express, washed with PBS, and later incubated with 10 μMof2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 20 min. Cells were later analysed with the Attune® Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA), and the results were treated with the Attune® Cytometric software. Ex ovo CAM assay The pro-/anti-angiogenic potential of the compounds was evaluated with the ex ovo chorioallantoic membrane (CAM) assay, as previously described in (Reigosa-Chamorro et al., 2021 26 ). 30,31 Chicken embryos were first incubated for 24 h and later exposed to the IC 50 concentrations of compounds 3a, 3b and 3c dissolved in PBS (in the centre of O-rings) or 0.1% (v/v) DMSO. The compounds’and DMSO’s distribution in the embryos were always performed in a different order. Then, embryos were incubated for another 48 h at 37 °C and its images were captured after 0, 24 and 48 h with a digital USB Microscope Camera (Opti-Tekscope OT-V1) to manually count the newly formed blood vessels via ImageJ software. The ex ovo CAM assay fulfils the Directive 2010/63/EU of the European Parliament to protect the animal models for scientific purposes. Statistical analysis The presented data are referred to as mean ± SEM from at least three biological independent experiments, unless otherwise stated. One-way ANOVA or Student’st-test were performed to determine the statistical significance (p< 0.05) with the GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Results and discussion The compounds and respective reactions are shown in Scheme 1 for simplification. The thiosemicarbazone ligands L1–L3,a,band cwere prepared by reaction of 4-acetylphenyl- boronic acid with the corresponding thiosemicarbazide as pure air-stable solids, as appropriate (see Experimental section). The NNHproton resonated ca. 10.2 ppm, whilst the NH 2 protons, a, gave rise to two characteristic resonances in the 1 H NMR spectrum which were attributed to the restricted rotation of the NH 2 group about the C(S)NH 2 bond axis. The NHR protons, b,c, showed a broad resonance at ca. δ8.5 ppm. The characteristics of the ligand spectra included two virtual doublets stemming from the aromatic AA′XX′spin system with an Nvalue at ca. 8.0–7.8 ppm. From them, the new cyclometallated compounds were obtained as described in Scheme 1. The preparative details and characteristic microanalytical and spectroscopic data are given in the Experimental section. Reaction of a,bor c, as appropriate, with potassium tetrachloroplatinate in water/ethanol gave clear solutions. These solutions were used to isolate the tetranuclear compounds 1a–1c as pure air-stable solids, with the ligand in the E,Zconfiguration. Absence of the NHresonance agreed with deprotonation at the hydrazine group. The 1 H NMR spectra showed the absence of the AA′XX′spin system upon metalation of the para-substituted phenyl ring, and distinct resonances were accordingly assigned to the H2, H3 and H5 nuclei (see Experimental). Reaction of [Pt(L1–3)] 4 1a–1c with tertiary phosphines PPh 3 , Ph 2 PCH 2 PPh 2 (dppm), and Ph 2 P(CH 2 ) 4 PPh 2 (dppb), in 1 : 4 or 1 : 2 molar ratios, as appropriate, gave the compounds [Pt(L1–3)(PPh 3 )] 2a–2c, [Pt(L1–L3)(Ph 2 PCH 2 PPh 2 -P)] 3a–3c, and [Pt(L1–L3) 2 {μ-Ph 2 P(CH 2 ) 4 PPh 2 }] 4a–4c as pure air-stable solids (Scheme 1). The microanalytical and spectroscopic data are presented in the Experimental section. The use of excess diphosphine only gave cleavage of the Pt–S bridging . Therefore, in compounds with diphosphines, coordination to the metal was only through one phosphorus atom. Usage of excess phosphine did not cleave the Pt–S chelate bond. The 1 H NMR spectra showed the high-field shift of the H5 resonance at ca. 1 ppm with respect to the corresponding tetranuclear compound, Paper Dalton Transactions Dalton Trans. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online owing to the shielding effect of the phosphine phenyl rings. The 31 P NMR spectra for the 3a–3c compounds showed two doublets assigned to the two non-equivalent phosphorus nuclei. The lower field resonance, at ca. 12.5 ppm, was assigned to the 31 P nucleus bonded to platinum, in agreement with the J(Pt–P) values. Meanwhile, for 4a–4c, the 31 P resonance was a singlet signal in accordance with the existence of equivalent phosphorus nuclei; the chemical shift values were consistent with a phosphorus to nitrogen trans geometry. 32,33 The resonance for the ABXY spin system of the PCH 2 P protons appeared at ca. at δ3.52. Attempts to produce mononuclear species analogous to 3a–3c with diphosphines other than Ph 2 PCH 2 PPh 2 were unsuccessful. Suitable crystals of 1b were grown by slowly evaporating an acetone solution of the complex. The ORTEP illustration of compound 1b is shown in Fig. 1. The tetranuclear compound crystallizes in the Pcan space group with two acetone molecules. Within each molecule, the metalated moieties are displayed as two sets of almost coplanar antiparallel pairs separated at ca. 3.5 Å. Each platinum atom of the internal Pt 4 S 4 nucleus is bonded to a tridentate C,N,S chelating ligand and to the sulfur atom, S bridging , of another metalated species. The longer Pt–S chelating bond lengths, as opposed to Pt–S chelating , reflect the differing trans influence of the phenyl carbon and nitrogen atoms of the ligands. Suitable crystals of 2c were grown by slowly evaporating a chloroform solution of the complex. The crystals were triclinic with P1 ˉspace group. The ORTEP illustration is shown in Fig. 2. The structure of compound 2c comprises a molecule with the platinum(II) atom bonded in a slightly distorted square planar coordination to four different donor atoms, a tridentate thiosemicarbazone through the aryl C(6) carbon, the imine N(1) nitrogen, and the thioamide S(1) sulfur atom, and to a phosphorus atom P(1) of the triphenylphosphine. The bond distances and angles are within the expected values for analogous structures, as was provided by Mogul 2020.3 from the CCDC program package. The angles at platinum are close to 90° with allowance for the somewhat smaller C(6)–Pt(1)–N (1) and S(1)–Pt(1)–N(1) angles, 79.81° and 83.4°, respectively, and larger C(6)–Pt(1)–P(1) and S(1)–Pt(1)–P(1) angles, 99.80° and 96.97°, also respectively, consequent upon chelation. The crystal packing shows the molecules are arranged in sheets held together by means of hydrogen bonding interactions between the boronic acid function, the chlorine atom of the solvent molecule and the amide nitrogen, as well as by π–π stacking. Antiproliferative activity The in vitro antiproliferative potential of compounds 1a–c,2a– c,3a–c,4a–cand the respective ligands a–cwas assessed using the CellTiter 96®Aqueous non-radioactive cell proliferation assay (MTS assay), as described in the Experimental section. 34 This antiproliferative activity was analysed by exposure of the ovarian carcinoma (A2780) and colorectal carcinoma (HCT116) cell lines and normal human primary dermal fibroblasts to 0.1–50 µM of all compounds for 48 h (ESI Fig. S1†). As shown in Fig. S1,†a reduction of the cell viability is observed upon increase of the compounds concentrations. Cell viability-con- centration curves (GraphPad software) allowed us to calculate Fig. 1 ORTEP drawing of the platinacycle 1b with thermal ellipsoid plot shown at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity. Selected bond distances (Å) and angles (°) for 10: Pt(1)–C(11) 2.013(8), Pt(1)–N(10) 1.988(7), Pt(1)–S(3) 2.287(2), Pt (1)–S(4) 2.347(2), S(4)–Pt(1)–S(3) 100.76(8), N(10)–Pt(1)–S(3) 174.5(2), N(10)–Pt(1)–S(4) 84.1(2), C(11)–Pt(1)–S(3) 94.6(3), C(11)–Pt(1)–S(4) 163.9 (3), and C(11)–Pt(1)–N(10) 80.8(3). Fig. 2 ORTEP drawing of the platinacycle 2c with thermal ellipsoid plot shown at the 50% probability level. Hydrogen atoms and solvent molecules have been omitted for clarity. Selected bond distances (Å) and angles (°) for 10: Pt(1)–C(6) 2.038(6), Pt(1)–N(1) 2.032(5), Pt(1)–S(1) 2.3558(17), Pt(1)–P(1) 2.2234(18), S(1)–Pt(1)–P(1) 96.97(6), N(1)–Pt(1)–S (1) 83.44(16), N(1)–Pt(1)–P(1) 179.24(18), C(6)–Pt(1)–S(1) 162.95(19), C(6)–Pt(1)–P(1) 99.79(19), and C(6)–Pt(1)–N(1) 79.8(2). Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2024 Dalton Trans. Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online the relative IC 50 values of each compound for each cell line (Table 1). Moreover, as positive controls, the antiproliferative activity of the Dox and Cis was evaluated through the exposure of A2780, HCT116 and Fibroblast cells to 0.1–50 µM of these antitumor drugs for 48 h (ESI Fig. S2†). The selectivity index (SI) of each compound –the ratio IC 50 in fibroblasts/IC 50 in the tumour cell line –was determined, and it is reported in Table 1 to evaluate the selectivity of the compound towards tumour cell lines. Higher SI values were correlated with higher selectivity of the compound for a particular tumour cell line compared to the normal cells. 35 Comparing the IC 50 values obtained for the HCT116 cancer cell line for all of the tested compounds (Table 1), compounds 3a–cpresented the lowest IC 50 values (2.1, 1.9 and 8.6 µM, respectively) with the following cytotoxicity order: 3b >3a >3c. Moreover, A2780 cells compounds 3a–calso presented the lowest IC 50 values (5.0, 1.9, 8.0, respectively), maintaining the same cytotoxicity order of 3b >3a >3c (Table 1). Interestingly, in the HCT116 cells, these 3 compounds presented a higher cytotoxic potential when compared to the chemotherapeutic agent, cisplatin (IC 50 value of 15.6 µM). All the other compounds do not demonstrate cytotoxicity in the HCT116 colorectal carcinoma cell line (IC 50 > 50 µM). This clearly indicates that coordination with dppm –Ph 2 PCH 2 PPh 2 -Pprovides a higher cytotoxicity compared to the other phosphines (PPh 3 or dppb) (Table 1). In A2780 ovarian carcinoma cells, compounds 1a–c,2b, and 4a–cshow moderate cytotoxicity values (10 µM < IC 50 < 50 µM) (Table 1). Among all compounds, it seems that compounds with substitution b(RvMe) show more cytotoxic effects, although ligands 1,2and 3did not present any cytotoxicity when tested separately (Table 1). We have yet to find a plausible explanation for the differing values for compounds 3a,3b as compared to 3c. The modification of the –NHR group was not thought to have a significant effect on the modes of action of the compounds, but more in the fine-tuning of internalization and their properties. It is possible that R groups with longer carbon chains affect interactions with the biological membranes and/or targets. This puts forward the need to examine distinct options when designing a metallodrug. One of the main reasons and interest to develop new platinum-based compounds is to circumvent the high cytotoxicity of approved platinum drugs, and to reduce the side effects in healthy tissues. Therefore, it is important that compounds 3a– chave much higher IC 50 values in a normal human cell line when compared to the values obtained for the studied tumor cell lines. Fibroblasts were used as healthy cells due to their importance in the tumor microenvironment, 36 and the IC 50 values obtained are reported in Table 1. Interestingly, when analysing the IC 50 values obtained for fibroblasts (Table 1), all of the compounds showed low cytotoxic potential (IC 50 values >50 µM), which is reflected in the high SI values. At the limit, if we consider the IC 50 of compounds 3a–cin fibroblasts as equal to 50 µM, compounds 3a and 3b showed the highest SI values (23.8 and 26.3, respectively) in the HCT116 cancer cell line, meaning that those com- Table 1 Relative IC 50 values and SI obtained for each of the platinum compounds and respective ligands in HCT116, A2780 and fibroblast cell lines. IC 50 values are expressed as the mean ± SEM of at least three biological independent assays Complex Cell line IC 50 (µM) SI 1a HCT116 >50 — A2780 10 < IC 50 <50 — Fibroblasts >50 — 1b HCT116 >50 — A2780 35.7 >1.4 Fibroblasts >50 — 1c HCT116 >50 — A2780 10 < IC 50 <50 — Fibroblasts >50 — 2a HCT116 >50 — A2780 >50 — Fibroblasts >50 — 2b HCT116 >50 — A2780 31.9 >1.6 Fibroblasts >50 — 2c HCT116 >50 — A2780 >50 — Fibroblasts >50 — 3a HCT116 2.1 >23.8 A2780 5.0 >10 Fibroblasts >50 — 3b HCT116 1.9 >26.3 A2780 1.9 >26.3 Fibroblasts >50 — 3c HCT116 8.6 >5.8 A2780 8.0 >6.3 Fibroblasts >50 — 4a HCT116 >50 — A2780 10 < IC 50 <50 — Fibroblasts >50 — 4b HCT116 >50 — A2780 31.0 >1.6 Fibroblasts >50 — 4c HCT116 >50 — A2780 10 < IC 50 <50 — Fibroblasts >50 — L1 HCT116 >50 — A2780 >50 — Fibroblasts >50 — L2 HCT116 >50 — A2780 >50 — Fibroblasts >50 — L3 HCT116 >50 — A2780 >50 — Fibroblasts >50 — Doxorubicin (Dox) HCT116 0.5 ± 0.10 24.2 A2780 0.1 ± 0.04 121 Fibroblasts 12.1 ± 0.20 — Cisplatin (Cis) HCT116 15.6 ± 5.30 0.6 A2780 1.9 ± 0.20 4.6 Fibroblasts 8.8 ± 2.90 — > indicates that the IC 50 is higher than the value indicated; –SI values not calculated. Paper Dalton Transactions Dalton Trans. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 23 August 2024. Downloaded on 11/12/2024 1:40:19 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online pounds are at least 23.8× more cytotoxic for HCT116 cancer cells than for fibroblasts. Considering the same approach, compounds 3a and 3b also show higher SI values in the A2780 cancer cell line (10 and 26.3, respectively). Compound 3c presented a SI value of 5.8 in the HCT116 cell line, and a slightly higher SI value (6.3) for the A2780 cell line. It is also interesting to note that all of the ligands (a,band c) do not show a cytotoxic effect in the tested cell lines (IC 50 > 50 µM in tumor and normal cells). Interestingly, our previous data 37 show high cytotoxicity for the free PPh 3 (a ligand that is present in compounds 2a,2b and 2c) and for other free diphenylphosphines in normal fibroblasts. In the present work, we show a high cytotoxicity for compounds, particularly those harbouring dppm, in tumor cell lines and no cytotoxicity in fibroblasts. This means that this effect is probably attributed to the effects of the compounds per se, and not of the free ligands, even though free phosphines also have high cytotoxicity in these tumor cell lines. 37 Thus, compounds 3a,3b and 3c show an overall very good therapeutic window and potential in the HCT116 cell line and from now on, all of the remaining stability and biological analysis will be assessed for these 3 compounds in the HCT116 cell line. Complex stability in biological media After selection of the best compounds for pursuing additional biological characterization, the stability of the compound (3a, 3b and 3c) in biological medium was analyzed using UV-Visible spectroscopy over a wavelength range of 220 to 700 nm for different incubation times (−0, 3 h, as shown in Fig. 3; 0, 24, 48 h in ESI Fig. S5†). Analysing the spectra of all compounds, high-energy absorption bands corresponding to π→π* and n →π* transitions with peaks in the 230–330 and 330–400 nm ranges, respectively, that are associated with the aromatic rings of terpyridines can be observed. 38 According to Fig. 3, for compound 3a, bands are observed at approximately 235 nm, 265 nm and 360 nm at 0 h. For compound 3b, four characteristic bands can be identified at approximately 293, 325, 352 and 371 nm. Finally, compound 3c presents bands at approximately 235 nm, 265 nm, 327 nm, 365 nm, 380 nm and 530 nm. As observed in Fig. 3, from 0 h to 3 h, there is an increase of absorbance that is most probably due to a better solubilization of the complexes in the medium, maintaining their characteristic bands. However, analysing the data from ESI Fig. S5,†it is possible to observe a change in the peak at 265 nm to 272 nm from 0 h to 48 h for compound 3c. However, for 3a at 24 h, the band at 360 nm is no longer observed. Furthermore, at 48 h there is a peak at approximately 325 nm, which may indicate that the complex is not stable in solution (ESI Fig. S5†). Furthermore, there is not a large decrease in the absorbance values throughout the spectrum between the different acquisition times. Regarding compound 3b, the four characteristic bands remained at the same wavelength throughout the 48 h. However, between 0 h and 24 h, there is a slight decrease in all absorbances throughout the spectrum (ESI Fig. S5†). Considering the stability of all compounds during the first three hours, we further accessed their internalization in HCT116 cells in this period. Nevertheless, for ensuring the best solubility and stability of the compounds in all biological assays, they were always performed with freshly prepared solutions of these compounds. Compound cellular internalization by ICP-AES The internalization of the platinum compounds 3a,3b and 3c in HCT116 cells was evaluated by ICP-AES technique, which allows a quantitative measurement of the amount of metal present in each sample. This assay allows us to understand if the compounds under study are indeed internalized by HCT116 cells. 39 To perform this technique, HCT116 cells were Fig. 3 Evaluation of the stability of compounds 3a (A), 3b (B) and 3c (C) via UV-visible spectroscopy for 3 h. 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