Investigation of the Anticancer Activity of Coordination-Driven Self-AssembledTwo-Dimensional Ruthenium Metalla-Rectangle

Coordination-driven self-assembly is an effective synthetic tool for the construction of spatially and electronically tunable supramolecular coordination complexes (SCCs), which are useful in various applications. Herein, we report the synthesis of a two-dimensional discrete metalla-rectangle [(η6-p-cymene)4Ru4(C6H2O4)2(2)2](CF3SO3)4 (3) by the reaction of a dinuclear half-sandwich ruthenium (II) complex [Ru2(η6-p-cymene)2(C6H2O4)Cl2] (1) and bis-pyridyl amide linker (2) in the presence of AgO3SCF3. This cationic ruthenium metalla-rectangle (3) has been isolated as its triflate salt and characterized by analytical techniques including elemental analysis, Fourier-transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), 1H-1H correlation spectroscopy (COSY), 1H-1H nuclear Overhauser effect spectroscopy (NOESY), diffusion ordered spectroscopy (DOSY), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). Significantly, the 2D cationic ruthenium metalla-rectangle showed better anticancer activity towards three different cell lines (A549, Caki-1 and Lovo) as compared with the parent ruthenium complex (1) and the commercially used drug, cisplatin.


Results
Half-sandwich ruthenium complex 1 ( Figure S1 and S2) is known for the formation of various discrete architectures based on the symmetry of organic linkers. The reflux reaction of 4aminopyridine with 1,1′-carbonyldiimidazole in dry tetrahydrofuran (THF) resulted in the formation of dipyridyl organic linker (2) as evidenced by Fourier-transform infrared spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance spectroscopy ( 1 H-NMR, 13 C-NMR) ( Figures S3 and  S4). FT-IR spectrum of 2 showed NH and CO vibrations stretching peaks at 2917 cm -1 and 1737 cm -1 respectively. 1 H-NMR spectrum of symmetrical organic linker exhibited one sharp singlet at 9.25 ppm (NH) along with two aromatic protons at 8.35 ppm (CHα), 7.42 (CHβ) ppm and a strong peak at 151.99 ppm in 13 C-NMR further approves the presence of amide functionalities. Self-assembly of 1 and 2 at Scheme 1. Synthesis of 2D arene ruthenium metalla-rectangle.

Results
Half-sandwich ruthenium complex 1 (Figures S1 and S2) is known for the formation of various discrete architectures based on the symmetry of organic linkers. The reflux reaction of 4-aminopyridine with 1,1 -carbonyldiimidazole in dry tetrahydrofuran (THF) resulted in the formation of dipyridyl organic linker (2) as evidenced by Fourier-transform infrared spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance spectroscopy ( 1 H-NMR, 13 C-NMR) ( Figures S3 and S4). FT-IR spectrum of 2 showed NH and CO vibrations stretching peaks at 2917 cm −1 and 1737 cm −1 respectively. 1 H-NMR spectrum of symmetrical organic linker exhibited one sharp singlet at 9.25 ppm (NH) along with two aromatic protons at 8.35 ppm (CH α ), 7.42 (CH β ) ppm and a strong peak at 151.99 ppm in 13 C-NMR further approves the presence of amide functionalities. Self-assembly of 1 and 2 at a 1:1 ratio using directional bonding approach led to the two-dimensional ruthenium metalla-rectangle (3) as its triflate salt ( Figure 1). Filtration and precipitation via the addition of diethyl ether yielded an analytically pure solid. Unfortunately, all attempts to grow single crystals for X-ray structural analysis failed. Furthermore, the planarity of urea functionalized bis-pyridyl amide linker and the nature of ruthenium complex 2 further approves the proposed structure of self-assembled metalla-rectangle 3 [64][65][66]. It is worth mentioning that the variation in symmetry elements of organic linkers and ligand to metal complex ratio afford different discrete architectures [67], for instance, self-assembly of pyridine based trigonal planar linker and half-sandwich ruthenium complex in 2:3 ratio led to ruthenium based metalla-prisms [68,69].
Molecules 2019, 24, 2284 3 of 12 a 1:1 ratio using directional bonding approach led to the two-dimensional ruthenium metallarectangle (3) as its triflate salt (Figure 1). Filtration and precipitation via the addition of diethyl ether yielded an analytically pure solid. Unfortunately, all attempts to grow single crystals for X-ray structural analysis failed. Furthermore, the planarity of urea functionalized bis-pyridyl amide linker and the nature of ruthenium complex 2 further approves the proposed structure of self-assembled metalla-rectangle 3 [64][65][66]. It is worth mentioning that the variation in symmetry elements of organic linkers and ligand to metal complex ratio afford different discrete architectures [67], for instance, selfassembly of pyridine based trigonal planar linker and half-sandwich ruthenium complex in 2:3 ratio led to ruthenium based metalla-prisms [68,69]. The synthesis of discrete arene-ruthenium architectures follows a well-established two-step strategy developed by Süss-Fink [66]. The infrared spectrum of 3 predominantly showed a strong absorption for C-F, C=O, and weak stretching C-H vibrations at 1254 cm -1 , 1733 cm -1 , and 3076 cm -1 , indicating the presence of trifluoromethanesulfonate anions, multiple carbonyl groups, and pcymene moieties, respectively ( Figure S5). Similar arene ruthenium based discrete metalla-rectangles, metalla-bowls and metalla-prisms exhibited stretching vibrations in a comparable range for C-F, C=O and C-H functionalities [70,71]. 1 H-NMR and 13 C-NMR spectra ( Figure S6 and S7) further confirms the successful synthesis of the ruthenium metalla-rectangle. As can be seen in Figure S7, the 13 C-NMR of 3 exhibited a peak around 184.45 ppm, validating the presence of amide functionality in the discrete structure. Moreover, the 1 H-NMR of 3 showed proton signal of both ligands and ruthenium complex implying the symmetric coordination driven self-assembly of both precursor units with the NH signal appears as a singlet at 9.11 ppm, while CHα and CHβ pyridine protons appear as doublets at 8.00 and 7.53 ppm, respectively. The p-cymene groups directly attached to 3 exhibited doublets at 5.92 and 5.70 ppm along with multiplet, singlet, and doublet in the range 2.90-1.30 ppm, which is somewhat similar to free ruthenium complex (1). In addition to this, 1 H-1 H correlation spectroscopy (COSY), 1 H-1 H nuclear overhauser effect spectroscopy (NOESY) were also performed to further confirm the successful synthesis of 2D ruthenium metalla-rectangle (3) as shown in Figure S8 and S9. Both 1 H-1 H COSY and 1 H-1 H NOESY spectrum exhibited the resonance cross peaks to NH, pyridine protons, p-cymene moieties in both NMR at specific shift not only corresponds to the successful preparation of only one symmetrical structure but, also discard any possibility of unwanted selfassembled units such as oligomers. The diffusion-ordered NMR (DOSY) spectrum of 3 was also recorded. This technique provides an estimation of the diffusion coefficient of a compound in a solution, which is directly related to the size and shape of the compound [72]. The DOSY experiment confirmed the presence of only one species in solution, with an approximate coefficient and hydrodynamic radius of 4.9 X 10 -10 m 2 s -1 and 0.5nm respectively (CD3NO2, 25 °C) ( Figure S10). The The synthesis of discrete arene-ruthenium architectures follows a well-established two-step strategy developed by Süss-Fink [66]. The infrared spectrum of 3 predominantly showed a strong absorption for C-F, C=O, and weak stretching C-H vibrations at 1254 cm −1 , 1733 cm −1 , and 3076 cm −1 , indicating the presence of trifluoromethanesulfonate anions, multiple carbonyl groups, and p-cymene moieties, respectively ( Figure S5). Similar arene ruthenium based discrete metalla-rectangles, metalla-bowls and metalla-prisms exhibited stretching vibrations in a comparable range for C-F, C=O and C-H functionalities [70,71]. 1 H-NMR and 13 C-NMR spectra (Figures S6 and S7) further confirms the successful synthesis of the ruthenium metalla-rectangle. As can be seen in Figure S7, the 13 C-NMR of 3 exhibited a peak around 184.45 ppm, validating the presence of amide functionality in the discrete structure. Moreover, the 1 H-NMR of 3 showed proton signal of both ligands and ruthenium complex implying the symmetric coordination driven self-assembly of both precursor units with the NH signal appears as a singlet at 9.11 ppm, while CH α and CH β pyridine protons appear as doublets at 8.00 and 7.53 ppm, respectively. The p-cymene groups directly attached to 3 exhibited doublets at 5.92 and 5.70 ppm along with multiplet, singlet, and doublet in the range 2.90-1.30 ppm, which is somewhat similar to free ruthenium complex (1). In addition to this, 1 H-1 H correlation spectroscopy (COSY), 1 H-1 H nuclear overhauser effect spectroscopy (NOESY) were also performed to further confirm the successful synthesis of 2D ruthenium metalla-rectangle (3) as shown in Figures S8 and S9. Both 1 H-1 H COSY and 1 H-1 H NOESY spectrum exhibited the resonance cross peaks to NH, pyridine protons, p-cymene moieties in both NMR at specific shift not only corresponds to the successful preparation of only one symmetrical structure but, also discard any possibility of unwanted self-assembled units such as oligomers. The diffusion-ordered NMR (DOSY) spectrum of 3 was also recorded. This technique provides an estimation of the diffusion coefficient of a compound in a solution, which is directly related to the size and shape of the compound [72]. The DOSY experiment confirmed the presence of only one species in solution, with an approximate coefficient and hydrodynamic radius of 4.9 × 10 −10 m 2 s −1 and 0.5 nm respectively (CD 3 NO 2 , 25 • C) ( Figure S10). The presence of single diffusion line suggests the formation of one discrete structure exclude the formation of any other side products such as polymers. The peaks are clearly allocated in Figure S10, in general, bigger the assembly, smaller the diffusion coefficient. Similar observations have been observed for analogous arene ruthenium architectures [40,73,74].
The successful formation of tetranuclear 3 was further supported by high resolution electrospray ionization mass spectra (HR-ESI-MS), as shown in Figure S11. Dicationic, tricationic, and tetracationic species corresponding to intact 3 with two, one, and no remaining trifluoromethane sulfonate anions were detected. The HR-ESI-MS spectrum of 3 displayed peak at m/z 972. 1283 4 , respectively. These peaks can be clearly attributed to the 2D metalla-rectangle. The peaks were isotopically resolved and were in good agreement with their theoretical distribution, as shown in Figure 2. More importantly, similar pattern and nature of parent/fragmented ions was also reported by Therrein, Nitschke, Mukherjee, Cook, Stang and Chi research groups for range of discrete ruthenium metalla-assemblies including metalla-rectangles [36,43,70,71,75,76]. We further conducted elemental analysis comparison between ruthenium triflate complex (complex 1 analogous) and complex 3 which in turn, showed good agreement due to presence of triflate counterions as expected (Table S1). In addition, the conductivity study showed a linear variation with increase in concentration of metalla-rectangle 3 and a drastic increase in conductivity of ruthenium triflate complex and metalla-rectangle 3 (168.9 µS) as compared to complex 1 (3.41 µS) was observed validate the presence of triflate counterions in specific proportion ( Figure S12) [77]. It is worth to note that these analytical results strongly favor the successful synthesis of proposed 2D discrete metalla-rectangle. Moreover, similar ruthenium-based architectures using amide based organic linker and ruthenium complex were reported with single crystal structure further approved our proposed structure [37,78,79]. presence of single diffusion line suggests the formation of one discrete structure exclude the formation of any other side products such as polymers. The peaks are clearly allocated in Figure S10, in general, bigger the assembly, smaller the diffusion coefficient. Similar observations have been observed for analogous arene ruthenium architectures [40,73,74]. The successful formation of tetranuclear 3 was further supported by high resolution electrospray ionization mass spectra (HR-ESI-MS), as shown in Figure S11. Dicationic, tricationic, and tetracationic species corresponding to intact 3 with two, one, and no remaining trifluoromethane sulfonate anions were detected. The HR-ESI-MS spectrum of 3 displayed peak at m/z 972. 1283 4 , respectively. These peaks can be clearly attributed to the 2D metalla-rectangle. The peaks were isotopically resolved and were in good agreement with their theoretical distribution, as shown in Figure 2. More importantly, similar pattern and nature of parent/fragmented ions was also reported by Therrein, Nitschke, Mukherjee, Cook, Stang and Chi research groups for range of discrete ruthenium metalla-assemblies including metallarectangles [36,43,70,71,75,76]. We further conducted elemental analysis comparison between ruthenium triflate complex (complex 1 analogous) and complex 3 which in turn, showed good agreement due to presence of triflate counterions as expected (Table S1). In addition, the conductivity study showed a linear variation with increase in concentration of metalla-rectangle 3 and a drastic increase in conductivity of ruthenium triflate complex and metalla-rectangle 3 (168.9 µS) as compared to complex 1 (3.41 µS) was observed validate the presence of triflate counterions in specific proportion ( Figure S12) [77]. It is worth to note that these analytical results strongly favor the successful synthesis of proposed 2D discrete metalla-rectangle. Moreover, similar ruthenium-based architectures using amide based organic linker and ruthenium complex were reported with single crystal structure further approved our proposed structure [37,78,79].    The absorption spectrum of 3 exhibited intense band at 287 and 500 nm. The absorption spectrum of 1 (λ abs = 310 and 501 nm) was similar to that of 3; however, hypsochromic shift was observed after coordination with the pyridine moieties of 2. These absorption bands are likely to be an amalgamation of the intramolecular π → π* transitions and metal-to-ligand charge transfer (MLCT) transitions. The complex containing hydrogen bonding synthons are able to interact with a range of anions via the non-covalent interactions. A similar trend has been observed for a range of discrete ruthenium-based architectures [37]. transitions. The complex containing hydrogen bonding synthons are able to interact with a range of anions via the non-covalent interactions. A similar trend has been observed for a range of discrete ruthenium-based architectures [37]. Importantly, the fact that discrete metalla-rectangle 3 contains two bis-amide moieties offers four potential bond donors that can interact, in a specific mode, with multi-carboxylate anions. The behavior of 3 toward biologically important multi-carboxylate anions such as sodium oxalate is shown in Figure S13. UV-Vis absorption spectra of 3 changes significantly upon the addition of increasing concentrations of sodium oxalate owing to the cooperative assistance provided by the hydrogen bonding synthons. As can be seen in Figure S13, metalla-rectangle 3 exhibited two strong absorption bands close to 287 and 502 nm, in which band at 287 nm underwent a bathochromic shift with decreasing intensity, along with the disappearance of a weak intensity shoulder at 404 nm, upon the addition of varying concentrations of sodium oxalate. Moreover, a 1:1 stoichiometry of anion binding to 3 was evident from Job's plot ( Figure S14). A similar observation was reported by Vardhan et. al, who showed the formation of discrete 2D and 3D ruthenium based metalla-rectangle, metallabowl, and metalla-prism upon 1:1 binding with multi-carboxylate anions [80,81].
Metal-based drugs are widely used in clinical applications. Due to the existing anticancer activity of O,O-bridged or N,N-bridged ruthenium supramolecular coordination complexes, the antiproliferative activity of coordination driven self-assembled 2D metalla-rectangle 3 was explored against different cancer cell lines such as A549 (lung cancer), Caki-1 (kidney cancer), and Lovo (colorectal cancer). All the three cancer cell lines were exposed for a particular time to increasing concentrations of 3, and their activities were determined using WST-1 cell proliferative reagent. The activity of 3 is summarized in Table 1. The IC50 values range from 3 to 4 µM for Lovo, Caki-1, A549 cell lines. Fascinatingly, 3 was observed to inhibit the proliferation of all the three cell lines, even at a very low concentration (Figure 4). This finding clearly established that metalla-rectangle 3 was profoundly more effective than cisplatin and ruthenium complex (1) in inhibiting the growth of different cancer cell lines and could be a potent candidate as a chemotherapeutic drug against cisplatin-resistant cancer cell lines [27,59,66,82,83]. Notably, the increase in drug concentration led to complete cell kills (Caki-1, Lovo). Importantly, the fact that discrete metalla-rectangle 3 contains two bis-amide moieties offers four potential bond donors that can interact, in a specific mode, with multi-carboxylate anions. The behavior of 3 toward biologically important multi-carboxylate anions such as sodium oxalate is shown in Figure S13. UV-Vis absorption spectra of 3 changes significantly upon the addition of increasing concentrations of sodium oxalate owing to the cooperative assistance provided by the hydrogen bonding synthons. As can be seen in Figure S13, metalla-rectangle 3 exhibited two strong absorption bands close to 287 and 502 nm, in which band at 287 nm underwent a bathochromic shift with decreasing intensity, along with the disappearance of a weak intensity shoulder at 404 nm, upon the addition of varying concentrations of sodium oxalate. Moreover, a 1:1 stoichiometry of anion binding to 3 was evident from Job's plot ( Figure S14). A similar observation was reported by Vardhan et. al, who showed the formation of discrete 2D and 3D ruthenium based metalla-rectangle, metalla-bowl, and metalla-prism upon 1:1 binding with multi-carboxylate anions [80,81].
Metal-based drugs are widely used in clinical applications. Due to the existing anticancer activity of O,O-bridged or N,N-bridged ruthenium supramolecular coordination complexes, the antiproliferative activity of coordination driven self-assembled 2D metalla-rectangle 3 was explored against different cancer cell lines such as A549 (lung cancer), Caki-1 (kidney cancer), and Lovo (colorectal cancer). All the three cancer cell lines were exposed for a particular time to increasing concentrations of 3, and their activities were determined using WST-1 cell proliferative reagent. The activity of 3 is summarized in Table 1. The IC 50 values range from 3 to 4 µM for Lovo, Caki-1, A549 cell lines. Fascinatingly, 3 was observed to inhibit the proliferation of all the three cell lines, even at a very low concentration (Figure 4). This finding clearly established that metalla-rectangle 3 was profoundly more effective than cisplatin and ruthenium complex (1) in inhibiting the growth of different cancer cell lines and could be a potent candidate as a chemotherapeutic drug against cisplatin-resistant cancer cell lines [27,59,66,82,83]. Notably, the increase in drug concentration led to complete cell kills (Caki-1, Lovo). Cell apoptosis is an important phenomenon responsible for destroying undesirable cells during the development and homeostasis of cellular organisms [84]. We minutely studied three metastatic cancer cell proliferations and compared them with normal Human Embryonic Kidney cells (HEK 293) as shown in Figure S15. It was observed that cell proliferation for A549, LoVo, and Caki-1 cells reduced to 58%, 66%, and 63%, respectively, at 5.0 µM 3. However, at the same concentration of 3, there was practically no reduction for HEK 293, indicating that this potential anticancer drug has almost no toxicity for the normal cells at this particular concentration. Experimental details are described in Materials and methods section. The cancer cells growing rapidly unwind DNA for replication and thus, leave the DNA strands available for drug binding. The drug-DNA complex inhibits the DNA replication and DNA repair, leading to cancer cell destruction and apoptosis. Normal cells have slower growth rate and are less affected by the drug. Therefore, the drug acts only on the rapidly proliferating cells, i.e., cancer cells and specifically inhibits the growth of cancer cells, leaving the normal cells unaffected at a certain concentration. The study of DNA ladder assay is very useful because of its easy availability for the quick screening of apoptotic changes in the cell population. The presence of similar self-assembled structures such as 3 shown DNA ladder assay studies further approve the existence of drug-DNA complex to inhibit DNA replication and repair [42,85,86]. Furthermore, it is expected that incubation with 5.0 µM can severely elevate the apoptotic population of cancer cells.

Materials and Methods
All chemicals, solvents, deuterated solvents used in this work were purchased from commercial sources and used without purification. Starting materials, arene-ruthenium chloride (1) and bent organic linker (2), were prepared according to the methods reported in literature [39,55]. 4-amino pyridine (98%; Oakwood Chemicals, West Columbia, SC, USA), 1,1-carbonyldiimidazole (97%; Sigma Aldrich, St. Louis, MI, USA), sodium oxalate (99.9%), dichloro(p-cymene)ruthenium(II)dimer (98%; Alfa Aesar, Heysham, Lancashire, UK), 2,5-dihydroxy-1,4-benzoquinone (98%; Sigma Aldrich). Cell apoptosis is an important phenomenon responsible for destroying undesirable cells during the development and homeostasis of cellular organisms [84]. We minutely studied three metastatic cancer cell proliferations and compared them with normal Human Embryonic Kidney cells (HEK 293) as shown in Figure S15. It was observed that cell proliferation for A549, LoVo, and Caki-1 cells reduced to 58%, 66%, and 63%, respectively, at 5.0 µM 3. However, at the same concentration of 3, there was practically no reduction for HEK 293, indicating that this potential anticancer drug has almost no toxicity for the normal cells at this particular concentration. Experimental details are described in Materials and methods section. The cancer cells growing rapidly unwind DNA for replication and thus, leave the DNA strands available for drug binding. The drug-DNA complex inhibits the DNA replication and DNA repair, leading to cancer cell destruction and apoptosis. Normal cells have slower growth rate and are less affected by the drug. Therefore, the drug acts only on the rapidly proliferating cells, i.e., cancer cells and specifically inhibits the growth of cancer cells, leaving the normal cells unaffected at a certain concentration. The study of DNA ladder assay is very useful because of its easy availability for the quick screening of apoptotic changes in the cell population. The presence of similar self-assembled structures such as 3 shown DNA ladder assay studies further approve the existence of drug-DNA complex to inhibit DNA replication and repair [42,85,86]. Furthermore, it is expected that incubation with 5.0 µM can severely elevate the apoptotic population of cancer cells.
Human cancer cell lines, Lovo, Caki-1, and A549 were purchased from ATCC: The Global Bioresource Center (Manassa, VA, USA). Cell proliferation reagent, WST-1, was used. Cells (3500 cells) were plated in triplicates on a 96-well microplate in McCoy's 5A (Logan, UT, USA) and F12K medium (Manassa, VA, USA). The cells were adhered after 24 h and were treated with different doses. The treatment was continued for 3 consecutive days, and after treatment on the 4 th day, 20 µL WST-1 reagent was added to the plates with 180 µL of media and mixed well. Three trials were performed for each cell lines, and IC 50 values were calculated. The prepared Ru based metalla-bowl (3) was tested for its anti-proliferative effect on three different metastatic cancer cell lines, namely, colorectal cancer (LoVo), kidney cancer (Caki-1) and lung cancer (A549). Effect on normal HEK 293 cells was also tested. Using the WST-1 assay, the effect of different drug concentrations on the cell proliferation was found for each cell line. In the 96-well microplate, 3500 cells were plated in each well, and 200 µL cell culture media was added. After 24 h, when the cells were adhered to the bottom of the wells, the media was replaced with fresh media. The cells were then treated with different doses (0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 7.50, and 10.0 µM) of the drug. The control for each cell line was treated with equal volume of DMSO. After 24 h of treatment, the media was again removed and replaced with 180 µL of fresh media. Additionally, 20 µL WST-1 reagents were added to the wells and mixed. The microplates were then incubated in an incubator (37 • C, 5% CO 2 ) and read at time t = 0 and t = 2 h. Three trials were performed for each cell line. Drug solutions were prepared using cell culture grade water from a stock 10 mM solution of the Ru-complex in DMSO.

Synthesis of Metalla-Rectangle (3)
A mixture of [Ru 2 (η 6 -p-cymene) 2 (C 6 H 2 O 4 )Cl 2 ] (1) (1 mmol) and 2 equivalents of AgCF 3 SO 3 (2 mmol) in 25 mL methanol was stirred in the dark for 3 h at room temperature. The white precipitate was filtered, and the corresponding red filtrate was added to 1,3-di(pyridin-4-yl)urea (2) (1 mmol) and stirred for 15 h; the solvent was removed under reduced pressure. The crude product thus obtained was redissolved in nitromethane and subjected to vapor diffusion of diethyl ether, resulting in a red colored product after several days. Yield (89%). Anal. Calcd for C 78

Stability of Metalla-Bowl in DMSO
For the stability analysis, 2D metalla-rectangle was dissolved in DMSO, and the sample was subjected to 1 H NMR spectroscopy immediately after dissolution and after 3 days. No change in proton NMR was observed, hence confirming the stability of the 2D ruthenium metalla-rectangle.

UV-Vis Binding Studies
A stock solution of metalla-rectangle (3) in methanol (1 × 10 −5 M) and sodium oxalate (1 × 10 −3 M) in water were prepared. The change in absorbance was recorded at room temperature after the addition of increasing concentrations of sodium oxalate to a constant volume of 3. The stoichiometry of the adduct with 3 (A-Å) was obtained from these spectra (Job's plot).

Conclusions
In conclusion, we have reported the synthesis of coordination driven self-assembled 2D ruthenium metalla-rectangle (3) by the reaction of half sandwich ruthenium complex (1) and bent organic linker (2). Wide range of structural and analytical tools such as FTIR and UV-Vis spectroscopy, elemental analysis, 1 H NMR, 13 C NMR, 1 H-1 H COSY, 1 H-1 H NOESY, and DOSY spectroscopy, HR-ESI-MS spectrometry all confirmed the successful formation of 3. This new metalla-rectangle showed powerful anticancer activity against three different cell lines (Lovo, Caki-1, A549), as compared with the parent ruthenium precursor and the commercial available cisplatin drug. Importantly, similar self-assembled discrete ruthenium architecture exhibit cell apoptosis therefore future studies are conducting in our laboratory with normal cells and DNA ladder assay provide insights into the cell apoptosis.