Synthesis and characterization of novel naphthalene-derivatized tridentate ligands and their net neutral rhenium tricarbonyl complexes and cytotoxic effects on non-small cell lung cancer cells of interest

Journal Pre-proofs Synthesis and characterization of novel naphthalene-derivatized tridentate li‐ gands and their net neutral rhenium tricarbonyl complexes and cytotoxic ef‐ fects on non-small cell lung cancer cells of interest Taniya Darshani, Frank R Fronczek, Varuni V Priyadarshani, Sameera R Samarakoon, Inoka C Perera, Theshini Perera PII: S0277-5387(20)30309-0 DOI: https://doi.org/10.1016/j.poly.2020.114652 Reference: POLY 114652 To appear in: Polyhedron Received Date: 28 March 2020 Revised Date: 2 June 2020 Accepted Date: 3 June 2020 Please cite this article as: T. Darshani, F.R. Fronczek, V. V Priyadarshani, S.R. Samarakoon, I.C. Perera, T. Perera, Synthesis and characterization of novel naphthalene-derivatized tridentate ligands and their net neutral rhenium tricarbonyl complexes and cytotoxic effects on non-small cell lung cancer cells of interest, Polyhedron (2020), doi: https://doi.org/10.1016/j.poly.2020.114652 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd. Synthesis and characterization of novel naphthalene-derivatized tridentate ligands and their net neutral rhenium tricarbonyl complexes and cytotoxic effects on non-small cell lung cancer cells of interest Taniya Darshani1, Frank R Fronczek2, Varuni V Priyadarshani3 , Sameera R Samarakoon3, Inoka C Perera4, Theshini Perera*1 1Department of Chemistry, University of Sri Jayewardenepura, Sri Lanka (*theshi@sjp.ac.lk) 2Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 3Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 4Department of Zoology and Environment Science, University of Colombo, Sri Lanka 1 Abstract Rhenium complexes have possible fluorescence imaging, radioimaging and therapeutic applications and also can serve as a model system for 99mTc. The quest for new pharmaceutical agents is thus an expanding area of research. In this study, we report two net neutral complexes, [Re(CO) (N(SO )(1-nap)dien)] and [Re(CO) (N(SO )(2-nap)dien)], 3 2 3 2 synthesized by treating fac-[Re(CO) (H O) ]+ with two novel tridentate ligands, (N(SO )(1- 3 2 3 2 nap)dienH and N(SO )(2-nap)dienH), derived from diethylenetriamine (dien) attached to a 2 sulfonamide group. The compounds were characterized by UV-Visible, FTIR, 1H NMR and fluorescence spectroscopies, together with X-ray crystallography. It is evident from the 1H NMR spectroscopic data recorded in DMSO-d as well as X-ray crystallography, supported 6 by FTIR data, that upon coordination to the metal centre, the sulfonamide nitrogen atom is deprotonated and the ligand binds with the metal in a tridentate fashion. Both the complexes have a distorted octahedral structure where the Re(I) metal is coordinated by three nitrogen atoms of the dien backbone and three CO ligands. Emission spectra were recorded in methanol and an enhanced fluorescence emission was observed in the 330-345 nm range for the ligands while the corresponding Re complexes showed no or quenched fluorescence intensities. The in vitro cytotoxic activity of the synthesized compounds was examined using NCI-H292 (non-small cell lung cancer cells) and MRC-5 (the human normal lung fibroblast cell line). The highest cytotoxic activity was observed for the [Re(CO) (N(SO )(1-nap)dien)] 3 2 complex on NCI-H292 cells with an IC value of 9.91 M at 48 h. However all four 50 compounds show acute cytotoxicity for MRC-5 cells at 24 h. The promising cytotoxic activity of the novel ligands and metal complexes indicate that these compounds may be good candidates to be utilized as anticancer drug leads against lung cancer. Keywords: rhenium tricarbonyl, naphthalene, sulfonamide, anticancer, fluorescence 2 1. Introduction Organometallic compounds serve as promising anti-cancer drug candidates [1-4] and cell imaging agents [5-10]. Technetium-99m (99mTc) is the most widely utilized radioisotope in diagnosis due to its ideal nuclear properties, low cost and widespread availability [11, 12]. In the past few decades, analogous fac-Re(CO) complexes have been widely studied as a 3 model system for 99mTc(CO) diagnostic agents [13]. However, the focus has now shifted to 3 rhenium complexes as anti-cancer agents in their own right [4, 14, 15]. Simpson et al recently investigated the anticancer properties of compounds against pancreatic cell lines and revealed the remarkable activity of the rhenium fragment as an anticancer agent [14]. Studies of physicochemical properties provide evidence for the activity of metal bound compounds in biological systems [3]. Rhenium complexes are favoured over other metals due to their kinetic inertness, long lifetime and large Stoke’s shift. [10, 16, 17]. Schibli et al. noted that pharmaceutical agents of the type fac-[99mTc(CO) L]+ where L is a tridentate 3 ligand system are more robust and possess better pharmacokinetics than those bearing bidentate ligands [18]. The charge and the overall shape influence the biological behavior [5]. Christoforou et al. investigated the chirality of diethylenetriamine (dien) moiety based NNN donor ligands [19-21] and noted that the position of the pendant group [19] as well as the linker group [21] affect the overall shape of the agent to be used as a potential radiopharmaceutical. Thus, it is of vital importance to select a suitable ligand system. Sulfonamide derivatives represent an important class of molecules, with a range of biological applications [22]. The sulfonamide group may also serve as a potential anchor for bioconjugation [19-21, 23]. Small nuclide pharmaceutical agents containing a net neutral core are possible when fac-[M(CO) (H O) ]+ (M = 99mTc, Re) is treated with a tridentate 3 2 3 monoanionic sulfonamido ligand. NNN donor ligands bearing terminal sulfonamides will be 3 useful in the development of pharmaceuticals with a 99mTc(CO) core. In this study, we 3 specifically opted to synthesize two naphthalene derivatized ligands. Compounds of naphthalene derivatives have been reported to exhibit various important biological activities, such as anticancer [24-26], antimicrobial [27, 28], anti-inflammatory [29] and anti- neurodegenerative [30, 31] activities. Naphthalene-based compounds, such as nafacillin, naftifine, tolnaftate and terbinafine, are currently being used as therapeutics [32]. Here we have utilized a three-pronged approach in designing ligands and complexes. Firstly, understanding the chemistry of rhenium complexes complements the development of 99mTc and 186/188Re radiopharmaceuticals. Secondly it is an added advantage if the compounds are fluorescent. Thirdly, the compounds themselves may possess anticancer activity if prudently designed. Our research has been fueled by the fact that although many organometallic compounds have been reported to possess various biological activities [33-42], alternative anti-cancer drugs are still being sought for platinum-based drugs to circumvent platinum resistance and the side effects [2, 43] associated with platinum based drugs. Lung cancer remains the leading cause of cancer deaths in the world. The survival rate of patients undergoing lung resection is reported to be over 80%, suggesting the importance of early cancer detection and treatment for specific cancer subtypes at an early stage [44]. In this study, two novel ligands, N(SO R)dienH (R = 1-naphthalene, 2-naphthalene), 2 derived from diethylenetriamine and containing a secondary sulfonamide group, together with their corresponding Re(I) complexes (Figure 1), were synthesized, characterized and their preliminary biological applications evaluated. We specifically report on the applicability of the Re(I) complexes as potential anti-cancer drug leads towards lung cancer. 4 Figure 1: Proposed ligands N(SO )(1-nap)dienH (L1), N(SO )(2-nap)dienH (L2) and the 2 2 complexes fac-[Re(CO) (N(SO )(1-nap)dien)] (C1), fac-[Re(CO) (N(SO )(2-nap)dien)] (C2) 3 2 3 2 used in this study. From this point on, the fac- designation is omitted for the metal complexes as both the complexes reported in the study have the facially coordinated geometry. 2. Experimental section 2.1. Starting materials 1-Naphthalenesulfonyl chloride ((1-nap)SO Cl), 2-naphthalenesulfonyl chloride ((2- 2 nap)SO Cl), diethylenetramine (dien), Re (CO) , dioxane, chromasolv water and 2 2 10 dichloromethane were used as received from Sigma-Aldrich. [Re(CO) (H O) ]OTf (OTf = 3 2 3 trifluoromethanesulfonate) was prepared by a known method [45]. MRC-5 (human lung fibroblast cell line) and NCI-H292 (non-small cell lung cancer cells) were obtained from the American Type Culture Collection. 2.2. Methodology TLC analysis was performed and visualized with ultraviolet light. UV-Vis absorption spectra were obtained using a GENESIS 10S UV-Vis spectrophotometer. Data were processed with UV WIN software. Spectra were obtained in methanol with baseline correction. FTIR spectra were recorded with a Thermo Scientific NICOLET iS10 5 spectrometer. Data were processed with OMNIC software. Fluorescence spectra were obtained on a Thermo Scientific Lumina spectrophotometer using a 150W Xenon arc lamp as the excitation source. Data were processed with Luminous software. Solutions were prepared by dissolving the analyte in methanol. 1H NMR spectra were recorded on a Bruker 400 MHz spectrometer in DMSO-d . Peak positions are relative to tetramethylsilane (TMS) and the 6 data were analysed with MestReNova software. X-ray data was collected using BrukerAPEX2 at 100 K. 2.2.1. Synthesis of the N(SO R)dienH ligands 2 A solution of sulfonyl chloride (5 mmol) in 100 ml of dioxane was added dropwise over 2 h to a solution of N(H)diene (50 mmol) in 100 ml of dioxane. The reaction mixture was stirred overnight at room temperature. The dioxane was completely removed by rotary evaporation and water (50 ml) was added. The product was extracted into CH Cl (2 x 100 2 2 ml) and the solvent was removed under rotary evaporation. 2.2.1.1. Synthesis of N(SO )(1-nap)dienH ligand (L1) 2 The general procedure described above with (1-nap)SO Cl (0.58 g, 2.5 mmol) and 2 N(H)diene (2.71 ml, 25 mmol) yielded the ligand as a deep red oil (0.46 g, 63% yield). Anal. calc. for C H N O S.2H O: C, 51.05; H, 7.04; N, 12.76; S, 9.73. Found: C, 51.73; H, 6.61; 14 19 3 2 2 N, 12.18; S, 9.48%. 1H NMR (DMSO-d , 400 MHz) δ (ppm): 8.66 (d, 1H), 8.23 (d, 1H), 8.14 6 (d, 1H), 8.09 (d, 1H), 7.63-7.74 (m, 3H), 2.84 (t, 2H, CH ), 2.43 (m, 4H, CH ), 2.28 (t, 2H, 2 2 CH ). 2 2.2.1.2. Synthesis of N(SO )(2-nap)dienH ligand (L2) 2 The general procedure described above with (2-nap)SO Cl (1.14 g, 5 mmol) and 2 N(H)diene (5.43 ml, 50 mmol) yielded the ligand as a deep red oil (1.44 g, 98% yield). Anal. calc. for C H N O S.H O: C, 54.00; H, 6.80; N, 13.49; S, 10.30. Found: C, 54.27; H, 6.25; 14 19 3 2 2 N, 12.11; S, 9.76%. 1H NMR (DMSO-d , 400 MHz): δ (ppm) 8.45 (s, 1H), 8.13-8.18 (m, 6 6 2H), 8.05 (d, 1H), 7.85 (d, 1H), 7.66-7.72 (m, 2H), 2.94 (t, 2H), 2.84-2.87 (m, 2H), 2.51-2.53 (m, 2H), 2.42 (t, 2H). 2.2.2. Synthesis of the [Re(CO) (N(SO R)dien)] complexes 3 2 An aqueous solution of [Re(CO) (H O) ]OTf (1.00 ml, 0.1 mmol) was treated with a 3 2 3 methanol solution of the ligand (0.1 mmol). The pH was adjusted to ~5, the reaction mixture was heated at reflux for 12 h and then allowed to cool at room temperature. The pH was increased to ~7. The obtained solid precipitate was collected on a filter. 2.2.2.1. Synthesis of [Re(CO) (N(SO )(1-nap)dien)] (C1) 3 2 Treatment of [Re(CO) (H O) ]OTf with the N(SO )(1-nap)dienH ligand (0.029 g, 0.1 3 2 3 2 mmol), as described above, yielded [Re(CO) (N(SO )(1-nap)dien)] as a white powder (0.049 3 2 g, 88% yield). 1H NMR (DMSO-d , 400 MHz) δ (ppm): 8.79 (d, 1H), 7.98-8.06 (m, 3H), 6 7.53-7.62 (m, 3H), 6.80 (s, 1H, N2H), 5.23 (b, 1H, N1H), 3.59 (b, 1H, N1H), 2.66-2.98 (m, 8H). 2.2.2.2. Synthesis of [Re(CO) (N(SO )(2-nap)dien)] (C2) 3 2 Treatment of [Re(CO) (H O) ]OTf with the N(SO )(2-nap)dienH ligand (0.029 g, 0.1 3 2 3 2 mmol), as described above, yielded [Re(CO) (N(SO )(2-nap)dien)] as a white powder (0.04 g, 3 2 71% yield). 1H NMR (DMSO-d , 400 MHz) δ (ppm): 8.31 (s, 1H), 7.96-8.02 (m, 3H), 7.85 6 (d, 1H), 7.59-7.61 (m, 2H), 6.69 (s, 1H, N2H), 5.16-5.18 (m, 1H, N1H), 3.50 (m, 1H, N1H), 2.64-2.90 (m, 8H). 2.2.3. In vitro cytotoxic effects The in vitro cytotoxic effects of the synthesized novel ligands and their rhenium complexes were evaluated on the non-small cell lung cancer cell line (NCI-H-292) and the human lung fibroblast cell line MRC-5 (normal lung fibroblast cells) by a Sulforhodamine B (SRB) assay. NCI H292 and MRC-5 cells (5 × 103 / well) were plated in 96-well cell culture plates with Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich D5648) supplemented with 10% Fetal Bovine Serum (FBS) and incubated for 24 h. Then, the medium 7 was removed and replaced with fresh medium containing different concentrations of the compound (1.25, 2.5, 5, 10, 20 μg/ml). The treated plates were then incubated for 24, 48 and 72 h. After the incubation period, cells were fixed with trichloroacetic acid (TCA) solution and incubated at 4 C for 1 h. The cells were then washed five times with tap water and stained with SRB solution for 15 min at room temperature. After incubation, the dye was removed by rinsing the cells five times with 1% acetic acid and the plate was air-dried. Next, unbuffered Tris-base was added to each well and the plate was placed on a shaker for 1 h at room temperature. The absorbance values were taken at OD 540 nm and the result expressed as the percentage cell viability (mean of control group – mean of treated group / control group × 100%). IC values were calculated using the software GraphPad Prism 6.0.1. 50 2.2.4. Fluorescence micrographs Allium cepa bulb cells were incubated in 1 mg ml-1 solutions of the ligands and the complexes for 10 min at room temperature and observed under optical and Olympus BX51epi-fluorescence microscopes. Fluorescent micrographs were obtained using Olympus DP70 and analyzed using Olympus Stream software. 3. Results and Discussion The two novel naphthalene derivatized ligands (L1 and L2) were synthesized in good yield by adapting a method by Krapcho and Kuell [46]. These ligands bind to the fac- Re(CO) core in a tridentate binding mode and deprotonation of the sulfonamido group gives 3 the metal complex a net neutral core, as elaborated by structural characterization. 3.1. X-ray characterization Crystal data and details of the structural refinement for C1 and C2 are summarized in Tables 1 and 2. The crystallographic data are deposited with the Cambridge Crystallographic Data Centre under deposition numbers CCDC 1828901 and 1828902. Both rhenium complexes reported here (Figure 2) possess a pseudo octahedral structure where one face of the octahedron is occupied by three carbonyl ligands and the other face is occupied by one sp2 8 nitrogen atom from the sulfonamide group and two sp3 nitrogen atoms. In both the C1 and C2 complexes, the Re-N(sp2) bond distance is shorter (Table 2) than the Re-N(sp3) bond distance and falls within the range reported [19-21, 47] for typical Re-N(sp2) bond lengths (2.14 to 2.18 Ǻ). Figure 2: ORTEP of [Re(CO) (N(SO )(1-nap)dien)] and [Re(CO) (N(SO )(2-nap)dien)]. 3 2 3 2 Thermal ellipsoids are drawn at 50% probability. The X-ray structures reveal that the Re atom is bound to the NNN donor ligand in a tridentate fashion, with one donor being the terminal nitrogen atom from the deprotonated sulfonamide group. The S-N bond lengths of the complexes C1 and C2 are 1.576(4) and 1.574(2) Å, which lie within the range (1.5-1.7 Å) for S-N bond lengths of deprotonated sulfonamides of similar fac-[Re(CO) L] complexes [19], as well as other relevant compounds 3 [48, 49]. Table 1: Summary of crystal data and refinement for [Re(CO) (N(SO )(1-nap)dien)] and 3 2 [Re(CO) (N(SO )(2-nap)dien)]. 3 2 C1 C2 Empirical formula C H N O ReS C H N O ReS 17 18 3 5 17 18 3 5 Formula weight 562.61 562.61 Radiation wavelength (Å) 0.71073 0.71073 Crystal system Monoclinic Triclinic Space group P2 /n P-1 1 Unit cell dimensions: a (Å) 8.0675(4) 8.4294(4) b(Å) 22.9977(12) 15.5265(7) 9 c(Å) 9.9692(5) 16.2990(7) α (deg) - 62.169(2) β (deg) 104.180(3) 89.907(2) γ (deg) - 78.689(3) V (Å3) 1793.27(16) 1839.83(15) T (K) 100 100 Z 4 4 density (Mg m-3) 2.084 2.031 1088 1088 F(000) abs coeff (mm-1) 6.93 6.75 crystal size (mm) 0.14×0.13×0.11 0.16×0.08×0.06 2θ (deg) 66.4 70.1 max R 0.028 0.038 int R[F2>2σ(F2)] 0.043 0.028 wR(F2) 0.082 0.056 res. dens (e Å-3) -5.38, 2.23 -1.01, 1.95 data/ param 6736/253 16170/505 Table 2: Selected bond lengths/Å and angles/° for [Re(CO) (N(SO )(1-nap)dien)] (C1) and 3 2 [Re(CO) (N(SO )(2-nap)dien)] (C2). 3 2 C1 C2 Re-N1 2.199(4) 2.205(2) Re-N2 2.228(4) 2.206(2) Re-N3 2.170(4) 2.174(2) S1-N3 1.576(4) 1.574(2) N1-Re-N2 77.56(14) 77.62(8) N2-Re-N3 76.01(13) 74.35(7) N1-Re-N3 87.22(14) 82.74(8) C7-N3-Re 109.2(3) 117.46(15) The ring pucker of the two chelate rings of the [Re(CO) (N(SO )(1-nap)dien)] and 3 2 [Re(CO) (N(SO )(2-nap)dien)] complexes have δδ and λλ chirality combinations, 3 2 10 respectively. These findings are in agreement with an IR data based study by Schmidtke and Garthoff, which reported that the same chirality combination for both rings is the most favoured configuration for facial coordinated Cr, Co and Rh metal complexes of diethylenetriamine [50]. We compared the effect of the position of pendant group on the shape and chirality of the ring pucker of the two complexes. An overlay of the two structures reveals a considerable effect on the ring pucker (Figure 3). An overlay of 14 atoms gave an rms value of 0.37 while an overlay of only 4 atoms gave an rms value of 0.0534. Nonetheless, it is evident that the position of the substituent in this case has an effect on the shape of the ring pucker of the complexes. Figure 3: Overlay of the C1 (red) and C2 (green) complexes. Left: rms = 0.37 from overlaying 14 atoms, Re, C and N of dien ring and CO atoms and right: rms = 0.0534 from overlaying Re, N1, N2 and N3 atoms. 3.2. 1H NMR characterization (b) (a()c) The ligands and the complexes were characterized by 1H NMR spectroscopy in Bon Bo DMSO-d . The proton peaks of the naphthalene groups of L1 and L2 appear in the aromatic d 6 nd Dist region of the spectra, while the peaks of the methylene protons of the dien backbone appear in Dis anc tan the range δ 2-3 ppm. The most downfield signal among the methylene protons is attributed to es ces the protons attached to the carbon atom which is adjacent to the sulfonamide nitrogen atom. R R e 2 Upon complexation, the N3 atom deprotonates and the proton signals of N1H and N2H can e 2 2 - . - . be clearly identified in the spectra of the complexes (δ 3.50-7.00 ppm, Table 3). We have N 1 N 1 1 9 11 1 9 9 9 ( 4 ( ) 4 R ) e 2 R - . e 2 N 2 - . 2 2 N 2 8 2 2 ( 8 4 ) ( R 4 e 2 ) - . R N 1 e 2 3 7 - . 0 N 1 ( 3 7 4 0 ) S ( 1 1 4 - . ) N 5 S 3 7 1 1 6 - . ( N 5 4 3 7 ) 6 Bon d ( Ang 4 les ) N Bo 1 7 nd - 7 An R . gle e 5 s - 6 N N ( 1 7 2 1 - 7 4 R . ) e 5 N - 6 2 7 N - 6 2 ( R . 1 e 0 4 - 1 ) N ( N 3 1 2 7 3 - 6 ) R . N e 0 1 8 - 1 - 7 N R . 3 ( e 2 1 - 2 3 N ( ) 3 1 N 4 1 8 ) - 7 C R . 7 1 e 2 - 0 - 2 N 9 N 3 . 3 ( - 2 1 R ( 4 e 3 ) ) C ) 7 1 - 0 N 9 3 . - 2 R e ( 3 ) ) exemplified the above using L1 and C1 (Figure 4). Since the two N1 protons of C1 orient toward (endo-NH) and away (exo-NH) from the CO ligands (Figure 2), the two protons are not magnetically equivalent as in the free ligand. As a result of this orientation, it gives an upfield signal (exo-NH) and a relatively downfield signal (endo-NH) in DMSO-d ,providing 6 evidence for complexation as previously observed for related compounds [19, 21]. The central NH peak appears at δ 6.80 ppm for C1. A similar pattern was observed for L2 and C2, indicating that these shifts fall in the normal range as reported for similar compounds (δ 3.30- 3.54 ppm for the exo-NH signal and δ 5.21-5.26 ppm for the endo-NH signal) [19, 21]. Figure 4: 1H NMR spectra of N(SO )(1-nap)dienH (a) and [Re(CO) (N(SO )(1-nap)dien)] (b) 2 3 2 in DMSO-d at 25 °C. 6 Table 3: Selected chemical shifts (δ, ppm) of [Re(CO) (N(SO )(1-nap)dien)] (C1) and 3 2 [Re(CO) (N(SO )(2-nap)dien)] (C2) in DMSO-d at 25 °C. 3 2 6 C1 C2 exo-N1H/Me 3.59 3.50 endo-N1H/Me 5.22 5.17 N2H 6.80 6.69 3.3. FT-IR spectroscopy The observed infrared bands of the synthesized compounds and their assignments are given in Table 4. The prominent and strong bands in the region 1800-2000 cm-1 in the IR spectra of the C1 and C2 complexes are due to the CO stretching vibrations [51]. In the free 12 ligands, the S-N stretching mode appears at 938 and 953 cm-1 and shifts towards higher frequencies in both complexes (Table 4). A strong band within the range 1143-1159 cm-1 is due to the symmetric stretching vibrations of the SO group, while a moderate peak within the 2 1269-1316 cm-1 range is due to asymmetric stretching vibrations of the SO group [51] in 2 each ligand and complex. Table 4: Selected IR bands/cm-1 of N(SO )(1-nap)dienH (L1), [Re(CO) (N(SO )(1-nap)dien)] 2 3 2 (C1), N(SO )(2-nap)dienH (L2) and [Re(CO) (N(SO )(2-nap)dien)] (C2) 2 3 2 Ligand/Complex νS-N ν (SO ) ν (SO ) ν(CO) as 2 s 2 L1 938 1316 1159 C1 961 1314 1158 2012, 1866 L2 953 1316 1152 C2 979 1269 1143 2009, 1867 3.4. UV-Visible and fluorometric analysis The UV-Visible spectra of the two ligands and their Re complexes were obtained in methanol at room temperature. The absorption spectra of the free ligands L1 and L2 show high energy bands between 200 and 300 nm due to intra ligand transitions (Figure 5). The absorption spectra of the two metal complexes show a hypsochromic shift, shifting to a shorter wavelength. 13 Figure 5: UV-Visible spectra of N(SO (1-nap)dienH, [Re(CO) (N(SO )(1-nap)dien)], 2 3 2 N(SO )(2-nap)dienH and [Re(CO) (N(SO )(2-nap)dien)] in methanol. 2 3 2 The ligands and complexes were excited in the UV range and the emission spectra were recorded in methanol (Table 5). L1 and L2 show high fluorescence intensities. However, the new metal complexes show lower or no fluorescence intensity in comparison to that of the ligands, indicating quenching of the fluorescence upon complexation (Figure S1, Supporting Information). Table 5: Excitation and emission wavelengths of L1, L2, C1 and C2 in methanol Test sample Excitation Emission wavelength/nm wavelength/nm N(SO (1-nap)dienH 280 342 2 [Re(CO) (N(SO )(1-nap)dien)] 280 - 3 2 N(SO (2-nap)dienH 280 333 2 [Re(CO) (N(SO )(2-nap)dien)] 280 340 3 2 3.5. In vitro cytotoxic effects The cytotoxic activity of a potential drug lead is initially analyzed by pre-clinical testing, administering the compound on different cancer cell lines. In this study, human normal lung fibroblast cells (MRC-5) and non-small cell lung cancer cells (NCI-H-292) were exposed to the four synthesized compounds and the half maximal inhibitory concentration (IC ) was determined for each compound (Table 6 and Supporting Information). 50 Table 6: IC values of the ligands and complexes described in this study, incubating for 24, 50 48 and 72 hours, on MRC-5 and NCI-H292 cells. IC (M) 50 Test compound MRC-5 NCI-H292 24 h 48 h 72 h 24 h 48 h 72 h N(SO2)(1-nap)dienH 50.04 134.13 161.53 209.38 1074.03 3325.04 [Re(CO)3(N(SO2)(1-nap)dien)] 30.61 54.62 94.67 17.33 9.91 18.72 N(SO2)(2-nap)dienH 48.81 199.19 151.07 230.25 1000> 1000> [Re(CO)3(N(SO2)(2-nap)dien)] 33.93 67.77 318.87 82.54 166.83 605.04 14 Cytotoxic effects were demonstrated by both the ligands and their respective rhenium complexes, where the metal complexes showed higher cytotoxicity against both cell lines. L1 and L2 show low cytotoxic capacity, where both cell lines recover from the acute toxicity in 24 h. The highest cytotoxic activity was observed for the [Re(CO) (N(SO )(1-nap)dien)] 3 2 complex against NCI-H292 cells, with an IC value of 9.91 at 48 h (Table 6). It can be 50 hypothesized that activation of cellular repair mechanisms are responsible for this increased IC value after 24 h [52]. However, the behavior of the response curve posits a secondary 50 cytotoxicity event taking place after 48 h for L2. Comparing the IC values at 24 h, both 50 metal complexes exhibit higher toxicity than the reported value (94.8 M) for cisplatin (https://www.cancerrxgene.org/translation/Drug/1005), a widely used anticancer drug and therefore they are much more potent than cisplatin. The morphology of the cells was observed under phase contrast light microscopy after treating with the compounds in a concentration gradient at 24, 48 and 72 h of incubation periods and the microscopic images were recorded (Figures 6-9). Upon analysis of the images, the NCI-H292 and MRC-5 cells treated with L1, C2 and L2 did not display significant cytomorphological changes at the tested concentrations and in the time series. Although the MRC-5 cells did not show any morphological changes with C1, NCI-H292 cells treated with C1 indicated clear morphological changes, such as cell shrinkage, reduction in cell volume and irregular cell shapes, which are indicative of apoptotic cell death. Triggering apoptotic cell death is an important feature of an anticancer drug lead and C1 shows a significant cytotoxic effect on non-small cell lung cancer cells (NCI-H292). Furthermore, C1 demonstrates specificity towards NCI-H292 cells, indicating the observed cytotoxicity and induction of apoptosis is highly specific to lung cancer cells as the compound did not display any cytotoxic effects on normal lung cells. Thus, C1 could be a potential drug lead to generate an effective chemotherapeutic agent to treat lung cancer due to its ability to induce apoptosis and having specificity towards lung cancer cells. Further analysis is warranted to 15 confirm the specificity and underlying molecular mechanisms for the cytotoxicity of C1 against NCI-H292. Figure 6: Morphology of human MRC-5 cells after 24, 48 and 72 hours incubating with N(SO )(1-nap)dienH (L1) and [Re(CO) (N(SO )(1-nap)dien)] (C1) at 20, 10, 5, 2.5 and 1.25 2 3 2 μg/ml concentrations. Scale bars given for the L1 treated cells (40 μm) apply to all treatments. 16 Figure 7: Morphology of human MRC-5 cells after 24, 48 and 72 hours incubating with N(SO )(2-nap)dienH (L2) and [Re(CO) (N(SO )(2-nap)dien)] (C2) at 20, 10, 5, 2.5 and 1.25 2 3 2 μg/ml concentrations. 17 Figure 8: Morphology of human NCI-H292 cells after 24, 48 and 72 hours incubating with N(SO )(1-nap)dienH (L1) and [Re(CO) (N(SO )(1-nap)dien)] (C1) at 20, 10, 5, 2.5 and 1.25 2 3 2 μg/ml concentrations. 18 Figure 9: Morphology of human NCI-H292 cells after 24, 48 and 72 hours incubating with N(SO )(2-nap)dienH (L2) and [Re(CO) (N(SO )(2-nap)dien)] (C2) at 20, 10, 5, 2.5 and 1.25 2 3 2 μg/ml concentrations. The cellular uptake of a cytotoxic compound can be studied by fluorescence microscopy images. Plant cells incubated with the synthesized compounds yielded a weak fluorescence signal upon imaging by epifluorescence microscopy (Figure 10). As reported in the literature on some cytotoxic Re organometallic complexes, their luminescence can be quenched in a biological system upon interaction with biological molecules [3, 53]. Furthermore, it may be due to poor uptake by the cells or due to photobleaching within cells. Cell walls as well as the nuclei were stained according to the microscopic images obtained, 19 suggesting higher accumulation of the compounds at those sites. Combined with previous studies, the cytotoxicity may arise from DNA association of the compounds. However, further experimentation is needed to better understand this phenomenon. Figure 10: (left) Bright-field microscopy, (middle) fluorescence microscopy images of Allium cepa bulb cells excited at 450 nm, and (right) excited at 550 nm, incubated with 1 mg ml-1 of N(SO )(1-nap)dienH (a, b, c), [Re(CO) (N(SO )(1-nap)dien)] (d, e, f), N(SO )(2-nap)dienH 2 3 2 2 (g, h, i, j) and [Re(CO) (N(SO )(2-nap)dien)] (k, l, m). 3 2 20 4. Conclusions Two novel ligands and their Re complexes were synthesized and characterized. Both the ligands showed high fluorescence intensities. Decreased fluorescence intensities of the two synthesized Re complexes may be due to a quenching effect upon direct binding of the ligands to the Re metal. All the tested compounds exhibited cytotoxicity against cultured cells, where higher cytotoxicity was shown by the metal complexes compared to their ligands. [Re(CO) (N(SO (1-nap)dien)] shows the most potent activity, where it shows more potency 3 2 than the widely known anticancer drug cisplatin. Interestingly, fluorescence microscopy images obtained for plant cells incubated with the compounds suggest that the compounds interact with the nuclei, indicating a possible mechanism of action via DNA interactions. Novel tridentate ligands will contribute towards developing useful novel pharmaceutical agents bearing the fac-[M(CO) ]+ core (M = 99mTc, 186/188Re) and we have 3 taken the first step towards this effort by structurally characterizing complexes of their non- radioactive congener. In this study, the structural characterization of two complexes of the type [Re(CO) (N(SO napdien)] where only the point of attachment of the R substituent to the 3 2 sulfonamide N differs was evaluated, revealing that the ring pucker was affected. The two new complexes are small in size, possess cellular uptake and possess remarkable cytotoxicity towards the tested lung cancer cell lines, which make them good leads towards such applications. Overall, [Re(CO) (N(SO (1-nap)dien)] is a promising compound with specific 3 2 cytotoxicity against NCI-H292 lung cancer cells and can be further studied as a promising anticancer drug lead. Declarations Ethics approval and consent to participate: Not applicable Data Availability: The following additional data are available with the online version of this paper. Fluorescence spectra of N(SO )(1-nap)dienH, [Re(CO) (N(SO )(1-nap)dien)], 2 3 2 21 N(SO )(2-nap)dienH and [Re(CO) (N(SO )(2-nap)dien)] in methanol and a plot of 2 3 2 percentage cell viability vs the concentration of the compounds N(SO )(1-nap)dienH (L1), 2 [Re(CO) (N(SO )(1-nap)dien)] (C1), N(SO )(2-nap)dienH (L2), [Re(CO) (N(SO )(2- 3 2 2 3 2 nap)dien)] (C2) obtained by a Sulforhodamine B assay. Conflicts of Interest: The authors declare that they have no competing interests. Acknowledgement: This work was supported by Grant no ASP/01/RE/SCI/2018/21 of the University of Sri Jayewardenepura with support for instrumentation from the Instrument Centre and Centre for Advanced Material Research of the University of Sri Jayewardenepura. The authors thank Prof Luigi Marzilli and Dr Kokila Ranasinghe of Louisiana State University for obtaining NMR data and for useful discussions. Authors’ contributions: TD carried out the synthesis, purification and characterization of the compounds as well as the initial writing of manuscript. TP designed and conceived the study and finalized the manuscript. ICP and SRS designed the biological experiments and together with VVP carried them out. All authors read and approved the final manuscript. Author’s information: TD recently obtained the Degree of Master of Philosophy at the Department of Chemistry, University of Sri Jayewardenepura, Sri Lanka under the supervision of TP where she currently works as a Research Assistant. FRF is a dedicated crystallographer spanning a very illustrious career. VVP has obtained the Degree of Master of Science and SRS is a senior lecturer at the Institute of Molecular Biology and Biochemistry, University of Colombo and ICP is a senior lecturer and researcher at the Department of Zoology, University of Colombo. TP is a senior lecturer and researcher at the Department of Chemistry, University of Sri Jayewardenepura with a special interest in synthesizing new metal complexes with biomedical relevance. 22 References [1] Rosenberg B, Vancamp L, TROSKO JE, MANSOUR VH. Platinum compounds: a new class of potent antitumour agents. Nature. 1969;222(5191):385. [2] Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer. 2007;7(8):573. [3] Leonidova A, Gasser G. Underestimated potential of organometallic rhenium complexes as anticancer agents. ACS Chem. Biol. 2014;9(10):2180-93. [4] Khan S, Malla AM, Zafar A, Naseem I. Synthesis of novel coumarin nucleus-based DPA drug-like molecular entity: In vitro DNA/Cu(II) binding, DNA cleavage and pro-oxidant mechanism for anticancer action. PloS one. 2017;12(8):e0181783. [5] Schibli R, Schubiger AP. Current use and future potential of organometallic radiopharmaceuticals. Eur. J. Nucl. Med. Mol. Imaging. 2002;29(11):1529-42. [6] Liu S. The role of coordination chemistry in the development of target-specific radiopharmaceuticals. Chem. Soc. Rev. 2004;33(7):445-61. [7] Lipowska M, Marzilli LG, Taylor AT. 99mTc(CO) -nitrilotriacetic acid: a new renal 3 radiopharmaceutical showing pharmacokinetic properties in rats comparable to those of 131I- OIH. J. Nucl. Med. 2009;50(3):454-60. [8] Lipowska M, He H, Xu X, Taylor AT, Marzilli PA, Marzilli LG. Coordination modes of multidentate ligands in fac-[Re(CO) (polyaminocarboxylate)] analogues of 99mTc 3 radiopharmaceuticals. Dependence on aqueous solution reaction conditions. Inorg. Chem. 2010;49(7):3141-51. [9] Dilworth J, Parrott S. The biomedical chemistry of technetium and rhenium. Chem. Soc. Rev. 1998;27(1):43-55. [10] Lo KK-W. Luminescent rhenium(I) and iridium(III) polypyridine complexes as biological probes, imaging reagents, and photocytotoxic agents. Acc. Chem. Res. 2015;48(12):2985-95. [11] Abram U, Alberto R. Technetium and rhenium: coordination chemistry and nuclear medical applications. J. Braz. Chem. Soc. 2006;17(8):1486-500. [12] Radioisotopes I, No RS. 1: Technetium 99m Radiopharmaceuticals: Status and Trends. Vienna; 2009. [13] He H, Lipowska M, Xu X, Taylor AT, Marzilli LG. Rhenium analogues of promising renal imaging agents with a {99mTc(CO) }+ core bound to cysteine-derived dipeptides, 3 including lanthionine. Inorg. Chem. 2007;46(8):3385-94. [14] Simpson PV, Casari I, Paternoster S, Skelton BW, Falasca M, Massi M. Defining the Anti‐Cancer Activity of Tricarbonyl Rhenium Complexes: Induction of G2/M Cell Cycle Arrest and Blockade of Aurora‐A Kinase Phosphorylation. Chem. Eur. J. 2017;23(27):6518- 21. [15] Knopf KM, Murphy BL, MacMillan SN, Baskin JM, Barr MP, Boros E, Wilson JJ. In Vitro Anticancer Activity and in Vivo Biodistribution of Rhenium(I) Tricarbonyl Aqua Complexes. J. Am. Chem. Soc. 2017;139(40):14302-14. [16] Guo X-Q, Castellano FN, Li L, Szmacinski H, Lakowicz JR, Sipior J. A long-lived, highly luminescent Re(I) metal–ligand complex as a biomolecular probe. Anal. Biochem. 1997;254(2):179-86. [17] Stephenson KA, Banerjee SR, Besanger T, Sogbein OO, Levadala MK, McFarlane N, Lemon NJ, Boreham JA, Maresca KP, Brennan JD, Babich JW, Zubieta J, Valliant JF. Bridging the gap between in vitro and in vivo imaging: isostructural Re and 99mTc complexes for correlating fluorescence and radioimaging studies. J. Am. Chem. Soc. 2004;126(28):8598-9. 23 [18] Schibli R, La Bella R, Alberto R, Garcia-Garayoa E, Ortner K, Abram U, Schubiger PA. Influence of the denticity of ligand systems on the in vitro and in vivo behavior of 99mTc (I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules. Bioconjugate Chem. 2000;11(3):345-51. [19] Christoforou AM, Fronczek FR, Marzilli PA, Marzilli LG. fac-Re(CO) L Complexes 3 Containing Tridentate Monoanionic Ligands (L-) with a Seldom-Studied Sulfonamido Group As One Terminal Ligating Group. Inorg. Chem. 2007;46(17):6942-9. [20] Christoforou AM, Marzilli PA, Fronczek FR, Marzilli LG. fac-[Re(CO) L]+ 3 Complexes with N−CH −CH −X−CH −CH −N Tridentate Ligands. Synthetic, X-ray 2 2 2 2 Crystallographic, and NMR Spectroscopic Investigations. Inorg. Chem. 2007;46(26):11173- 82. [21] Christoforou AM, Marzilli PA, Fronczek FR, Marzilli LG. fac-[Re(CO) L] 3 Complexes Containing Tridentate Monoanionic Ligands (L−) Having a Terminal Amido and Two Amine Ligating Groups. J. Chem. Crystallogr. 2008;38(2):115-21. [22] Abhayawardhana PL, Marzilli PA, Fronczek FR, Marzilli LG. Complexes possessing rare “tertiary” sulfonamide nitrogen-to-metal bonds of normal length: fac- [Re(CO) (N(SO R)dien)]PF complexes with hydrophilic sulfonamide ligands. Inorg. Chem. 3 2 6 2014;53(2):1144-55. [23] Perera T, Abhayawardhana P, Marzilli PA, Fronczek FR, Marzilli LG. Formation of a metal-to-nitrogen bond of normal length by a neutral sufonamide group within a tridentate ligand. A new approach to radiopharmaceutical bioconjugation. Inorg. Chem. 2013;52(5):2412-21. [24] De Groot FM, Loos WJ, Koekkoek R, van Berkom LW, Busscher GF, Seelen AE, Albrecht C, De Bruijn P, Scheeren HW. Elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release. J. Org. Chem. 2001;66(26):8815-30. [25] Valente S, Trisciuoglio D, De Luca T, Nebbioso A, Labella D, Lenoci A, Bigogno C, Dondio G, Miceli M, Brosch G, Del Bufalo D, Altucci L, Mai A. 1, 3, 4-Oxadiazole- containing histone deacetylase inhibitors: anticancer activities in cancer cells. J. Med. Chem. 2014;57(14):6259-65. [26] Abate C, Niso M, Lacivita E, Mosier PD, Toscano A, Perrone R. Analogues of σ receptor ligand 1-cyclohexyl-4-[3-(5-methoxy-1, 2, 3, 4-tetrahydronaphthalen-1-yl) propyl] piperazine (PB28) with added polar functionality and reduced lipophilicity for potential use as positron emission tomography radiotracers. J. Med. Chem. 2011;54(4):1022-32. [27] Laverty G, McCloskey AP, Gilmore BF, Jones DS, Zhou J, Xu B. Ultrashort cationic naphthalene-derived self-assembled peptides as antimicrobial nanomaterials. Biomacromolecules. 2014;15(9):3429-39. [28] Chen Y-Y, Gopala L, Bheemanaboina RRY, Liu H-B, Cheng Y, Geng R-X, Zhou C- H. Novel naphthalimide aminothiazoles as potential multitargeting antimicrobial agents. ACS Med. Chem. Lett. 2017;8(12):1331-5. [29] Tay F, Erkan C, Sariozlu NY, Ergene E, Demirayak S. Synthesis, antimicrobial and anticancer activities of some naphthylthiazolylamine derivatives. Biomed. Res. 2017;28(6):2696-703. [30] Biswas S, Zhang S, Fernandez F, Ghosh B, Zhen J, Kuzhikandathil E, Reith MEA, Dutta AK. Further structure–activity relationships study of hybrid 7-{[2-(4-phenylpiperazin- 1-yl) ethyl] propylamino}-5, 6, 7, 8-tetrahydronaphthalen-2-ol analogues: identification of a high-affinity D3-preferring agonist with potent in vivo activity with long duration of action. J. Med. Chem. 2007;51(1):101-17. [31] Mao G-J, Wei T-T, Wang X-X, Huan S-y, Lu D-Q, Zhang J, Zhang X-B,Tan W, Shen G-L,Yu R-Q. High-sensitivity naphthalene-based two-photon fluorescent probe suitable for direct bioimaging of H2S in living cells. Anal. Chem. 2013;85(16):7875-81. 24 [32] Makar S, Saha T, Singh SK. Naphthalene, a versatile platform in medicinal chemistry: Sky-high perspective. Eur. J. Med. Chem. 2018. [33] Gasser G, Metzler-Nolte N. The potential of organometallic complexes in medicinal chemistry. Curr. Opin. Chem. Biol. 2012;16(1-2):84-91. [34] Gasser G, Ott I, Metzler-Nolte N. Organometallic anticancer compounds. J. Med. Chem. 2010;54(1):3-25. [35] Hartinger CG, Metzler-Nolte N, Dyson PJ. Challenges and opportunities in the development of organometallic anticancer drugs. Organometallics. 2012;31(16):5677-85. [36] Hartinger CG, Dyson PJ. Bioorganometallic chemistry—from teaching paradigms to medicinal applications. Chem. Soc. Rev. 2009;38(2):391-401. [37] Bruijnincx PC, Sadler PJ. New trends for metal complexes with anticancer activity. Curr. Opin. Chem. Biol. 2008;12(2):197-206.b [38] Jaouen G, Metzler-Nolte N. Medicinal organometallic chemistry: Springer; 2010. [39] Peacock AF, Sadler PJ. Medicinal organometallic chemistry: designing metal arene complexes as anticancer agents. Chem. Asian J. 2008;3(11):1890-9. [40] Smith GS, Therrien B. Targeted and multifunctional arene ruthenium chemotherapeutics. Dalton Trans. 2011;40(41):10793-800. [41] Süss-Fink G. Arene ruthenium complexes as anticancer agents. Dalton Trans. 2010;39(7):1673-88. [42] Patra M, Gasser G. Organometallic compounds: an opportunity for chemical biology? ChemBioChem. 2012;13(9):1232-52. [43] Jung Y, Lippard SJ. Direct cellular responses to platinum-induced DNA damage. Chem. Rev. 2007;107(5):1387-407. [44] Hoseok I, Cho J-Y. Lung cancer biomarkers. Adv. Clin. Chem. 72: Elsevier; 2015. p. 107-70. [45] He H, Lipowska M, Xu X, Taylor AT, Carlone M, Marzilli LG. Re(CO) Complexes 3 Synthesized via an Improved Preparation of Aqueous fac-[Re(CO) (H O) ]+ as an Aid in 3 2 3 Assessing 99mTc Imaging Agents. Structural Characterization and Solution Behavior of Complexes with Thioether-Bearing Amino Acids as Tridentate Ligands. Inorg. Chem. 2005;44(15):5437-46. [46] Krapcho AP, Kuell CS. Mono-Protected Diamines. N-tert-Butoxycarbonyl-α, ω- Alkanediamines from α, ω-Alkanediamines. Synth. Commun. 1990;20(16):2559-64. [47] Wei L, Babich JW, Ouellette W, Zubieta J. Developing the {M(CO) }+ core for 3 fluorescence applications: rhenium tricarbonyl core complexes with benzimidazole, quinoline, and tryptophan derivatives. Inorg. Chem. 2006;45(7):3057-66. [48] Goodwin JM, Olmstead MM, Patten TE. Identification and characterization of monoanionic tripodal tetradentate ligand complexes of copper(I) and copper(II) involved in halogen atom transfer reactions. J. Am. Chem. Soc. 2004;126(44):14352-3. [49] Congreve A, Kataky R, Knell M, Parker D, Puschmann H, Senanayake K, Wiley L. Examination of cobalt, nickel, copper and zinc (II) complex geometry and binding affinity in aqueous media using simple pyridylsulfonamide ligands. New J. Chem. 2003;27(1):98-106. [50] Schmidtke H, Garthoff D. The infrared spectra and conformational analysis of geometric isomers of diethylenetriamine complexes. Inorg. Chim. Acta. 1968;2:351-6. [51] Socrates G. Infrared and Raman characteristic group frequencies: tables and charts: John Wiley & Sons; 2001. [52] Basu A, Krishnamurthy S. Cellular responses to Cisplatin-induced DNA damage. J. Nucleic Acids. 2010;2010. [53] Filipovic L, Arandelovic S, Gligorijevic N, Krivokuca A, Jankovic R, Srdic-Rajic T, Rakic G, Tesic Z, Radulovic S. Biological evaluation of transdichloridoplatinum (II) complexes with 3-and 4-acetylpyridine in comparison to cisplatin. Radiol. Oncol. 2013;47(4):346-57. 25 Taniya Darshani: Investigation, Formal analysis, Methodology, Writing - original draft Frank R. Fronczek: Investigation, Resources, Formal analysis, Data curation. Varuni V Priyadarshani: Investigation, Formal analysis, Data curation. Sameera R Samarakoon: Investigation, Resources, Formal analysis, Data curation. Inoka C Perera : Investigation, Resources, Formal analysis, Data curation. Theshini Perera: Methodology, Conceptualization, Supervision, Writing - review & editing Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: 26 Two novel ligands and their rhenium(I) tricarbonyl complexes were synthesized and characterized towards utilizing the compounds as potential anticancer drug leads against lung cancer.  Synthesis of two novel sulfonamide derivatized diethylenetriamine ligands  Synthesis of Re(I) tricarbonyl complexes bearing a sulfonamide linkage  The compounds showed promising cytotoxic activity against NCI-H292 lung cancer cells  The reported compounds may be utilized as anticancer drug leads against lung cancer 27 28 29