Synthesis, characterization and biological evaluation of dipicolylamine sulfonamide derivatized platinum complexes as potential anticancer agents

Three new Pt complexes, [PtCl2(N(SO2(2-nap))dpa)], [PtCl2(N(SO2(1-nap))dpa)] and [PtCl2(N(SO2pip)dpa)], containing a rare 8-membered ring were synthesized in good yield and high purity by utilizing the ligands N(SO2(2-nap))dpa, N(SO2(1-nap))dpa and N(SO2pip)dpa, which contain a dipicolylamine moiety. Structural studies of all three complexes confirmed that the ligands are bound in a bidentate mode via Pt–N(pyridyl) bonds forming a rare 8-membered ring. The intense fluorescence displayed by the ligands is quenched upon coordination to Pt. According to time dependent density functional theory (TDDFT) calculations, the key excitations of N(SO2(2-nap))dpa and [PtCl2(N(SO2(1-nap))dpa)] involve the 2-nap-ligand-centered π → π* excitations. While all six compounds have shown antiproliferative activity against human breast cancer cells (MCF-7), the N(SO2pip)dpa and N(SO2(2-nap))dpa ligands and [PtCl2((NSO2pip)dpa)] complex have shown significantly high cytotoxicity, directing them to be further investigated as potential anti-cancer drug leads.


Introduction
Metals play a major role in therapeutic and diagnostic 1 applications of cancer, prompting the exploration of novel metal organic compounds towards this end. Clinically important organometallic complexes have also been used for the synthesis of effective diagnostic agents. 2 In fact, [Tc(CNR) 6 ] + is the rst example of an organometallic complex to be clinically used in nuclear medicine. 3 99m Tc and other short lived isotopes are used as radiopharmaceuticals in disease diagnosis and treatment. 4 Such radionuclide cationic complexes containing 123 I nuclide 5 and 99m Tc nuclide 6 are broadly used as potential diagnostic agents while complexes bearing the 188 Re tricarbonyl core are used as therapeutic agents. 7 In addition to that, inorganic compounds have found use in chemotherapeutic agents such as the gold-containing antiarthritic drug auranon and antibacterial, antiviral, antiparasitic and radiosensitizing agents. 8 The development of modern medicinal inorganic chemistry was stimulated by the discovery of serendipitous cisplatin, the rst ever anticancer drug. 9,10 To address the drawbacks of the severe toxicity 9 and drug resistance of cisplatin, a large number of cisplatin analogues have been synthesized and evaluated over three generations of platinum drugs (cisplatin, carboplatin and oxaliplatin). 11,12 Pt(II) complexes have the advantage of low coordination number, preferential binding to the more limited so centers in proteins and nucleic acids 13 and avoid unwanted covalent reactions with nucleotides. 14 In fact, a key factor explaining why platinum is most useful comes from the ligand exchange kinetics, which for platinum complexes of the type cis-[PtX 2 (amine) 2 ] (X ¼ anionic group and amine ¼ primary or secondary amine) are in the order of a few hours, thereby preventing rapid equilibration reactions. [15][16][17] Many Pt(II) complexes with ligands having a diene backbone have been synthesized and studied. 18 For an example, the dansyldiene (DNSH-dienH) ligand which can act as a bidentate, tridentate or quadridentate forms a complex with platinum metal. 18 N(SO 2 R)dpatype tridentate ligands and their Pt(II) compounds with chloride leaving ligands have been synthesized and studied. 19 These N(SO 2 R)Me n dpa (R ¼ Me, Tol; n ¼ 2, 4) ligands are coordinated in a bidentate fashion in [PtCl 2 (N(SO 2 R)3,3 0 ,5,5 0 -Me 4 dpa)] complexes, forming a rare eight-membered chelate ring. 19 The recently reported dinuclear platinum-amine compounds appeared to give rise to DNA binding at two different positions, thereby enhancing the antitumor effect. 20 Multinuclear platinum drugs that can contain two, three or four Pt centers with both cis and trans congurations have become an attractive strategy to develop potent cisplatin analogues. 15 Breast cancer is one of the most abundant types of cancer in recent times. Among various types of chronic human diseases, certain types of cancers are widely treated by organometallic complexes. 4 However, only a few metallopharmaceuticals are available for diagnostic and therapeutic purposes for human breast cancer. 21 We note that sigma receptors are a specic class of membrane-bound proteins, 21 classied as N-allylnormetazocine link receptors, a type of opiate receptors. 22 These receptors, their subtypes, transmitters and enzymes are mostly found in the central nervous system, liver, kidney and can serve as attractive targets for radio-selective molecules in oncology. 22 Sigma receptors are over expressed in vastly dividing cells such as breast, lung and prostate cancer cells 23 and there is adequate evidence that expression of sigma receptors is down-regulated in quiescent cells. 21 Furthermore, a few ligands labeled with 123 I, 22 99m Tc, 23 21 We ourselves have reported on the therapeutic potential of a novel sulfonamide ligand bearing a piperidinyl group and its rhenium complex, [Re(CO) 3 (N(SO 2 pip)dpa)]BF 4 for therapy of human breast cancer, mainly because these types of compounds preferentially bind with sigma receptors. 25 Sulfonamide groups are considered as a pharmacophore because they possess many biological activities such as antimicrobial, 26 anticarbonic anhydrase, 27 antihypersensitive, 28 hypoglycemic 29 activities as well as, most signicantly, anticancer activity. 30 Sulfonamide appended rhenium complexes, for example fac-[Re(CO) 3 (NSO 2 Rdien)]PF 6 (R ¼ dmb, tol) have also been proposed as model systems for radiopharmaceuticals. 31 Symmetrically complexed radiopharmaceuticals derived from dipicolylamine, such as glucosamine-dpa 32 have been synthesized and evaluated. Previous studies revealed that the replacement of the amine proton of N(SO 2 R)dpa by various substituents facilitates the formation of metal to nitrogen bonds within the normal range of bond lengths arising exceptional biochemical properties. 33 The main objective of this study was to synthesize platinum complexes containing dipicolylamine sulfonamides and investigate their activity as anticancer agents. In this study, we specically opted to synthesize 1-naphthalene sulfonyl, 2naphthalene sulfonyl and piperidine-1-sulfonyl derivatized compounds based on bioactivities reported for these groups; naphthalene derivatized compounds, such as nafcillin, nai-ne, tolnaate and terbinane, are currently being used as therapeutics 34 while naphthalene compounds have been reported to exhibit numerous promising pharmaceutical properties, such as anticancer, [35][36][37] antimicrobial, 38 anti-inammatory 39 and antineurodegenerative 40,41 activities. As noted earlier, piperidinyl derivatized compounds have been used as radiotracers towards targeting breast cancer cells. 21 Thus, we report on the synthesis and characterization of three novel platinum complexes bearing 1-naphthalene sulfonyl, 2naphthalene sulfonyl and piperidine-1-sulfonyl groups (Scheme 1) and evaluate their cytotoxic activity towards human breast cancer cells. This study explores the binding of Pt(II) towards dpa derivatized sulfonamides and presents a novel concept in terms of opening up new avenues for bioconjugation of Pt in rare eight-membered rings. It also explores the differences in coordination mode of ligands containing this scaffold towards platinum and rhenium. 4 ], DMSO, di(2-picolyl)amime, piperidine-1-sulfonyl chloride, 1-naphthalene sulfonyl chloride, 2-naphthalene sulfonyl chloride, analytical grade dioxane, analytical grade methanol, chromasolv water, dichloromethane, anhydrous sodium sulphate and acetonitrile were used as received from Sigma Aldrich, USA. Human breast cancer cell line, MCF-7, was obtained from American Type Culture Collection.

NMR measurements
1 H NMR spectra were recorded in DMSO-d 6 using a Bruker 400 MHz spectrometer. Peak positions are relative to trimethylsilane (TMS) or solvent residual peak, with TMS as reference. All NMR data were processed with Top Spin 3.2 and Mestre-Nova soware.

X-ray data collection and structure determination
Crystal data were collected using a Bruker Kappa APEX-(11) DUO diffractometer. Data were collected at low temperature and data reduction was done on Bruker SAINT and includes absorption by multi scan method, using SHELXS97. Molecular graphics were drawn using ORTEP-3 for windows.

UV-visible spectroscopy
Electronic spectra for ligands and metal complexes were obtained within the spectral range of 200-800 nm using Spectro VIS auto version 3.16, UV-2602 spectrometer. Methanol was used to obtain the spectra with baseline correction. Spectral data were processed with UV WIN soware.

FTIR analysis
ATR spectra for ligands and metal complexes were obtained within the spectral range of 4000-600 cm À1 using Thermo Scientic NICOLET iS10 spectrometer. Spectral data were processed with OMNIC soware.

Fluorometric analysis
Emission spectra for ligands and metal complexes were obtained in methanol on a Thermo Scientic Lumina spectrophotometer. A 150 W xenon lamp was used as the excitation source. Spectral data were processed with Luminous soware.

Melting point determination
Melting points were manually determined in open capillaries.

Synthesis
In order to synthesize metal complexes, [PtCl 2 (DMSO) 2 ] precursor was prepared as the starting material using K 2 [PtCl 4 ] according to a known procedure. 42 2.8.1. N(SO 2 (2-nap))dpa ligand. A solution of 2-naphthalene sulfonyl chloride (2.5 mmol, 0.572 g) was added to a solution of N(H)dpa (5 mmol, 0.92 ml) according to a known procedure to obtain N(SO 2 (2-nap))dpa ligand. 43 Brownish yellow colour, plate like crystals suitable for X-ray diffraction were obtained (0.662 g, 68%). 1 H NMR, UV-vis and FTIR data were matched with previously reported data. Melting point: 80.5 C.

Computational methods
Geometry optimizations were performed using density functional theory (DFT) as implemented in the Gaussian 16 program. 44 The PBE1PBE 45 density functional, including the Grimme's dispersion 46 and the Becke-Johnson damping, was employed for ground state calculations. The SDD 47,48 basis set and associated effective core potentials were used for Pt, and the det2-TZVP 49,50 basis sets were applied for the other atoms. The polarizable continuum model (PCM) 51-53 was used as the implicit solvation model, where methanol (3 ¼ 32.613) was the solvent. Excited-state calculations were performed using the time dependent density functional theory (TDDFT), where the PBE1PBE functional with the Grimme's dispersion and the Becke-Johnson damping, PCM solvation, and the basis sets described above were employed. The nonequilibrium PCM was applied for calculating the singlet vertical excitations, while the equilibrium PCM was used for optimizing the excited states. The "UltraFine" integration grid was applied for TDDFT calculations, where the two-electron integral accuracy parameter was set to 12. All geometry optimizations were full with no restrictions. Vibrational frequency calculations (at 298.15 K and 1 atm) conrmed that the optimized ground structures were minima (i.e. no imaginary frequencies).
2.10. Biological studies 2.10.1. Anticancer activity. The ligands and corresponding metal complexes were investigated for their cytotoxicity against MCF-7 (breast cancer) cells and MCF-10A (normal human breast) cells. Cells were cultured in 96-well culture plates and exposed to 12.5 mg ml À1 , 25 mg ml À1 , 50 mg ml À1 and 100 mg ml À1 concentrations of ligands and complexes for 24 hours and cytotoxicity was assessed by sulforhodamine B assay. All exposures were carried out in triplicate. Briey, the cell supernatant was completely removed and washed with phosphate buffer solution. Trichloroacetic acid (50%, 25 ml) was added on top of fetal bovine serum-free fresh medium (200 ml) to make nal concentration of 10% trichloroacetic acid and was incubated at 4 C for one hour prior to the SRB assay. The plates were then washed with ve washing cycles with water and dried completely. An aliquot of 100 ml of 0.4% sulforhodamine B dissolved in 1% trichloroacetic acid, was added to each well, and was allowed to stain for 15 minutes. The plates were again washed with ve washing cycles to remove unbound dye using 1% (vol/vol) acetic acid aer removing the stain. The protein bound dye was solubilized with trisbase (10 mM, pH 7.5, 200 ml), aer air drying. The plates were then shaken for 60 minutes to homogenize the dye solution. The absorbance was then measured at 540 nm using Synergy HTBioTek microplate reader. The percentage viability was calculated by the equation given below.  (1-nap)) dpa)] and [PtCl 2 (N(SO 2 pip)dpa)] are given in Fig. 1. Key structural parameters of the X-ray structures and fully optimized ground state structures are summarized in Table 2. In general, calculated structures are in agreement with the X-ray structures. The S-N bond length (1.6277 (6) A) of the N(SO 2 (2-nap))dpa ligand is comparable with the S-N bond lengths of 1.6194 (11) A for N(SO 2 pip)dpa 25 and 1.602 (9) A for N-methyltoluene-psulfonamide and 1.641 (2) A for N,N-dimethyltoluene-p-sulfonamide. 54 However, that has not been depicted by the shortening of S-N bond length in N(SO 2 (2-nap))dpa as that of the most common cases. [55][56][57] In the ligand, the bond distances between methylene carbon and pyridyl nitrogen (1.4752 (9) (4) 54 Since the O-S-O angle is as wide as to 121.56 (16) A and the other angles around sulfur atom are slightly smaller in the range of 106.10-108.17 A (Table 3) than the ideal tetrahedral angle (109.5 ), the sulfonyl moiety represents a distorted tetrahedral arrangement around the S atom.

1 H NMR analysis
The newly synthesized platinum complexes, [PtCl 2 (N(SO 2 (2nap))dpa)], [PtCl 2 (N(SO 2 (1-nap))dpa)] and [PtCl 2 (N(SO 2 pip)dpa)] were characterized by 1 H NMR spectroscopy in DMSO-d 6 at 298 K. All the peaks were assigned related to the structure of metal complex, based on the chemical shis, splitting patterns as well as the integration of corresponding peaks. Peaks related to the residual solvents were also identied. 59 1 H NMR data for the ligands utilized in this study have been previously reported. 25,43 Therefore, the signals related to the platinum complexes were assigned accordingly. 1 H NMR spectra for all compounds are depicted in Fig. 2 Table 3. In the 1 H NMR spectrum of N(SO 2 (2-nap)) dpa, the aromatic region (7.66-8.13 ppm) is crowded with signals due to magnetically inequivalent protons present in 2-  naphthalene group of the ligand. The signal at 7.63 ppm corresponds to the H4/4 0 proton that is in the para position of the pyridyl nitrogen. Then in the range of 7.66 ppm to 8.13 ppm, 2-naphthalene ring protons can be identied according to the previously reported data for the naphthalene ring. 60 Within that region, the proton attached to C14 (1H nap in Fig. 2) at 8.47 ppm can be clearly identied due to the fact that it is a singlet and is the most deshielded proton in the naphthalene ring. Two protons in each methylene group are sterically similar. Hence, their apparent magnetic equivalence is depicted in 1 H NMR spectra by a singlet found at 4.60 ppm.

. Comparison of 1 H NMR shis (ppm) of dipicolylamine units of ligands and complexes in DMSO-d 6 is reported in
In the [PtCl 2 (N(SO 2 (2-nap))dpa)] complex, the peak attributed to methylene protons has split into two doublets (6.06 ppm and 5.33 ppm) due to magnetic inequivalence and are shied downeld. Upon coordinating to Pt, the H6/6 0 doublet has moved downeld depicting coordination of N1 and N3 to Pt. Furthermore, the proton (attached to C14) of [PtCl 2 (N(SO 2 (2nap))dpa)] appears as a singlet at 8.74 ppm.
In a 1 H NMR spectrum of the N(SO 2 (1-nap))dpa ligand, 1naphthalene ring protons can be identied in the range of 7.65 ppm to 8.21 ppm. Similar to N(SO 2 (2-nap))dpa, the proton of 1-naphthalene ring (attached to C14, Fig. 1) at 8.58 ppm can be clearly identied. Two protons of methylene group are sterically similar. Therefore a singlet for those methylene protons was found at 4.72 ppm (Fig. 2). Methylene protons of [PtCl 2 (N(SO 2 (1-nap))dpa)] complex has split into two doublets (6.42 ppm and 5.32 ppm) due to magnetic inequivalence and are shied downeld. Upon coordinating to Pt, the H6/6 0 doublet and 1H singlet of [PtCl 2 (N(SO 2 (1-nap))dpa)] have moved down-eld (9.29 ppm and 8.74 ppm, respectively).
In the [PtCl 2 (N(SO 2 pip)dpa)] complex, a higher downeld shi (0.77 ppm) is observed vs. 0.39 ppm for the rhenium tricarbonyl complex bearing the same ligand 25 showing the greater inductive of Pt vs. Re. A similar pattern was observed for Pt vs. Re complexes carrying the naphthyl derivatives. 43 For all the signals in the ligand upon coordination to Pt, signicant downeld chemical shis are observed (Table 3). These chemical shis provide a strong evidence for the formation of metal complex. Downeld chemical shis are due to inductive effect resulting from the direct Pt-N bonds which are formed between Pt metal and N atoms of the pyridyl ring.

FTIR analysis
As evident from literature, the short absorption band at 3052 cm À1 represents the asymmetric stretching vibrations of C-H bonds in aromatic ring whereas the short absorption peaks around 2900 cm À1 are attributed to the C-H symmetric stretching vibrations in aliphatic systems in N(SO 2 (2-nap))dpa ligand and its [PtCl 2 (N(SO 2 (2-nap))dpa)] complex. The spectra exhibited the decrease of intensities of the bonds at 1426 cm À1 to 1608 cm À1 which may be attributed to the C]C groups in the naphthalene ring. Consequently, the stretching vibrational peak due to C]N, in the pyridyl ring may be present in the range between 1426 cm À1 to 1590 cm À1 . The absorption peaks at 928 cm À1 (N(SO 2 (2-nap))dpa), 890 cm À1 ([PtCl 2 (N(SO 2 (2-nap)) dpa)]), 920 cm À1 (N(SO 2 (1-nap))dpa), 899 cm À1 ([PtCl 2 (N(SO 2 (1-nap))dpa)]) and 923 cm À1 ([PtCl 2 (N(SO 2 pip)dpa)]) are due to S-N stretching vibrational modes for sulfonamide groups. Most of the ligand peaks also appear in the spectra of new complexes. The strong peak at 1334 cm À1 and 1332 cm À1 were attributed to S-C stretching vibrations of the bond between S atom of sulfonyl group and the C atom of naphthalene group of N(SO 2 (2-nap))dpa and [PtCl 2 (N(SO 2 (2-nap))dpa)], respectively. Similar to N(SO 2 (2-nap))dpa, the short absorption band at 3071 cm À1 represents the asymmetric stretching vibrations of C-H bonds in aromatic ring of 1-naphthalene. C]C groups in the naphthalene ring exhibits peaks at 1590 cm À1 and 1473 cm À1 . The stretching vibrational peak due to newly formed S-C may be present at 1276 cm À1 . Most of the other peaks are similar to the previously discussed ligand, N(SO 2 (2-nap))dpa. According to the results obtained, most of the ligand peaks also appear in the spectrum of new [PtCl 2 (N(SO 2 (1-nap))dpa)] complex. The peaks at 1457 cm À1 and 1598 cm À1 are due to C]C stretching vibrations of naphthalene ring. The peaks at 1276 cm À1 and 1132 cm À1 were attributed to S-C stretching vibrations of N(SO 2 (1-nap))dpa and [PtCl 2 (N(SO 2 (1-nap))dpa)], respectively. The obtained sharp peaks at 1157 cm À1 and 1134 cm À1 of metal precursor due to the S-Pt bond were not observed in the spectrum of [PtCl 2 (N(SO 2 pip)dpa)] complex providing evidence that N(SO 2 pip)dpa ligand and metal precursor have completely reacted to give the product of [PtCl 2 (N(SO 2 pip)dpa)].

Fluorometric analysis
Fluorescence spectra were obtained for all six compounds (Table S3 †) in methanol. The concentration of the test samples were approximately 0.01 mol dm À3 for the analysis. Fluorescence spectrum for N(SO 2 pip)dpa was previously reported. 25 The compounds were excited in the UV range and emission spectra of Pt complexes were obtained with l max ¼ 275 nm (Fig. S1 †). In addition to that, a small peak was observed at 757 nm for the three complexes (Table S3 †).
We have calculated the lowest excited singlet state minima for N(SO 2 (2-nap))dpa system, showing the emission at 354 nm. This is in agreement with the experimental value (343 nm). Computed electron density difference between the singlet excited state minima and its ground state is shown in Fig. 5, which can be assigned as the 2-nap ligand-centered emission (i.e. 1 LC).
3.6. Bio assays 3.6.1. Antiproliferative activity. Three ligands and three novel complexes were assayed for their antiproliferative activity in human breast cancer cells (MCF-7) and normal human breast cells (MCF-10A). In this study, the cell lines were exposed to synthesized compounds in a concentration gradient up to 100 mg ml À1 and the half maximal inhibitory concentration (IC 50 ) was determined for each compound. Cytotoxicity was measured with sulforhodamine B assay. The results are provided in Fig. 6.
Excitingly, all ligands and complexes show dose dependent antiproliferative activity against the MCF-7 human breast cancer cell line where N(SO 2 (1-nap))dpa, N(SO 2 (2-nap))dpa and N(SO 2 pip)dpa show IC 50   Interestingly, platinum complexation changes the dynamics of the ligands' behavior against the cancer cells as well as the normal cells. [PtCl 2 (N(SO 2 (1-nap))dpa)] compared to its ligand has low cytotoxicity against the MCF-7 but higher cytotoxicity against MCF-10A. Furthermore, N(SO 2 (1-nap))dpa shows a biphasic cytotoxicity curve suggesting the involvement of multiple pathways in eliciting cytotoxic activity (Fig. S3 †). The highest perturbation of ligand activity was observed with N(SO 2 (2-nap))dpa complexation with Pt where cytotoxicity against cancerous cells was lowered and a high cytotoxicity was mounted against MCF-10A non-cancerous cells. It can be hypothesized that complexation with Pt would lock and make the ligand conformationally rigid. Hence, its dynamic binding to the proteins or DNA/RNA can be perturbed. However, some ligands may get locked in the correct conformation, rendering them to be more effective once complexed with Pt acting synergistically. It is interesting to note that the N(SO 2 pip)dpa ligand and its Pt complex display a high level of cytotoxicity against MCF-7 cells. Comparing the IC 50 values at 24 h against MCF-7 breast cancer cell lines, [PtCl 2 (N(SO 2 pip)dpa)], N(SO 2 (2-nap))dpa, N(SO 2 pip)dpa and [PtCl 2 -N(SO 2 (1-nap))dpa] compounds exhibit higher toxicity than the reported value for cisplatin (97.86 mM) (https://www.cancerrxgene.org/ translation/Drug/1005) which is a widely used anticancer drug. Further investigations are warranted to decipher the cellular mechanisms of cell death due to these compounds.
High antiproliferative activity implies the need of low concentrations of effective doses that may result in lower sideeffects in treatments. This assay system does not test for recurring cell proliferation aer the drug pressure is lied. However, at higher doses cells do not appear in the treatments indicating efficient removal of cancer cells. These ndings emphasize that the above compounds have the potential to be promising anticancer drug leads.

Conclusions
Three novel Pt complexes were synthesized in good yield in high purity and characterized by various spectroscopic techniques. It is noteworthy that the splitting of the methylene (-CH 2 ) signal to doublets in NMR spectra is not indicative of the denticity of ligands containing the dpa sulfonamide moiety in platinum and rhenium complexes. Structural results of all three complexes established that a rare 8-membered chelate ring is formed in each of the three complexes where the central sulfonamide N is not bound to Pt. X-ray structures are consistent with the optimized structures from DFT. TDDFT calculations indicated that, the key vertical excitations of N(SO 2 (2-nap)) dpa and [PtCl 2 (N(SO 2 (2-nap))dpa)] involved the 2-nap-ligandcentered p / p* excitation ( 1 LC). In the case of N(SO 2 (2nap))dpa, strong 1 LC emission leads to orescence.
We have reported here the rst experimental results for anticancer activity for ligands containing the naphthyl dpa sulfonamide derivatives as well as for three novel platinum complexes where all compounds displayed positive anticancer activity. Low IC 50 values obtained for ligands and complexes conrm that this moiety could indeed be explored towards nding new drugs which possess promising anticancer properties.