Design, Synthesis, and Anticancer Studies of a <i>p</i>-Cymene-Ru(II)-Curcumin Organometallic Conjugate Based on a Fluorescent 4-Amino-1,8-naphthalimide Tröger's Base Scaffold.

pubs.acs.org/IC Article Design, Synthesis, and Anticancer Studies of a p‑Cymene-Ru(II)Curcumin Organometallic Conjugate Based on a Fluorescent 4‑Amino-1,8-naphthalimide Tröger’s Base Scaffold Binduja Mohan, Sandra Estalayo-Adrián, Deivasigamani Umadevi, Bjørn la Cour Poulsen, Salvador Blasco, Gavin J. McManus, Thorfinnur Gunnlaugsson,* and Sankarasekaran Shanmugaraju* Downloaded via SOONCHUNGYANG UNIV on November 8, 2022 at 05:45:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: Inorg. Chem. 2022, 61, 11592−11599 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: A unique V-shaped “chiral” supramolecular scaffold, N-(4-pyridyl)-4-amino-1,8-naphthalimide Tröger’s base (TBNap), was synthesized in good yield from a precursor N-(4-pyridyl)-4-amino-1,8-naphthalimide (Nap). TBNap was characterized using different spectroscopic methods and the molecular structure was elucidated by diffraction analysis. A new pcymene-Ru(II)-curcumin conjugate (TB-Ru-Cur) was designed by reacting TBNap dipyridyl donor and ruthenium-curcuminato acceptor [RuCur = (p-cymene)Ru-(curcuminato)Cl] in the presence of silver triflate. TB-Ru-Cur was isolated in quantitative yield and characterized using Fourier transform infrared (FT-IR), NMR (1H, 13C, and 19F), and electrospray ionization mass spectrometry (ESI-MS), and the molecular structure has been predicted using a computational study. Both TBNap and TB-Ru-Cur exhibited intramolecular charge transfer (ICT)-based fluorescence emission. Furthermore, the anticancer properties of TBNap, Ru-Cur, and TB-Ru-Cur were assessed in different cancer cell lines. Gratifyingly, the conjugate TB-Ru-Cur displayed fast-cellular internalization and good cytotoxicity against HeLa, HCT-116, and HepG2 cancer cells and the estimated IC50 value was much lower than that of the precursors (TBNap and Ru-Cur) and the well-known chemotherapeutic drug cisplatin. ■ limit their long-term usage for cancer treatments.8 Recently, ruthenium metal complexes have emerged as a viable substitute to Pt(II)-based metallodrugs because of their comparable ligand-exchange kinetics. As of now, three ruthenium metal complexes, namely, NAMI-A, KP1019, and (N)KP1339, are in clinical observations.9,10 Recently, arene-Ru(II) organometallic complexes have been realized for their anticancer properties because of their high solubility, easy functionalization, and improved cancer selectivity.11−14 Further, the arene-capping unit helps to maintain the structural stability of complexes under various physiological conditions. 14 Some known examples are [(C6H5Ph)Ru-(ethylenediamine)Cl][PF6] (RM175) and [(p- INTRODUCTION The design and synthesis of therapeutically active new organic ligands and metal complexes for cancer treatment is an active interdisciplinary field of research.1,2 While significant achievements have been made to date, it remains that the early-stage detection and effective treatment of cancer are still challenging, particularly because of multidrug resistance and the limited availability of cancer-targeting theragnostic agents.3,4 Hence, there exists the need to develop novel candidates to overcome such drawbacks. Metal complexes have prominent significance for cancer chemotherapy as metal ions are crucial for several biological processes.5 In particular, luminescent transitionmetal complexes have been identified as promising theragnostic probes, such as Ru(II)−polypyridyl complexes, because of their facile synthesis, easy structural modifications, and functional tunability.6 Furthermore, complexes such as cisplatin and its derivatives oxaliplatin and carboplatin are wellknown Pt(II)-based cancer drugs widely used to effectively inhibit several types of cancer cell growth.7 However, poor selectivity, dose-dependent toxicity, and multidrug resistance © 2022 American Chemical Society Received: March 26, 2022 Published: July 20, 2022 11592 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry pubs.acs.org/IC cymene)Ru(1,3,5-triaza-7-phosphotricyclo[3.3.1.1]-decane)Cl2] (RAPTA-C).15,16 These complexes selectively inhibit metastasis in vivo and exhibit poor in vitro anticancer activity. Nevertheless, the functionalization of such arene-Ru(II) complexes with therapeutically known bioactive ligands would enhance the antitumor efficacy.7,17 Curcumin is a naturally available bioactive ligand that is extracted from the plant Curcuma longa (turmeric) and is famous for its various medicinal properties such as anti-inflammatory, anti-Alzheimer’s disease effect, antitumoral effect, antiviral property, and so on.17−21 Considering the anticancer properties of curcumin, the Ru-Cur complex was developed by reacting the commercial curcumin powder with [(p-cymene)RuCl2]2 in methanol.22 It has been demonstrated that Ru-Cur is an effective anticancer agent against ovarian A2780 (IC50 = 23.4 μM) and breast MCF7 (IC50 = 19.6 μM) cancer cell lines. Later, the Ru-Cur complex was modified by replacing the ancillary chloride with the 1,3,5-triaza-7-phosphaadamantane ligand to enhance the cytotoxic action against A2780R and A2780 ovarian carcinoma cell lines.23 We have developed several Ru(II)-based luminescent metal complexes and organometallic conjugates based on TBNap and demonstrated their anticancer properties against different cancer cell lines.24−26 TBNaps are strongly emissive due to the “push−pull” type of intramolecular charge transfer (ICT) transition and their unique cleft-shaped structure, making them an attractive building block in supramolecular chemistry.25,26 We have developed a plethora of TBNap structures and successfully employed them as bifunctional scaffolds to generate multifunctional supramolecular structures, functional materials, and porous polymers for applications in biomedicine and environmental sciences.26−33 For instance, these have been used as imaging probes for live-cell staining, DNA targeting agents,24,27,28 and fluorescence sensors for chemical explosives and biologically important analytes.32 In this article, we report a new TBNap-based luminescent p-cymene-Ru(II) conjugate (TB-Ru-Cur) (see Scheme 1) and demonstrated its anticancer properties in different cancer lines. We foresaw that the cationic nature of TB-Ru-Cur would facilitate fast-cellular uptake, and it was found to be the case. The anticancer activity of TB-Ru-Cur was checked in HeLa, HCT-116, and HepG2 Article cancer cell lines, and cell viability studies established the high anticancer activity for TB-Ru-Cur, which is significantly prominent than Ru-Cur and TBNap precursors, and much more potent than cisplatin drug. These results demonstrate the importance of combining multiple anticancer ligands within TB-Ru-Cur. ■ EXPERIMENTAL SECTION Synthesis of TBNap. N-(4-Pyridyl)-4-amino-1,8-naphthalimide (Nap, 200 mg, 0.69 mmol, 1 equiv) and paraformaldehyde (31 mg, 1.04 mmol, 1.5 equiv) were taken in a 50 mL round-bottom flask. Trifluoroacetic acid (TFA, 4 mL) was added at 0 °C and the mixture was stirred at room temperature for 12 h under N2 atmosphere. The reaction mixture was neutralized by adding NH4OH (100 mL) until a pH > 10 was reached. The organic layer was extracted in dichloromethane (DCM) (200 mL) and washed with saturated NaHCO3 (2 × 100 mL), brine (2 × 100 mL), and H2O (2 × 100 mL). The solution was dried over MgSO4 and the solvents were removed and triturated with cold diethyl ether to isolate TBNap (230 mg, 0.37 mmol, 54%). Anal. calcd (%) for C37H22N6O4.CH2Cl2: C, 65.24; H, 3.46; N, 12.01. Found: C, 65.73; H, 3.62; N, 11.79. Melting point: 319−321 °C (decomp.). HRMS (APCI) m/z: calcd for C37H23N6O4 [M + H+] 615.1775, found 615.1760; 1H NMR (400 MHz, (CD3)2SO) δ 8.82−8.80 (2H, d, J = 8.0 Hz, Ar−H), 8.74−8.72 (4H, d, J = 8.0 Hz, pyridyl-Hα), 8.52−8.50 (2H, d, J = 8.0 Hz, Ar−H), 8.16 (2H, s, Ar−H), 8.03−7.99 (2H, t, J = 8.0 Hz, Ar−H), 7.42−7.40 (4H, d, J = 8.0 Hz, pyridyl-Hβ), 5.25−5.20 (2H, d, J = 20.0 Hz, NCH2), 4.79 (2H, s, NCH2), 4.74−4.69 (2H, d, J = 20.0 Hz, NCH2); 13 C NMR (101 MHz, (CD3)2SO) δ 150.56, 148.38, 143.91, 130.76, 130.38, 129.49, 128.00, 127.22, 126.81, 126.23, 124.57, 122.79, 117.90, 64.90, 56.91, 54.90, 15.15; FT-IR υmax (ATR, cm−1) 3326, 31.62, 3053, 2163, 1699, 1666, 1586, 1572, 1507, 1459, 1405, 1356, 1300, 1244, 1188, 1126, 1088, 1038, 931, 781, 691, 659, 626, 587. Synthesis of TB-Ru-Cur. A mixture of Ru-Cur (30 mg, 0.047 mmol, 2 equiv) and AgCF3SO3 (13 mg, 0.052 mmol, 2.2 equiv) was stirred in 1:1 DCM-CH3OH (8 mL) at room temperature for 1 h in dark and then filtered through celite to remove AgCl. TBNap (14 mg, 0.023 mmol, 1.0 equiv) was added to the filtrate, which was stirred overnight at room temperature. Then, the reaction mixture was filtered and the solvent was stripped under vacuum. The residue was redissolved in 3 mL of DCM-CH3OH (1:1) and diethyl ether was slowly diffused to isolate the product TB-Ru-Cur (20 mg, 0.011, 48%) as an orange-brown solid. Anal. calcd (%) for C101H88F6N6O22Ru2S2·6CH2Cl2: C, 48.91; H, 3.84; N, 3.20. Found: C, 48.93; H, 3.52; N, 3.17. Melting point: 285−288 °C (decomp.); HRMS (ESI+) calcd for [M − (RuCur + SO3CF3−)]+ m/z = 1217.3036 (C68H55N6O10Ru), found m/z = 1217.3016. 1H NMR (400 MHz, CDCl3−CD3OD) δ 9.24−9.22 (d, J = 8.0 Hz, 2H, TBNap-H3), 9.05−9.04 (d, J = 4.0 Hz, 4H, pyridyl-Hα), 8.97−8.95 (d, J = 8.0 Hz, 2H, TBNap-H5), 8.46 (s, 2H, TBNap-H6), 8.08−8.04 (t, J = 8.0 Hz, 2H, TBNap-H4), 7.91−7.90 (d, J = 4.0 Hz, 4H, CurH16), 7.88−7.86 (m, 12H, Cur-H17−19), 7.27−7.26 (d, J = 4.0 Hz, 4H, pyridyl-Hβ), 6.84−6.80 (d, J = 16.0 Hz, 4H, Cur-H15), 6.18−6.16 (d, J = 8.0 Hz, 4H, cymene-H10), 5.98−5.96 (d, J = 8.0 Hz, 4H, cymeneH9), 5.81 (s, 2H, Cur-H14), 5.62−5.57 (d, J = 20 Hz, 2H, TBNapH7), 5.03−5.00 (d, J = 12 Hz, 2H, TBNap-H7′), 4.95 (s, 2H, TBNapH8), 4.32 (s, 12H, Cur-OCH3), 3.41−3.35 (m, 2H, cymene-H13), 2.66 (s, 6H, cymene-H11), 1.86−1.84 (d, J = 8.0 Hz, 12H, cymeneH12); 13C NMR (101 MHz, CDCl3-CD3OD) δ 178.34, 163.18, 162.58, 152.31, 150.37, 148.66, 147.68, 145.99, 140.38, 131.56, 131.31, 130.10, 128.33, 127.25, 126.92, 126.29, 125.67, 123.19, 122.82, 121.76, 117.28, 115.18, 109.76, 102.91, 98.46, 83.56, 81.88, 65.64, 56.94, 55.43, 30.60, 21.86, 16.92, 14.51. 19F NMR (377 MHz, CDCl3−CD3OD) δ −79.71 (s, 6F, SO3CF3); FT-IR υmax (ATR, cm−1) 2950, 1706, 1669, 1623, 1593, 1507, 1400, 1375, 1341, 1241, 1218, 1158, 1128, 1027, 991, 967, 932, 808, 777, 638, 578. Scheme 1. Synthesis of TB-Ru-Cur 11593 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry pubs.acs.org/IC ■ RESULTS AND DISCUSSION Synthesis and Characterization of TBNap. As shown in Scheme 2, TBNap, (bis-[N-(4-pyridyl)]-9,18-methano-1,8- Article shaped confirmation due to the methano-1,5-diazocine ring with the bridgehead angle of 112.25°, and the mean interplanar angle between two 1,8-naphthalimide moieties is 94.68° (Figure 2A). The packing view of TBNap showed face-to- Scheme 2. Synthesis of TBNap from Precursor Nap naphthalimide-[b,f ][1,5]diazocine), was synthesized as a racemic mixture in 54% yield from the precursor Nap, N-(4pyridyl)-4-amino-1,8-naphthalimide, which was obtained from N-(4-pyridyl)-4-nitro-1,8-naphthalimide by Pd/C catalytic hydrogenation.29,33 Various spectroscopic techniques were used to characterize TBNap (for details, see the Supporting Information). In the 1H NMR spectrum, two doublets and a singlet appeared in the range of δ = 5.16−4.63 ppm due to the −CH2− proton resonances of the diazocine moiety and the peaks corresponding to the 1,8-naphthalimide and 4-pyridyl moieties appeared at δ = 8.82−7.41 ppm (Figure 1).26 The HRMS analysis showed an intense peak at m/z = 615.176 ascribed to the molecular ion [M + H]+ (Figure S6). Further, the diffraction analysis was used to elucidate the molecular structure of TBNap. The diffraction quality crystals were obtained from the saturated DCM-CH3OH (2:1) solution of TBNap by evaporating at room temperature. Crystallographic details are provided in Table S1. The diffraction analysis revealed that TBNap crystallized in monoclinic space group P21/c, and the structural model evidenced that the 1,8-naphthalimide units are placed in cleft- Figure 2. (A) Molecular structure of TBNap obtained using diffraction analysis. (B) π−π stacking interactions (3.73 Å) between two 1,8-naphthalimide moieties. Hydrogen atoms are excluded for clarity (CCDC no. 2121304). face π−π stacking interactions (dcentroid−dcentroid = 3.73 Å) between the adjacent ligands, which resulted in head-to-tail arrangements of naphthalimide moieties (Figure 2B). Further, Figure 1. 1H NMR spectrum of TBNap (400 MHz, DMSO-d6) with peak assignment. 11594 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry pubs.acs.org/IC Article Figure 3. 1H NMR of TB-Ru-Cur {400 MHz, CDCl3-CD3OD (3:1 volume ratio)} with peak assignment. *Peaks of diethyl ether cocrystallized in the recrystallization process. the solid-state packing diagram of TBNap evidenced the presence of multiple C−H···O hydrogen bonding and C−H···π interactions between the adjacent molecules (Figure S7). Synthesis and Characterization of TB-Ru-Cur. As shown in Scheme 1, TB-Ru-Cur was synthesized in a single step via metal−ligand coordination assembly. To facilitate the anion exchange, first, Ru-Cur (acting as an acceptor) was treated with silver triflate in CH2Cl2−CH3OH (1:1) solution and the anion exchanged Ru-Cur was subsequently reacted with the dipyridyl TBNap (acting as a donor) in a 2:1 stoichiometry ratio to obtain the expected TB-Ru-Cur in 48% yield. TB-Ru-Cur was air-stable and highly soluble in common solvents. The p-cymene capping moieties engender high structural stability and also limit any further coordination. The coordinated curcuminato ligand further enhances the structural stability via the chelate effect. TB-Ru-Cur was characterized using Fourier transform infrared (FT-IR), NMR, and electrospray ionization mass spectrometry (ESI-MS) analyses. The FT-IR spectrum showed several intense bands due to the functional groups present in it (see Figure S1). In the 1H NMR spectrum recorded in CDCl3−CD3OD, the signals for the methylene (−CH2N−) group of the diazocine moiety appeared as two doublets in the range of 5.62−5.00 ppm, which confirmed the presence of Tröger’s base unit (Figure 3). Notably, the peaks corresponding to the 4-pyridyl unit of coordinated TBNap were slightly downfield (∼0.3 ppm) shifted compared to the free TBNap, which is because of the decrease of electron density upon metal−ligand coordination bonding. The proton signals of p-cymene moiety appeared as two sharp doublets at 6.18−5.96 ppm and all the proton resonances due to the curcuminato fragments appearing as sharp signals (Figure 3). The 19F NMR spectrum showed a single peak at δ = 79.71 ppm assigned to the triflate counter anions and the appearance of a single peak also suggested that two triflate anions are chemically equivalent (Figure S9). Furthermore, the appearance of an isotopically well-resolved prominent peak at m/z = 1217.3036 for the charged fragment [M − (RuCur + OTf−)]+ confirmed the formation of TB-RuCur conjugate (Figure S10). All attempts to obtain the diffraction quality single crystals of TB-Ru-Cur were unsuccessful and therefore, computational calculations were conducted to predict the energy-minimized structure. The calculations were conducted using the semiempirical PM6 method on the Gaussian 09 program.34 The optimized structure of TB-Ru-Cur established a cleft-shaped conformation with the mean interplanar angle between two 1,8-naphthalimide moieties being 103.96°, which is slightly higher than the interplanar angle (94.68°) in free TBNap (Figure 4). The two curcuminato units are flanked outward from the cleft and slightly tilted away from the plane of TBNap to minimize the steric influence. The two Ru(II) ions adopt a pseudo-octahedral coordination geometry and the distance between them is 22.3 Å. Three coordination sites on each Ru(II) are occupied by the capped p-cymene, while the curcuminato ligand coordinates through its two oxygen atoms and the 4-pyridyl “N” occupies the sixth coordination site. The two pyridyl-N donor sites are coordinated to two different Ru(II) centers, which gives rise to a cleft conformation. The Ru−N bond distance is 2.08 Å, which is slightly less than the Ru−O bond distance of 2.10 Å. Photophysical Study. We next studied the photophysical properties of TBNap and TB-Ru-Cur at room temperature (see Figure 5). The absorption and emission spectra were recorded using a 2.5 μM CH2Cl2 stock solution. The ultraviolet−visible (UV−vis) absorption spectrum of TBNap showed a band at λ = 346 nm (ε = 19.3 × 103 M−1 cm−1) corresponding to π−π* transition and a band at λ = 387 nm (ε = 23.7 × 103 M−1 cm−1) assigned to the ICT transition (Figure 5A). Likewise, the absorption spectrum of TB-Ru-Cur displayed four typical bands at λ = 347−470 nm (Figure 5A).26−33 The characteristic metal-to-ligand charge transfer (MLCT) absorption band (due to the transition from the 11595 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry pubs.acs.org/IC Article from the computational calculations.34 It is noticed that the highest occupied molecular orbital (HOMO) density was localized on the Ru(II) centers, while the lowest unoccupied molecular orbital (LUMO) density was largely on the 1,8naphthalimide moieties (Figure S11). These results strongly supported the existence of the proposed MLCT process. Biological Studies. We next assessed the cellular uptake and antiproliferative activity of TBNap and TB-Ru-Cur in three different cancer cell lines such as HeLa (cervical cancer), HCT-116 (colon carcinoma), and HepG2 (hepatocellular carcinoma). ICT-based strong fluorescence emission facilitates the visualization of the cellular internalization of compounds using confocal laser scanning microscopy (CLSM). At first, both TBNap and TB-Ru-Cur were treated separately with HeLa cells and the cells were incubated at different incubation times of 3, 30, and 60 min at 37 °C. Then, the incubated HeLa cells were dosed with a DRAQ5 nuclear stain, and the cells were imaged using CLSM. Due to its nonemissive nature, the cellular internalization of Ru-Cur was not able to be visualized. A few representative confocal images of HeLa cells with TBNap and TB-Ru-Cur at different incubation times are presented in Figure 6. The obtained microscopy images Figure 4. (A) Front and (B) top view of the optimized structure of TB-Ru-Cur. Hydrogen atoms are excluded for clarity. Ru(II) filled 4d orbitals to the empty π* orbitals of the ligand) appeared at λ = 470 nm (ε = 76.4 × 103 M−1 cm−1) and the two intense bands at λ = 347 (ε = 55.0 × 103 M−1 cm−1) and 443 (ε = 86.7 × 103 M−1 cm−1) are due to the inter/intramolecular π−π* transitions.24 Notably, the ICT-based absorption band at λ = 400 nm (ε = 105.4 × 103 M−1 cm−1) was red-shifted by ∼13 nm compared to TBNap; this red shift is presumably due to the influence of coordination interactions (Figure 5A). Upon excitation at λ = 387 nm, TBNap exhibited an intense ICT emission band at λ = 504 nm, while ICT-based emission of TB-Ru-Cur was observed at λ = 511 nm when it was excited at λ = 400 nm (Figure 5B). The ICT-based emission of TB-Ru-Cur was slightly red-shifted by 7 nm due to the effect of coordination bonding. The calculated quantum yield for TBNap is Φ = 0.242 and 0.135 for TB-Ru-Cur. Notably, the emission intensity of TB-Ru-Cur was lower than that of TBNap emission; this is due to the excited-state energy transfer from TBNap to the curcumin moiety, which quenches the emission intensity. The proposed energy transfer process is supported by the absorption and emission spectra overlap of TB-Ru-Cur. The existence of an energy transfer in TB-Ru-Cur was further supported by the frontiers molecular orbital (highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)) diagram obtained Figure 6. Confocal live-cell images of TBNap (5 μM) and TB-RuCur (5 μM) within HeLa cells at different incubation times. Green emission = TBNap or TB-Ru-Cur and red emission = DRAQ5. exhibited that both TBNap and TB-Ru-Cur were quickly taken up into cells, even after 3 min of incubation, and localized within the cytoplasm or on the edges of the nucleus. Both TBNap and TB-Ru-Cur exhibited green emission and the red emission in the nucleus corresponds to the DRAQ5 nuclear stain. However, both TBNap and TB-Ru-Cur were almost undetectable inside HCT-116 and HepG2 cancer cells probably because of their poor cellular uptake or week emissivity at the concentration of study (Figures S12−S15). Furthermore, the addition of TB-Ru-Cur to HepG2 cells seems to harm the cell integrity, where the extracellular membrane is “blebbing” up, suggesting that cells undergo Figure 5. (A) Absorption and (B) emission spectra of TBNap and TB-Ru-Cur recorded in CH2Cl2 (2.5 × 10−6 M) at room temperature. 11596 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry pubs.acs.org/IC apoptosis and necrosis (Figure S15).35 The fast-cellular uptake of TB-Ru-Cur can be attributed to its cationic nature, which facilitates enhanced passive diffusion through the cancer cell membrane.36 The quick cellular internalization and strong fluorescence emission within the HeLa cells endow the application of TBNap and TB-Ru-Cur as cancer diagnostic probes. Inspired by the fast-cellular uptake in HeLa cells and cytoplasmic localization, we next assessed the cytotoxicity potential in HCT-116, HeLa, and HepG2 cancer cells for the precursors TBNap and Ru-Cur as well for the conjugate TBRu-Cur using the Alamar Blue viability assay. The cancer cells were incubated with different compounds at varying concentrations in the dark for 48 h and then the cell viability was evaluated. The estimated IC50 is summarized in Table 1. significantly more potent than that seen for the individual components. Furthermore, as TB-Ru-Cur is highly emissive and easily identifiable within the cellular environment, it makes TB-Ru-Cur a potential theragnostic agent in cancer therapy. ■ CONCLUSIONS In conclusion, we have synthesized a unique Tröger’s base naphthalimide dipyridyl scaffold, TBNap, and successfully used it to develop a new p-cymene-Ru(II)-curcuminato luminescent conjugate TB-Ru-Cur. The structures of TBNap and TB-RuCur were characterized using standard analytical methods. Further, the molecular structure of the TBNap scaffold was elucidated by diffraction analysis. Due to the ICT-based transition, both TBNap and TB-Ru-Cur were emissive, and thus, using confocal fluorescence microscopy, we have demonstrated the cellular uptake for TBNap and TB-Ru-Cur both being localized within the cytoplasm of cancer cells. Furthermore, their cytotoxicity potentials against three cancer cell lines were assessed, and cell viability studies showed significantly more anticancer potential for TB-Ru-Cur than for the corresponding precursors, with TBNap being nontoxic and Ru-Cur exhibiting moderate cytotoxicity. In summary, the work discussed herein evidences the versatility of TBNaps as potential scaffolds that can be used to generate potential therapeutically active ligands and metal complexes. Further works are in progress to functionalize TB-Ru-Cur to improve its selectivity for cancer cells and enhance the anticancer efficacy via conjugating the arene-Ru(II) complex to various known bioactive ligands for their applications in cancer therapy. Table 1. IC50 Values for TBNap, Ru-Cur, and TB-Ru-Cur in HeLa, HCT-116, and HepG2 Cancer Cells IC50 ± SEM (μM) 48 h incubation in the dark ligands and complexes HeLa HCT-116 HepG2 TBNap Ru-Cur TB-Ru-Cur cisplatin >100 15 ± 3 2.0 ± 0.3 13 >100 16 ± 1 2.7 ± 0.1 >100 16.6 ± 0.6 1.9 ± 0.1 Article For comparison, the IC50 value of the cisplatin drug was measured under identical conditions and included in Table 1. As shown in Figure 7, up to 100 μM concentration of TBNap did not induce any cytotoxicity in three cell lines and thus IC50 values for TBNap were not determined. Ru-Cur exhibited moderate cytotoxicity against the three different cell lines and the IC50 values estimated were 15 ± 3, 16 ± 1, and 16.6 ± 0.6 μM in HeLa, HCT-116, and HepG2 cells, respectively. Gratifyingly, the conjugate TB-Ru-Cur showed significant cytotoxicity against all three cancer cell lines, being on all occasions significantly more potent than the individual components, with the calculated IC50 values of 2.0 ± 0.3, 2.7 ± 0.1, and 1.9 ± 0.1 μM being determined in HeLa, HCT-116, and HepG2, respectively. Notably, the estimated IC50 value of TB-Ru-Nap was found significantly lower compared to cisplatin (IC50 = 13 μM), which was similar to that seen for Ru-Cur alone.37 As Table 1 demonstrates, the anticancer potency of TB-Ru-Nap measured in three different cancer cells was significantly higher than that seen for TBNap and Ru-Cur. The estimated higher anticancer potency of TB-Ru-Cur is presumably due to the combined activity of two anticancer agents Ru-Cur and TBNap motifs, as TB-Ru-Cur is ■ ASSOCIATED CONTENT sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c01005. Details of characterization data {FT-IR, NMR (1H, 13C, and 19F), ESI-MS}, X-ray crystallography, computational simulation, and additional confocal cellular images (PDF) Accession Codes CCDC 2121304 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. Figure 7. Toxicity profile of TBNap, Ru-Cur, and TB-Ru-Cur in (A) HeLa, (B) HCT-116, and (C) HepG2 cancer cells. 11597 https://doi.org/10.1021/acs.inorgchem.2c01005 Inorg. Chem. 2022, 61, 11592−11599 Inorganic Chemistry ■ pubs.acs.org/IC AUTHOR INFORMATION (5) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen, Z.-S. The Development of Anti-cancer Ruthenium(II) Complexes: From Single Molecule Compounds to Nanomaterials. Chem. Soc. Rev. 2017, 46, 5771−5804. 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Corresponding Authors Thorfinnur Gunnlaugsson − School of Chemistry and Trinity Biomedical Sciences Institute, Trinity College Dublin, The University of Dublin, Dublin 2 D02 PN40, Ireland; orcid.org/0000-0003-4814-6853; Email: gunnlaut@ tcd.ie Sankarasekaran Shanmugaraju − Department of Chemistry, Indian Institute of Technology Palakkad, Palakkad 678557 Kerala, India; orcid.org/0000-0002-3283-7847; Email: shanmugam@iitpkd.ac.in Authors Binduja Mohan − Department of Chemistry, Indian Institute of Technology Palakkad, Palakkad 678557 Kerala, India Sandra Estalayo-Adrián − School of Chemistry and Trinity Biomedical Sciences Institute, Trinity College Dublin, The University of Dublin, Dublin 2 D02 PN40, Ireland Deivasigamani Umadevi − Department of Chemistry, Indian Institute of Technology Palakkad, Palakkad 678557 Kerala, India; orcid.org/0000-0001-9732-2556 Bjørn la Cour Poulsen − School of Chemistry and Trinity Biomedical Sciences Institute, Trinity College Dublin, The University of Dublin, Dublin 2 D02 PN40, Ireland Salvador Blasco − Instituto de Ciencia Molecular, Universidad de Valencia, 46980 Paterna, Spain Gavin J. McManus − School of Biochemistry and Immunology and Trinity Biomedical Sciences Institute, Trinity College Dublin, The University of Dublin, Dublin 2 D02 PN40, Ireland Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.2c01005 Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The authors are grateful to the Science and Engineering Research Board (EMEQ Award EEQ/2018/000799 to S.S.) and Science Foundation Ireland (SFI PI Awards 10/45 IN.1/ B2999 and 13/IA/1865 to T.G.) for the financial support. S.B. thanks ERC for a Marie Curie grant. B.M. and S.S. are thankful to the Department of Chemistry and CIF-CMFF, IIT Palakkad, for providing the characterization facilities. ■ Article REFERENCES (1) (a) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly, J. M.; Gunnlaugsson, T. The Development of Ruthenium(II) Polypyridyl Complexes and Conjugates for In-vitro Cellular And Invivo Applications. Chem. Soc. 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Design, Synthesis, and Anticancer Studies of a p-Cymene-Ru(II)-Curcumin Organometallic Conjugate Based on a Fluorescent 4-Amino-1,8-naphthalimide Tröger's Base Scaffold.