Enhanced G-Quadruplex DNA Stabilization and Telomerase Inhibition

Feb 6, 2017 - Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we ha...
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Enhanced G‑Quadruplex DNA Stabilization and Telomerase Inhibition by Novel Fluorescein Derived Salen and Salphen Based Ni(II) and Pd(II) Complexes Asfa Ali,† Mohini Kamra,† Soma Roy,† K. Muniyappa,§ and Santanu Bhattacharya*,†,‡ †

Department of Organic Chemistry and §Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Director’s Research Unit, Indian Association for the Cultivation of Science, Kolkata 700 032, India

‡

S Supporting Information *

ABSTRACT: Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we have synthesized nickel(II) and palladium(II) salen and salphen complexes by using readily available fluorescein as the backbone to provide an extended aromatic surface. The metal complexes exhibit affinity toward the human telomeric G-quadruplex DNA with promising inhibition of telomerase activity. This has been ascertained by their efficiency in the long term cell proliferation assay which showed significant cancer cell toxicity in the presence of the metal complexes. Confocal microscopy showed cellular internalization followed by localization in the nucleus and mitochondria. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway.

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INTRODUCTION Metal based complexes have gained importance with the discovery of cis-platin, a Pt(II)-drug, which entered into the clinical trials in 1971 and has been in use in chemotherapy since then.1,2 While some Pt(II)-based drugs have been used widely in restricting cancer, they have several disadvantages like applicability to limited types of cancers, acute nephrotoxicity, and drug resistance problems.3 This necessitated the research on non-platinum-based metal complexes toward inhibition of cancer cell proliferation. Transition metal ions, in general, have varying oxidation states and can extend their coordination numbers and, therefore, span a large number of geometries.4 As a result, various complexes with transition metal centers have been synthesized over the years to bind DNA, which serve as the prime target for the inhibition of cancer.5 Depending upon the nature of metal ions and their corresponding oxidation states, a wide variety of metal complexes with varying geometries is known. Several square planar complexes have been reported, especially in the case of d8-systems [Ni(II), Pd(II), and Pt(II)], which interact with duplex DNA (ds-DNA) either by noncovalent associations (intercalation,6 groove binding,7,8 stacking4,9,10) or via covalent interactions (interstrand/intrastrand cross-linking).11−13 On the other hand, many complexes having octahedral, square © 2017 American Chemical Society

pyramidal, and other nonplanar geometries have also been synthesized with Fe(III),14,15 Zn(II),16,17 Mn(III),18 Ru(II) etc.19,20 as the core metal ion. Various classes of ligands and metal complexes have been reported to interact with ds-DNA and the mode of interaction of such metal complexes on DNA may be varied (electrostatic, noncovalent, or sometimes by damaging the DNA via cleavage mechanisms).14−21 G-quadruplex (G4) DNA is a morphologically distinct, higher-order DNA structure with characteristic planar aromatic surfaces called the G-tetrads which are linked by a variety of loops and grooves.22−24 The abundance of these structures at the telomeres and promoter regions of several oncogenes makes them putative anticancer targets.22−28 Also, the telomerase enzyme is overexpressed in cancer cells compared to the normal ones.29,30 Extensive research has resulted in several G4 stabilizers and telomerase inhibitors31−39 with a prominent role played by a wide variety of metal based complexes. Among various metal complexes, Ru(II) complexes have been reported to have affinity toward G4 DNA with substantial telomerase inhibition.40−44 Certain phthalocyanines Received: August 2, 2016 Revised: December 18, 2016 Published: February 6, 2017 341

DOI: 10.1021/acs.bioconjchem.6b00433 Bioconjugate Chem. 2017, 28, 341−352

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Bioconjugate Chemistry Scheme 1a

Reagents, conditions, and yields: (a) CHCl3, MeOH, 15-crown-5, NaOH, 55 °C, 2 h, 33%; (b) ethylene diamine, EtOH, reflux, 12 h, 80%; (c) NiCl2·6H2O/Pd(OAc)2, MeOH, 65 °C, 2 h, 74−82%; (d) o-phenylenediamine, EtOH, reflux, 12 h, 78%; (e) NiCl2·6H2O/Pd(OAc)2, MeOH, 65 °C, 2 h, 78−80%. a

DNA. This has been substantiated by the UV−vis absorption, circular dichroism (CD), and fluorescence spectral titration experiments. The DNA melting data showed the extent of stabilization by these metal complexes. Besides, the metal complexes also showed efficiency toward telomerase inhibition. The metal complexes exhibited high activity specifically toward cancer cell lines over the long term which ascertains their telomerase inhibition pathway. Detailed analysis showed cellular internalization, nuclear and mitochondrial targeting, and apoptotic mechanism responsible for the cell toxicity.

and porphyrazines having Zn(II) as the central metal ion have been reported to induce and stabilize the G4 DNA over dsDNA.45,46 Pt(II) is another metal ion which has been used extensively for selectively targeting the G4 DNA for its capability of adopting square planar geometry, thus having an additional advantage for identifying the planar G-tetrads.4,47,48 Numerous metal based salen/salphen compounds have been reported to interact with the ds-DNA;49−54 however, recently it has been shown that the square planar Ni(II) complexes possessing the salen scaffold exhibit better human G4 DNA stabilization and telomerase inhibition.9,10,17,55,56 Herein, we present novel salen/salphen based series of fluorescein derivatives with Ni(II) and Pd(II) centers to attain the planarity required for efficient interaction with the G4 DNA structures. Fluorescein has an intrinsic fluorescent property and we have designed a salen scaffold which bridges the two fluorescein moieties. Extensive structural analyses by several groups have indicated that the presence of a large planar aromatic moiety is quite significant in binding to the guanine tetrads.9,23,57−59 Although the size of the metal complexes (18.9−19.2 Å) is larger than that of the tetrad core (10.9 Å × 13.6 Å), it has been observed that the planar core moiety of the complex stacks considerably well over the guanine tetrads (SI, Figure S1). The terminal rings of the extended aromatic system of the complex do not participate in the binding interaction with the G4 DNA. However, their presence is essential to maintain the aromaticity and hence the emissive nature of the complexes. The enhanced planarity along with the fluorescent handle makes the molecules apt for stabilization of the G4

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RESULTS AND DISCUSSION Chemistry. The fluorescein based salen/salphen metal complexes were designed with the idea of extending the aromatic planar surface while retaining the core salen/salphen scaffold. In addition, the presence of fluorescein moiety resulted in an intrinsic fluorescence of the metal complexes without requiring the attachment of any extra fluorescent tag. Recently, fluorescein hydrazones have been used as topoisomerase (Topo I and IIα) inhibitors.60 However, to the best of our knowledge, fluorescein salens and salphens have not been prepared and investigated toward the G4 stabilization and telomerase inhibition to restrain cancer. Fluorescein monoaldehyde was synthesized from readily available fluorescein using the Reimer-Tiemann reaction,61 and the same was treated with ethylene diamine or o-phenylenediamine to yield the salen (6) or salphen (7) ligands, respectively (Scheme 1). Further reaction with the corresponding metal salts gave the desired Ni(II) and Pd(II) complexes (Figure 1). 342

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Bioconjugate Chemistry

Figure 1. Molecular structures of the ligands (A) DFSP, (B) DFS, and their corresponding Ni(II) and Pd(II) complexes (C) DFSP-Ni, (D) DFSNi, (E) DFSP-Pd, and (F) DFS-Pd under study, shown as CPK model. The structures have been optimized at the B3LYP/6-31G* level of theory.

however, exhibited an insignificant effect on titration with the G4 DNAs. As perhaps expected,17 the salphen complexes (DFSP-Ni and DFSP-Pd) exhibited better association with the G4 DNAs compared to the corresponding metallosalens. Salphens possess extended π-aromatic surface and significantly greater planarity due to the presence of the phenyl bridge between the two fluorescein moieties in comparison with the aliphatic ethylene diamine linker comprising the salens. The ionic radius of the central metal ion also plays an important role in the stabilization of the G-quartet62 which might be one of the factors for better stabilization capacity of Ni(II) complexes compared to the relatively larger Pd(II) complexes. However, the metal complexes showed negligible interaction with a telomeric duplex DNA and calf-thymus (CT) DNA. The binding constants of the metal complex-G4 DNA association were determined from the linear fitting of the Scatchard plots (Table 1). Circular dichroism (CD) spectroscopy is an important experimental tool to determine the interaction of each metal complex toward various DNAs. A considerable change in the CD spectra was observed on addition of the metal complexes to the G4 DNAs with substantial increase observed in the case of DFSP-Ni and DFSP-Pd when titrated with Hum21(KCl) and Hum21(NaCl). However, the interaction of the corresponding

The formation of the Schiff base has been confirmed by the distinct changes in the chemical shift (δH, ppm) values as the aldehyde in compound 5 gets converted into the corresponding imines in compounds 6 and 7. All new compounds were fully characterized by 1H NMR, 13C NMR, IR, mass spectral, and elemental analysis. Although it is well established from literature that Ni(II) and Pd(II) salen complexes result in a square planar geometry,17,54,55 we still ascertained the same from electron paramagnetic resonance (EPR) spectroscopy which showed the metal complexes to be EPR-silent confirming them to be diamagnetic square planar complexes. DNA Binding Studies. Various biophysical experiments were performed to evaluate and quantify the binding affinity of the metal complexes to the human telomeric G4 DNA. UV−vis absorption spectral titrations of the metal complexes were carried out with the Hum21(KCl) (mixed hybrid-type) and Hum21(NaCl) (antiparallel) G4 DNAs (SI, Figure S2). A strong hypochromism along with a bathochromic shift was observed on titration of the salphen complexes with the human telomeric G4 DNAs (SI, Figure S2). This indicates a binding involving considerable π−π stacking interaction between the planar aromatic core of the complexes and the tetrads of the G4 DNA. The extent of hypochromism was maximum for DFSPNi with Hum21(KCl) G4 DNA. The corresponding salens, 343

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Bioconjugate Chemistry Table 2. Summary of the DNA Melting Studiesa

Table 1. Binding Constants (Ka) of the Metal Complexes with the Preformed G-Quadruplex DNA and CT DNA as Determined from UV−vis Spectral Titrations Ka (106 M−1) metal complex DFS-Ni DFS-Pd DFSP-Ni DFSP-Pd

Hum21(KCl)a 0.4 0.6 5.0 2.9

± ± ± ±

0.05 0.01 0.4 0.2

Hum21(NaCl)a

ds-DNAb

± ± ± ±

0.02 ± 0.004 -

2.6 0.7 3.6 1.2

0.2 0.01 0.3 0.2

metal complex

Tm (°C)

ΔTm (°C)

DFS-Ni DFS-Pd DFSP-Ni DFSP-Pd

70 68.5 76 72

4 2.5 10 6

ΔTm values were determined from the difference in melting temperatures ligand-G4 DNA complex and G4 DNA [Hum21(KCl)] using CD melting studies. A buffer having 10 mM Tris-HCl, 100 mM KCl, pH 7.4 was used. The results are the average of two independent experiments and the errors are within ±0.5 °C. a

a

UV−vis spectral titrations were performed with the Hum21(KCl) and Hum21(NaCl) G4 DNA in a buffer having 10 mM Tris-HCl, 100 mM KCl/NaCl, pH 7.4. bUV−vis spectral titrations were performed with CT DNA in a buffer having 10 mM Tris-HCl, 40 mM NaCl, pH 7.4.

mode of association of the metallosalphens which make them better candidates as G4 binders than the corresponding salens, we performed molecular modeling studies using the G4 DNAs in the K+ and Na+ ion medium (PDB ID 1KF1 and 143D). Here, we have taken the energy minimized structures of both the Ni(II) salen and salphen complexes (DFS-Ni and DFSPNi) and subsequently docked them to the morphologically distinct human telomeric G4 DNAs. While both have identical fluorescein moieties, the difference lies in the bridging linker constituting the salen/salphen scaffold, which, in turn, modulates the extent of their planarity. The more planar salphen complex, DFSP-Ni, stacked on the G-quartet quite efficiently compared to the salen complex (DFS-Ni). This is reflected in the much lower binding energy of the former (−11.15 kcal mol−1) with a high clustering pattern. On the other hand, the metal complexes exhibited comparatively less efficient binding with the antiparallel G4 DNA, thereby justifying the earlier data (Figure 3). Fluorescence Titrations. An important aspect of designing the salen/salphen scaffold with fluorescein moieties is the presence of their inherent fluorescence. On titration with the ds- and G4 DNAs, a quenching in the fluorescence of the metal complexes around the peak ∼520−530 nm was observed in all the cases. However, the extent of quenching differs with the nature of the metal complexes and their interaction with specific DNA morphology (ds- or G4) (Figure 4, SI Figure S7). Fluorescein, in general, is reported to quench its fluorescence in the vicinity of nucleobases.62 Guanine, with its highest electron donating capacity among all the nucleobases, quenches the fluorescence intensity of fluorescein moiety due to the formation of complex between them.64 Although quenching was observed for all kinds of DNA investigated, the extent was greater in the case of G4 DNA in the presence of DFSP-Ni, which comes in close vicinity of the G-tetrads due to efficient end-stacking (Figure 4). The relatively lower decrease in the fluorescence emission with DFSP-Pd may be due to its less efficient stacking owing to the larger size of Pd(II) compared to that of corresponding Ni(II) complex. However, an enhancement in the fluorescence ∼390−450 region of the metal complexes also occurred upon titration with the G4 DNAs (Figure 4). This increase in fluorescence could be due to the binding of the metal complexes to the G4 DNA, leading to their likely occupation inside a hydrophobic environment. On the other hand, in the case of CT-DNA, there is fluorescence quenching of the metal complexes in the vicinity of nucleobases (Ka ∼ 104) but negligible extent of enhancement in the fluorescence intensity which suggests an inefficient or “loose” association of the metal complexes toward them (SI, Table S1). Thus interaction of the metal complexes with G4 DNA resulted in an interesting dual effect of

metallosalens with the G4 DNAs was weaker, which is consistent with the results obtained from the absorption spectral titrations. A moderate shift in the band (∼295 to 288 nm) was observed during the titration of Hum21(NaCl) with DFS-Ni. A characteristic positive peak at 295 and 260 nm signifies the presence of the antiparallel and parallel G4 DNAs, respectively. Also, the peak at 295 nm mainly corresponds to the guanine stacks in the G4 DNA.63 So, the band shift in this case might indicate that the compound interacts with the Gtetrad of the antiparallel G4 DNA (Figure 2, SI Figure S3).

Figure 2. CD spectral titrations of 4 μM Hum21(KCl) G4 DNA with increasing concentration of DFSP-Ni in a buffer having 10 mM TrisHCl, 100 mM KCl/NaCl, pH 7.4.

To further investigate the stabilization of the G4 DNA with metal complexes, thermal denaturation studies were performed by CD spectroscopy. Preformed G4 DNA (4 μM) was incubated with the metal complexes (8 μM) in a buffer having 10 mM Tris-HCl, 100 mM KCl/NaCl, pH 7.4 and the temperature was varied from 25 to 90 °C. The change in CD was monitored and the melting curve was determined at 295 nm (SI, Figures S4−S6). Elevation in the melting temperature (ΔTm) indicates the extent of stabilization of the G4 DNA by the metal complexes. Here, DFSP-Ni showed the maximum increase in the melting temperature followed by DFSP-Pd. On the other hand, the salens (DFS-Ni and DFS-Pd) stabilized the G4 DNA but to a significantly lower extent (Table 2). The above experiments conclusively show the better binding efficiencies of the metallosalphens toward G4 DNA (DFSPNi>DFSP-Pd) than the corresponding metallosalens. Mode of Binding. The metallosalphens preferred binding to the human telomeric G4 DNA. However, to investigate the 344

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Figure 3. Docked structures of DFSP-Ni (blue) with (A) parallel propeller type (PDB: 1KF1), (B) antiparallel basket type (PDB: 143D) G4 DNAs and DFS-Ni (red) with (C) parallel propeller type (PDB: 1KF1), (D) antiparallel basket type (PDB: 143D) G4 DNAs.

Figure 4. Fluorescence titrations of 0.5 μM DFSP-Ni with (A) Hum21(KCl) G4 DNA with saturation at [G4 DNA]/[metal complex] ratio of ∼40 in a buffer having 10 mM Tris-HCl, 100 mM KCl, pH 7.4, and (B) CT DNA in a buffer having 10 mM Tris-HCl, 40 mM NaCl, pH 7.4.

345

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Bioconjugate Chemistry fluorescence enhancement as well as quenching depending on the nature of the core moiety and the central metal ion used. Telomerase Enzyme Inhibition. As cancer cells witness a marked overexpression of the telomerase enzyme (>85%) in contrast with negligible activity in normal somatic cells,65 we examined the efficiency of the metal complexes toward the inhibition of the enzyme. TRAP-LIG assay66,67 showed considerable inhibition of the telomerase action which is evident from the decrease in the ladder intensity even at low concentrations of DFSP-Ni. However, such inhibition was observed only at a relatively higher concentration of other metal complexes. The TS primer elongates by the addition of TTAGGG repeats by the telomerase enzyme and this phenomenon is restricted to the formation of the G4 DNA structures. Hence, the metal complexes that effectively stabilize the G4 DNA are capable of inhibiting this elongation step leading to a decrease in the number of bands in the ladder. Here, the efficient G4 DNA stabilizing complex DFSP-Ni proved to be highly active in the inhibition of the telomerase action (IC50 value of 0.9 ± 0.02 μM) while DFS-Ni failed to do so (Figure 5).

However, noticeable toxicity was not observed with the metal complexes on HeLa cell line. Accordingly, the metal complexes showed marked concentration dependent changes in the cell viability resulting in clumping and debris formation, as seen in HEK 293T cells in comparison to A549 cells (Figure 6). The biophysical experiments have shown the efficiency of the metal complexes toward their stabilization of the G4 DNA. However, the short-term (72 h) cell viability assay cannot be correlated with the telomerase inhibitory activity of the metal complexes.69 So long-term cell proliferation experiment (15 days) was performed on human embryonic kidney transformed cells (HEK 293T) and human cervical cancer cells (HeLa) with subcytotoxic metal complex concentration (3 μM). The metal complexes showed sufficient antiproliferative activity toward the cell lines with DFSP-Ni which was found to be the most efficient one (Figure 7). It is to be noted that the metal complexes showed prominent inhibition in the cancer cell proliferation over the long term while sufficiently less toxic activity on the same cell lines over the short term (72 h). This ascertains that the pronounced long-term cytotoxicity of the metal complexes may be due to the inhibition of telomerase activity. Mechanism of Cell Death. We then investigated the nature of the effect of metallosalen/salphen treatment on the cancer cells (HEK 293T and A549). As the cell viability assays revealed marked changes in the cellular morphology consistent with apoptosis (characterized by rounding of cells, debris formation, etc.), we quantified the apoptotic cells by the cell cycle analysis. Propidium iodide is quite impermeable to the viable cell membranes; however, it is reported to identify the dead cells which are apoptotic in the sub-G1 region of the cell cycle.70 On treatment of HEK 293T cells with the metal complexes at near IC50 values for a period of 48 h, we observed ∼33−42% cells in the sub-G1 (apoptotic) region of the cell cycle (Figure 8, SI Table S3). With the significantly higher subG1 population in the treated cells compared to the untreated ones, it can be concluded that the metal complexes resulted in cell death via apoptosis. Cellular Internalization. The ability of the metal complexes to penetrate and internalize into the cancer cells was investigated using confocal microscopy experiments. Upon the treatment of metallosalen/salphens on HEK 293T and A549 cells, considerable internalization was observed in the case of DFSP-Ni and DFSP-Pd after incubation for a period of ∼24 h (Figure 9, SI, Figure S8). Co-staining experiments have been carried out by staining both the nucleus and mitochondria (the DNA rich regions in the cell). The images indicate that the metal complexes internalize well in both mitochondria and nucleus (shown by white arrows in SI, Figure S8). Thus besides G4 DNA targeting and telomerase inhibition, these fluorescein based metallosalens/salphens resulted in easy cellular penetration and internalization as demonstrated by the inherent fluorescence of the probes. Fluorescence intensity of the DFSP-Ni complex (green channel) was measured by selecting equally sized regions of interest (ROI 1, ROI 2, and ROI 3) in the nucleus, mitochondria, and cytoplasm, respectively (SI, Figure S8). Based on these intensity values, relative intensity profiles were plotted (SI, Figure S9) which indicate the time-dependent accumulation of the complex in the nucleus and mitochondria. This preferential accumulation of the metal complex in the DNA rich regions of the cells testifies its DNA binding affinity. G-quadruplex DNA motifs are known to be present in

Figure 5. Telomerase inhibition assay with (A) DFSP-Ni; lane 1: positive control; lanes 2, 3, and 4: increasing concentrations of DFSPNi (0.4, 0.8, and 1.2 μM); lane 5: negative control and (B) DFSP-Pd; lane 1 and 2: negative and positive control, respectively; lanes 3, 4, and 5: increasing concentrations of DFSP-Pd (0.4, 0.8, and 1.2 μM).

Toxicity toward Cancer and Normal Cells. We have checked the effect of the metal complexes on various cancer cells, namely, human embryonic kidney transformed cells (HEK 293T), human cervical cancer cells (HeLa), and adenocarcinoma human alveolar basal epithelial cells (A549). Normal cells like telomerase negative human foreskin fibroblast normal cells (HFF)68 and human embryonic kidney cells (HEK 293) were also used for the study under identical conditions. We determined the growth inhibition profile of these cell lines after metal complex treatment for a period of 72 h (short-term) using MTT based cell viability assay (SI, Table S2). Although the metal complexes exhibited concentration-dependent toxicity toward cancer cell lines, significantly lower toxicity was obtained for the noncancer cell lines (HFF and HEK 293). 346

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Figure 6. Representative bright field images of the cell morphology of human embryonic kidney transformed cells (HEK 293T) with increasing concentrations of (A) DFSP-Ni, (B) DFSP-Pd, (C) DFS-Ni, and (D) DFS-Pd.

Figure 7. Effect of metal complexes on the cell proliferation upon their long-term exposure (15 days) with (A) human embryonic kidney transformed cells (HEK 293T) and (B) human cervical cancer cells (HeLa) as determined using Trypan Blue cell counting assay.

Figure 8. Cell cycle analysis to determine the percentage of cells in sub-G1 (apoptotic) region in (A) HEK 293T cells alone and on treatment with (B) DFS-Ni and (C) DFSP-Ni.

titrations. While the CD and fluorescence spectroscopic studies exhibited better binding of the salphens to the human telomeric G4 DNA [Hum21(KCl)/Hum21(NaCl)], the same is also reflected in the DNA melting studies in the presence of the corresponding metal complexes. Computational studies showed the principal binding mode of the metal complexes to be endstacking where, in the case of DFSP-Ni, the extended aromatic core of the metal complex stacks on the π-surface of the guanine tetrads. Significant telomerase inhibition was obtained at a very low metal complex concentration (0.9 μM). The poor activity of the metal complexes at the short-term (72 h) cell viability assays and high efficiency of the same at long-term (15

fundamental regulatory regions of the human genome such as telomeres, gene bodies, UTRs, and promoters.71−74.

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CONCLUSIONS In the present study, we have synthesized salen and salphen metal complexes based on fluorescein as the backbone. The highly planar and extended aromatic core of the salphen complexes made them excellent G4 DNA stabilizers with the maximum efficiency exhibited by DFSP-Ni. The Pd(II) complexes, on the other hand, showed relatively weaker association with the G4 DNA. This is clearly evident from the binding constant data obtained from the UV−vis spectral 347

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Figure 9. Representative confocal microscopic images of HEK 293T cells on treatment with (A) DFSP-Ni and (B) DFSP-Pd for 24 h at near IC50 concentration. DAPI and MitoTracker Red CMXRos were used as the nuclear and mitochondrial counterstains, respectively. Panels (left to right) represent bright field, metal complex fluorescence (green), DAPI nuclear (blue) and MitoTracker Red CMXRos mitochondrial (red) counterstains, and, finally, overlay of the previous two images, respectively. Scale bar = 20 μm.

pound 5 (2 g, 5.55 mmol) was taken in 15 mL of ethanol and heated to dissolve. To it, freshly distilled ethylenediamine (133.2 mg, 2.22 mmol) was added and the resulting mixture was heated at 65 °C for 14 h. A light orange precipitate was filtered. The residue was dried, washed thoroughly with cold methanol, and finally dried again. (3.31 g, 80%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 14.73 (s, 2H), 10.20 (s, 2H), 9.22 (s, 2H), 7.98 (d, J = 7.6, 2H), 7.75−7.80 (m, 2H), 7.68−7.73 (m, 2H), 7.26 (d, J = 7.6, 1H), 7.18 (d, J = 7.2, 1H), 6.80 (d, J = 10.8 Hz, 2H), 6.54−6.60 (m, 6H), 6.47−6.50 (m, 2H), 4.14 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 168.8, 168.7, 161.9, 159.4, 152.2, 152.1, 151.1, 150.7, 135.6, 132.9, 130.1, 128.9, 126.2, 126.1, 124.6, 124.0, 116.0, 113.2, 109.5, 105.1, 104.9, 102.5, 82.2, 55.5; IR (KBr): 3440, 2928, 1748, 1634, 1461, 1399, 1285, 1250, 1221, 1163, 1115, 1094, 1014, 850 cm-1; HRMS: m/z = 745.1829 [M + H]+; Calcd =745.1822 [M + H]+; mp >250 °C. Anal. (C44H28N2O10) calcd: C 70.96, H 3.79, N 3.76; found: C 71.11, H 3.82, N 3.73. 2-{5-[(E)-N-{2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]methylide- ne}amino]phenyl}carboximidoyl]-6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid (7). Compound 5 (1.5 g, 4.17 mmol) was taken in 12 mL of ethanol and heated to dissolve. To it, freshly crystallized ophenylenediamine (180.5 mg, 1.67 mmol) was added and the resulting mixture was heated at 65 °C for 14 h. A light orange precipitate was filtered. The residue was dried, washed thoroughly with cold methanol, and finally dried again (3.31 g, 78%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 14.55 (s, 2H), 10.18 (s, 2H), 9.54 (s, 2H), 8.01 (d, J = 7.6 Hz, 2H), 7.78−7.82 (m, 2H), 7.70−7.74 (m, 4H), 7.53−7.56 (m, 2H), 7.29−7.32 (m, 2H), 6.93 (d, J = 1.2 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.68− 6.72 (m, 2H), 6.60−6.61 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 168.8, 159.5, 152.5, 151.9, 135.6, 130.1, 129.1, 126.2, 124.6, 124.1, 112.7, 109.6, 102.3, 83.1; IR (KBr): 3437, 2922, 2852, 1751, 1622, 1455, 1336, 1286, 1225, 1160,

days) indicate the affinity of these complexes toward the G4 DNA which, in turn, inhibit the telomerase action. Although fluorescein itself is cell permeable, the metal complexes effectively localize in the mitochondria and nucleus which is quite prominent from the cellular internalization studies. Moreover, sufficient population at the sub-G1 region of the cell cycle in the case of metal complex treated cells exhibits that the observed cytotoxicity may be due to apoptotic pathway followed by the metal complexes. Thus, we have shown that inherently fluorescent fluorescein-based molecules selectively target the G4 DNA, leading to inhibition of the telomerase activity, which results in apoptosis and cancer cell toxicity.

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EXPERIMENTAL SECTION Materials. All starting materials were procured from the best known commercial sources. The solvents were purchased from Merck and were distilled and/or dried before use whenever necessary. General Spectrometric Characterization. 1H (400 MHz) and 13C NMR (100 MHz) spectra were recorded on Bruker AMX spectrometer. IR spectra were recorded on FT-IR PerkinElmer Spectrum GX spectrometer. Mass spectra were recorded on Micromass Q-TOF Micro TM spectrometer. Synthesis. 2-(5-Formyl-6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (5). Compound 5 was synthesized given in %yield following a reported procedure.10 1 H NMR (400 MHz, DMSO-d6) δ ppm 6.63 (s, 2H), 6.68 (d, J = 8.8, 1H), 6.85 (s, 1H), 6.96 (d, J = 9.2, 1H), 7.26 (d, J = 7.6, 1H), 7.71−7.75 (m, 1H), 7.79−7.82 (m, 1H), 8.04 (d, J = 7.6, 1H), 10.31 (s, 1H), 10.70 (s, 1H); IR (KBr): 3378, 2927, 1727, 1613, 1460, 1288, 1256, 1217, 1189, 1113, 895 cm−1; HRMS: m/z = 361.0713 [M + H]+; Calcd = 361.0712, [M + H]+. 2-{5-[(1E)-({2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]-methyldene }amino]ethyl}imino)methyl]6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid (6). Com348

DOI: 10.1021/acs.bioconjchem.6b00433 Bioconjugate Chem. 2017, 28, 341−352

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Bioconjugate Chemistry 1093, 1108, 1010, 958, 860 cm−1; HRMS: m/z = 815.1647 [M + H]+; Calcd =815.1642 [M + H]+; mp >250 °C. Anal. (C48H28N2O10) calcd: C 72.72, H 3.56, N 3.53; found: C 72.58, H 3.59, N 3.53. 2-{5-[(1E)-({2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]methylid- ene}amino]ethyl}imino)methyl]6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid-nickel(II) (DFS-Ni, 1). Compound 6 (200 mg, 0.269 mmol) was taken in 3 mL methanol to which 1 drop of saturated NaHCO3 solution in water was added. To it, methanolic solution (1 mL) of NiCl2·6H2O (64 mg, 0.269 mmol) was added and the resulting solution was heated at 60 °C for 12 h. The reaction mixture was reduced to dryness and the crude solid obtained was then washed with methanol. The final product was then dried and characterized (159 mg, 74%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.30 (s, 1H), 8.04 (d, J = 7.6 Hz, 2H), 7.45−7.49 (m, 5H), 7.05−7.09 (m, 3H), 6.8 (d, J = 8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 3H), 6.28 (s, 2H), 6.14 (d, J = 8.8 Hz, 1H), 6.08 (d, J = 9.6 Hz, 1H), 6.03 (s, 1H), 4.02 (d, J = 6.4 Hz, 2H), 3.90 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm 188.4, 181.2, 181.1, 170.0, 169.4, 161.7, 142.5, 133.7, 132.9, 130.0, 130.0, 128.8, 128.1, 128.0, 125.2, 124.3, 112.5, 111.1, 110.3, 108.2, 102.7, 59.7 ; IR (KBr): 3430, 2928, 1737, 1637, 1589, 1459, 1376, 1341, 1182, 1118, 1018, 922, 836, 645 cm−1; HRMS: m/z = 801.1017 [M + H]+; Calcd = 801.1019 [M + H]+; mp >250 °C. Anal. (C44H26N2NiO10) calcd: C 65.95, H 3.27, N 3.50; found: C 66.01, H 3.25, N 3.49. 2-{5-[(1E)-({2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]methylidene }amino]ethyl}imino)methyl]-6hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid-palladium(II) (DFS-Pd, 2). Compound 6 (150 mg, 202 mmol) was taken in 2 mL methanol to which 1 drop of saturated NaHCO3 solution in water was added. To it, methanolic solution (1 mL) of Pd(OAc)2 (45 mg, 0.202 mmol) was added and the resulting solution was heated at 60 °C for 12 h. The reaction mixture was reduced to dryness and the crude solid obtained was then washed with methanol. The final product was then dried and characterized (141 mg, 82%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (s, 2H), 7.99− 8.02 (m, 2H), 7.49−7.54 (m, 2H), 7.42−7.47 (m, 3H), 7.01− 7.04 (m, 1H), 6.74−6.76 (m, 2H), 6.66−6.69 (m, 2H), 6.57− 6.64 (m, 1H), 6.44−6.46 (m, 1H), 6.39 (s, 1H), 6.33−6.35 (m, 1H), 6.15 (s, 1H), 6.09−6.13 (s, 1H), 4.09 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 188.7, 181.4, 181.2, 169.3, 168.8, 161.9, 141.1, 133.6, 133.2, 130.2, 130.1, 128.9, 128.3, 128.2, 128.1, 125.1, 124.6, 111.3, 110.7, 109.3, 107.5, 103.0, 48.7; IR (KBr): 3498, 3119, 2921, 2851, 2687, 2391, 2310, 1974, 1580, 1192, 1072, 1017 cm−1; HRMS: m/z = 849.0567 [M + H]+; Calcd, 849.0520 [M + H] + ; mp >250 °C. Anal. (C44H26N2O10Pd) calcd: C 62.24, H 3.09, N 3.30; found: C 62.05, H 3.10, N 3.32. 2-{5-[(E)-N-{2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]methylid- ene}amino]phenyl}carboximidoyl]-6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid-nickel(II) (DFSP-Ni, 3). Compound 7 (100 mg, 0.126 mmol) and NiCl2·6H2O (30 mg, 0.126 mmol) were treated by following the same procedure as for compound 1 to obtain the product 3 (83 mg, 78%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.21 (s, 2H), 9.71 (s, 1H), 8.29 (br s, 2H), 7.99 (d, J = 6.4 Hz, 2H), 7.72−7.79 (m, 4H), 7.33 (s, 4H), 7.14 (s, 2H), 6.61 (s, 2H), 6.53−6.57 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ ppm 168.5, 166.4, 159.3, 152.0, 152.0, 151.1, 150.7, 142.0, 135.5, 135.5, 130.0,

126.1, 124.5, 123.9, 109.1, 103.6, 103.1, 83.0, 83.0, 79.1; IR (KBr): 3442, 2929, 1741, 1633, 1463, 1456, 1362, 1241, 1122, 1102, 922, 826, 798 cm−1; HRMS: m/z = 849.1018 [M + H]+; Calcd, 849.1019 [M + H] + ; mp >250 °C. Anal. (C48H26N2NiO10·H2O) calcd: C 66.46, H 3.25, N 3.23; found: C 66.59, H 3.27, N 3.21. 2-{5-[(E)-N-{2-[(E)-{[9-(2-Carboxyphenyl)-6-hydroxy-3-oxo3H-xanthen-5-yl]methylide-ne}amino]phenyl}carboximidoyl]-6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid-palladium(II) (DFSP-Pd, 4). Compound 7 (100 mg, 0.126 mmol) and Pd(OAc)2 (28 mg, 0.126 mmol) were reacted by following the same procedure as for compound 2 to obtain the product (90 mg, 80%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.32 (s, 2H), 8.02− 8.05 (m, 2H), 7.59 (d, J = 6.8 Hz, 1H), 7.53−7.56 (m, 2H), 7.45−7.48 (m, 2H), 7.21 (d, J = 10, 1H), 7.12−7.13 (m, 1H), 7.05 (d, J = 8 Hz, 1H), 6,71 (s, 1H), 6.69 (d, J = 4 Hz, 1H), 6.66 (d, J = 3.6 Hz, 1H), 6.62−6.64 (m, 2H), 6.57−6.60 (m, 2H), 6.31−6.37 (m, 2H), 6.20−6.23 (m, 1H), 6.11−6.16 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm 181.4, 181.0, 169.6, 158.1, 157.9, 140.8, 133.7, 133.2, 130.3, 130.2, 128.9, 128.6, 128.4, 125.1, 124.7, 111.4, 110.8, 107.6, 103.1, 79.3; IR (KBr): 3791, 3395, 2924, 1582, 1462, 1309, 1121 cm−1; HRMS: m/z = 896.0622 [M]+; Calcd, 896.0622 [M]+; mp >250 °C. Anal. (C48H26N2O10Pd) calcd: C 64.26, H 2.92, N 3.12; found: C 64.16, H 2.93, N 3.14. Fluorescence Titration. The fluorescence experiments were recorded on a Carey Eclipse Varian spectrophotometer using quartz cells of path length of 4 mm. The temperature of the sample solutions were maintained at 20 °C using a Peltier temperature controller. 0.5 μM of metal complex solution was taken in the cell and G4 DNA was added successively from a 30 μM stock solution in 10 mM Tris-HCl, 0.1 M KCl or NaCl, pH 7.4. Similar procedure was followed for the ds-DNA (CT DNA) in 10 mM Tris-HCl, 40 mM NaCl, pH 7.4. All spectra were recorded at 25 °C with an incubation time of 20 min after each addition and continued until saturation. Cell Viability Assay. HeLa (human cervical cancer transformed cells), HEK 293T (human embryonic kidney transformed cells), and A549 (adenocarcinoma human alveolar basal epithelial cells) cells were seeded in 96-well plates (5.0 × 103 cells/well) and allowed to adhere and attain 70% confluency over a period of 24 h prior to their exposure to different concentrations of the various salen complexes in the presence of 0.2% FBS containing DMEM. After an incubation of 72 h, 25 μL of a 4 mg/mL solution of methyl thiazolyl tetrazolium bromide (MTT) reagent was added to each well and incubated with the cells for an additional 4 h. Following this, the supernatants were discarded leaving behind Formazan crystals in each well which were dissolved in DMSO, and absorbance read at a λmax of 570 nm. The % cell viability was calculated from the absorbance values. Long-Term Cell Proliferation Experiment. HEK 293T, HeLa, and A549 cells were grown in six-well tissue culture plates at a seeding density of 5.0 × 104 per cells well. Subcytotoxic concentration of 3 μM of each compound under study were added and incubated with the cells for 5 days. At the fifth day, the cells were counted using Trypan blue, reseeded in new wells with half the population of cells following which, treatment was given for another 5 days. In this way, the proliferation of the cells was monitored over a period of 15 days. Plots of cell population versus time (days) depict the antiproliferative activity. 349

DOI: 10.1021/acs.bioconjchem.6b00433 Bioconjugate Chem. 2017, 28, 341−352

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Cell Cycle Analysis. The cell cycle analysis was performed on human embryonic kidney transformed cells (HEK 293T) stained with propidium iodide (PI). The metal complex treated cells were fixed with 70% ethanol, treated with RNase, and stained with PI (1 μg/mL) for a period of 30 min followed by analysis on a FACS machine. Categorization of the cell population into sub-G1 (G0), G1, S, and G2/M phases were done where the population at the sub-G1 region (% apoptotic cells) was determined by WinMDI public domain software. Confocal Microscopy. HEK 293T cells were plated on 18 mm coverslips, placed in a NuncTM 12-well plate, at a seeding density of 15 000 cells per well. After 24 h, these were treated with the different metal complexes at a concentration nearly equal to the IC50. After 24 h of incubation with the metal complexes, the live cells were first stained with MitoTracker Red CMXRos, washed with growth medium followed by 50 mM PBS. The cells were then fixed using 4% paraformaldehyde, washed with 50 mM PBS, stained with DAPI, washed again with 50 mM PBS, and the coverslips were mounted on the glass slides using Prolong Gold Antifade. These were sealed on to the glass slide using nail paint and then viewed under Lieca Confocal Microscope at 63× oil immersion objective. Zstack over 4 μm thickness was taken to cover the entire cell volume. The ROI intensity profiling was carried out using Leica LAS AF version 2.6.0 and normalized values (obtained from three different images for each time point) were plotted using GraphPad Prism 5.

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ABBREVIATIONS G4 DNA, G-quadruplex DNA; CD, circular dichroism; ICD, induced circular dichroism; TRAP, telomerase repeat amplification protocol; FI, fluorescence intensity; DMSO, dimethyl sulfoxide; MTT, methyl thiazolyl tetrazolium bromide; NMR, nuclear magnetic resonance; IR, infrared; ODN, oligonucleotides; PAGE, polyacrylamide gel electrophoresis

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00433. Characterization of metal complexes, additional UV−vis titration, CD spectroscopic titration, fluorescence titration, cytotoxicity, cell cycle data, cellular internalization data, and additional methods (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (91)-80-22932664. Fax: (91)-80-22930529. ORCID

Santanu Bhattacharya: 0000-0001-9040-8971 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by J. C. Bose Fellowship, DST to Prof. S. Bhattacharya. A.A. thanks CSIR for a senior research fellowship and for a Research Associate-ship supported by the grant CSIR 0385 inter-institutional project (Advanced Drug Delivery Systems). We would like to acknowledge Dr. Santosh Podder and other staff at the Central Confocal Facility, Division of Biological Sciences, IISc for the confocal imaging. The human foreskin fibroblast (HFF) cells were a kind gift from the Manipal University, Manipal. 350

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DOI: 10.1021/acs.bioconjchem.6b00433 Bioconjugate Chem. 2017, 28, 341−352

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DOI: 10.1021/acs.bioconjchem.6b00433 Bioconjugate Chem. 2017, 28, 341−352