RHENIUM(I) METALLOCYCLE AND METHOD OF PREPARATION THEREOF

Abstract

The present disclosure relates to a rhenium(I) metallocycle of Formula (I) (I) wherein R is H or F. The present disclosure also relates to its method of preparing the rhenium(I) metallocycle of Formula (I) and the metallocycles are found to display cytotoxic activity towards cervical cancer and skin cancer.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to organometallic complexes. The present disclosure particularly relates to Rhenium(I) tricarbonyl core-based dinuclear metallocycles as anticancer drugs displaying cytotoxicity towards cervical and skin cancer. The present disclosure also relates to a method of preparing the same.

BACKGROUND OF THE INVENTION
[0002] Cancer, being one of the deadliest diseases, continues to increase worldwide despite constant and breakthrough research. The design and synthesis of clinical Pt(II)-based metallodrugs, including cisplatin and its derivatives, are well-known for their significant chemotherapeutic efficacy in cancer diagnosis. These simple and classical organometallic complexes have proven to be the most effective agents in enhancing patient longevity. However, there is a rising need for alternative anti-cancer metal complexes not only to overcome the drawbacks of these drugs, including their drug resistance and toxic side effects on healthy organelles, but also to possess ideal mechanisms of action, high selectivity, and stability. In this regard, several other metal complexes containing copper, gallium, palladium, ruthenium, titanium, gold, and silver as central metal ions have proven to address the limitations of platinum-based drugs and exhibit anti-cancer activity (Helmin-Basa et al., Coord. Chem. Rev. 2022, 452, 214307).
[0003] Recently, a series of dinuclear Re(I) complexes containing nitrogen donor ditopic motif bridged with bis-chelating naphthacenedione and anthraquinone moiety exhibited cytotoxic activity against various cancer cell lines (Kargese et al., Chem. Eur. J. 2024, 30, e202400217). Therefore, there is a need for potential anti-cancer active metallodrug which can show anticancer activity at micromolar range and show less toxic side effects.








OBJECTS OF THE INVENTION
[0004] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0005] It is the primary objective of the present disclosure to provide robust and biocompatible Rhenium(I) metallocycles of formula (I) as potent anti-cancer therapeutic agents.

(I)
[0006] It is another objective of the present disclosure to provide neutral nitrogen donor ligand motif for preparing Rhenium(I) metallocycles.
[0007] It is yet another objective of the present disclosure to provide a simple one-step synthetic approach for the preparation of Rhenium(I) metallocycles.

SUMMARY OF INVENTION
[0008] The present disclosure relates to a rhenium(I) metallocycle of Formula (I)


(I)
wherein R is H or F

[0009] The present disclosure also relates to a process of preparing a metallocycle of formula (I), comprising the steps of:

(I)

wherein R is H or F

(a) mixing dirhenium decacarbonyl (Re2(CO)10), sodium bisulphite, ligand of formula (II), in a solvent in a sealed Teflon-lined stainless-steel bomb; and



(II)

wherein R is H or F;

(b) heating the mixture at a temperature range of 100 °C to 200°C for 48 h and then cooling to 25 °C to obtain the metallocycle of Formula (I).



BRIEF DESCRIPTION OF DRAWINGS
[0010] The present disclosure contains the following drawings that simply illustrates certain selected embodiments of the nanocarrier composition and processes that are consistent with the subject matter as claimed herein, wherein:
[0011] Figure 1 depicts ATR-IR spectra of metallocycles C1 (A) and C2 (B).
[0012] Figure 2 depicts 1H-NMR spectrum of L1 in DMSO-d6 (# = DMSO-d6, & = residual H2O).
[0013] Figure 3 depicts partial 19F NMR spectra of L1 (blue) and C1 (red) in DMSO-d6
[0014] Figure 4: depicts Partial 1H NMR spectra of L1 (blue) and C1 (red) in DMSO-d6 ( = toluene).
[0015] Figure 5 depicts partial 1H-NMR spectra of L2 (blue) and C2 (red) in DMSO-d6 ( = toluene).
[0016] Figure 6 depicts molecular structures of C1 and C2. (H atoms are removed for clarity). Color code: C = grey; O = red, N = blue, F = green, S = yellow, Re = teal.
[0017] Figure 7 depicts IC50 value determination of ligands L1, L2, and complexes C1, C2,
[0018] Figure 8 depicts biocompatibility study and determination of IC50 values of ligands L1, L2, and complexes C1, C2, using MTT assay on non-malignant cells L929 and C2C12 cell line.
[0019] Figure 9 depicts Live-dead cell images of HeLa cell lines treated with C1 and Cisplatin using (Scale bar: 100 μm). Untreated groups (negative control) and Cisplatin (positive control) (Scale bar: 100 μm). FDA and PI dyes stain the live and dead cells, respectively.
[0020] Figure 10 depicts (A) Images showing ROS generation (green fluorescence) after HeLa cell lines were treated with C1 and Cisplatin (Scale bar: 100 μm). Untreated groups (negative control) and Cisplatin (positive control). (B) The quantitative analysis of DCFDA is represented in the graph
[0021] Figure 11 depicts (A) Assessment of mitochondrial membrane potential (ΔΨm) by JC-1 dye on HeLa cells in the presence of C1. Green fluorescence (JC-1 monomer) and red fluorescence (JC-1 aggregates). Untreated cells were used as a negative control, and cells treated with Cisplatin were used as a positive control. (Scale bar: 50 μm) (B) Quantification data of JC-1 monomers and JC-1 aggregates.
[0022] Figure 12 depicts assessment of fluorescence intensity after displacement of ethidium bromide intercalated in CT-DNA following the addition of C1. CT-DNA (negative control) and Cisplatin (positive control).
[0023] Figure 13 depicts (A) Images displaying colony formation after treatment with C1. (B) Quantitative data of clonogenic assay.
[0024] Figure 14 depicts (A) detection of apoptosis using Acridine Orange (AO) and Ethidium Bromide (EtBr) on HeLa cells in the presence of C1. Living cells are stained green, while apoptotic cells are stained red. Cells without treatment were used as a negative control, and cells treated with Cisplatin served as a positive control. (Scale bar: 50 μm) (B) Quantification data of fluorescence intensity of AO and EtBr.

DESCRIPTION OF THE INVENTION:
[0025] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated vartions of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0026] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" or "has" and/or "having" when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
[0027] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0028] As used herein, the term "cancer" refers to a cell that displays uncontrolled growth and division, invasion of adjacent tissues, and often metastasizes to other locations of the body. Herein the cancer is cervical cancer or skin cancer.
[0029] As used herein the term room temperature is defined as the temperature between 25 to 35 °C.
[0030] In an embodiment, the present disclosure relates to a rhenium(I) metallocycle of Formula (I)


(I)
wherein R is H or F.
[0031] In an embodiment of the present disclosure, the rhenium(I) metallocycles of Formula (I) are heteroleptic, cage like metallocycles comprising two functionalized fac-Re(CO)3 core containing metallocycles, two neutral flexible fluorobenzimidazolyl/benzimidazolyl-based ditopic N donor ligands (L1 or L2) and one anionic sulphate motif.
[0032] In an embodiment of the present disclosure, the metallocycle is selected from the group consisting of:

C1


C2
[0033] The present disclosure also relates to a process of preparing a metallocycle of formula (I), comprising the steps of:

(I)

wherein R is H or F;

(c) mixing dirhenium decacarbonyl (Re2(CO)10), sodium bisulphite, ligand of formula (II), in a solvent in a sealed Teflon-lined stainless-steel bomb; and

(II)

wherein R is H or F.
(d) heating the mixture at a temperature range of 100 °C to 200°C for 48 h and then cooling to 25 °C to obtain the metallocycle of Formula (I).
[0034] In an embodiment of the present disclosure, a one-pot solvothermal approach is utilized to prepare the metallocycle of formula (I), wherein the technique involves the addition of all the starting reactant materials in a Teflon vial, sealed in a stainless steel autoclave, and the reaction is carried out at a controlled high temperature (higher than the boiling point of the solvent) and high-pressure conditions in a high boiling aromatic solvent. This makes the solvothermal reaction more efficient than the traditional multi-step synthesis of compounds
[0035] In an embodiment of the present disclosure, the amount of the Re2(CO)10 is in range of 50-100 mg.
[0036] In another embodiment of the present disclosure, the amount of the ligand is in the range of 50mg-1g.
[0037] In yet another embodiment of present disclosure, the ligand of formula (II) is selected from the group consisting of

(L1), and

(L2)

[0038] In an embodiment of the present disclosure, the ligand of formula (II) is prepared by the process comprising the steps of:
a) mixing and stirring a compound of formula (L), Sodium hydride, and a solvent for 2 to 4 h at room temperature;


b) adding and stirring 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene for 36 to 52 h;
c) evaporating the solvent and quenching by adding water to obtain the ligand of formula(II)
[0039] In another embodiment of the present disclosure, the concentration of 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene is in range of 50mg-1g.
[0040] In another embodiment of the present disclosure, the solvent is selected from the group consisting of tetrahydrofuran, acetone, toluene, or a combination thereof.
[0041] In another embodiment of the present disclosure, the heating is carried out for 36 hours to 52 hours.
[0042] In another embodiment of the present disclosure, the metallocycle C1 exhibits anti-cancer activity towards specific breast and cervical cancer cell lines that is notably higher than the classical cisplatin drug molecule. The uniqueness of this invention also lies in the coordinated anionic sulfate motif (SO42-) in both the metallocycles, which feature two uncoordinated oxygen atoms. These oxygen atoms can act as hydrogen bond acceptors within the overall framework, interacting with the DNA of cancer cells, leading to apoptosis.

ADVANTAGES OF THE PRESENT INVENTION
[0043] In accordance with the present disclosure, the rhenium(I) metallocycle of Formula (I)
has the following advantages:
• Kinetic inertness,
• Thermal stability,
• 3D-geometry
• Outwardly directed metal-coordinated carbonyl groups,
• Rich photophysical properties
• One step-synthesis
[0044] The present disclosure will be explained using the following examples:

EXAMPLE
Materials and Methods

[0045] Dirhenium decacarbonyl (Re2(CO)10), 1,3-bis(bromomethyl)benzene, 4,5-difluoro-1,2-phenylenediamine, paraformaldehyde, 30-33% HBr in acetic acid, glacial acetic acid, formic acid, sodium bisulphite and sodium hydride, toluene, mesitylene, acetone, and dimethylfomamide (DMF) were obtained from commercial sources and used as received.

1,3-Bis(bromomethyl)-2,4,6-trimethylbenzene and 5,6-difluorobenzimidazole were prepared by using the procedure given in
N. E. Kanitz, M. Fresia, P. G. Jones, T. Lindel, Eur. J. Org. Chem. 2021, 3573.,
J. Sun, D. Sun, S. Yuan, D. Tian, L. Zhang, X. Wang, D. Sun, Chem. Eur. J. 2012, 18, 16525.
THF and hexane were distilled using conventional procedures.
NMR spectra were recorded on a BrukerAvance III 500 MHz spectrometer.

Example 1
Synthesis of fac-[{Re(CO)3}2(µ-SO4)(L1)2] (C1).
The compound C1 is prepared by scheme 1 and scheme 2 as given below:
Step 1: Synthesis of L1
[0046] A mixture of 5,6-difluorobenzimidazole (100 mg, 0.653 mmol), NaH (40 mg, 0.98 mmol), and THF (10 ml) was stirred for 3 h at room temperature. To this mixture, 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene (100 mg, 0.326 mmol) was added and allowed to stir for 48 h. The solvent was evaporated to half and quenched by adding water (~200 mL). The resulting white powder was collected by filtration, washed several times with water, and air-dried. Yield: 97% (143.00 mg). 1H NMR (500 MHz, DMSO-d6): δ 7.81 (s, 2H, Ha), 7.71 (m, 2H, Hb), 7.63 (m, 2H, Hc), 7.15 (s, 1H, He), 5.42 (s, 4H, Hd), 2.27 (s, 6H, Hf) and 2.08 (s, 3H, Hg). 19F NMR (500 MHz, d6-DMSO): -142.85 (d, J = 25 Hz) and -145.01 (d, J = 25 Hz).

Step 2: Synthesis of fac-[{Re(CO)3}2(µ-SO4)(L1)2] (C1).
[0047] A mixture of Re2(CO)10 (51.23 mg, 0.078 mmol), NaHSO3 (8.22 mg, 80.1 mmol), L1 (63.00 mg, 0.153 mmol), toluene (6 mL), and acetone (1 mL) was sealed in a Teflon-lined stainless steel bomb and heated at 160 °C for 48 h. After cooling the bomb to room temperature, light brown crystals were obtained. The crystals were washed with hexane and air-dried. Yield: 71 % (87 mg). 1H NMR (500 MHz, DMSO-d6): δ 8.70 (s, 2H, Ha), 8.30 (m, 2H, Hb), 8.24 (m, 2H, Hc), 7.70 (m, 2H, Hb'), 7.37 (s, 1H, Hd), 6.71 (s, 1H, Hd'), 6.61 (s, 2H, Ha'), 6.25 (m, 2H, Hc'), 5.71 (d, J = 14.18 Hz, 2H, CH2), 5.47 (d, J = 14.15 Hz, 4H, CH2), 4.87 (d, J = 14.45 Hz, 2H, CH2), 2.33 (s, 6H, CH3), 2.21 (s, 3H, CH3), 1.79 (s, 3H, CH3), and 1.22 (s, 6H, CH3). ATR-IR (cm1 ): 2020 (CO), 1908 (CO). and 1871 (CO).
Example 2
Synthesis of fac-[{Re(CO)3}2(µ-SO4)(L2)2] (C2).
Step 1: Synthesis of L2
[0048] Ligand L2 (1,1'-((2,4,6-trimethyl-1,3-phenylene)bis(methylene))bis(1H-benzo[d]imidazole)) was synthesized using Scheme 1 as shown below
Step 2: Synthesis of fac-[{Re(CO)3}2(µ-SO4)(L2)2] (C2).
[0049] A mixture of Re2(CO)10 (51.7 mg, 0.076 mmol), NaHSO3 (8.00 mg, 79.2 mmol), L2 (59.31 mg, 0.155 mmol), toluene (5 mL), and acetone (1 mL) was sealed in a Teflon-lined stainless steel bomb and heated at 160 °C for 48 h as shown in scheme 2. After cooling the bomb to room temperature, light brown crystals were obtained. The crystals were washed with hexane and air-dried. Yield: 72 % (80.0 mg). 1H NMR (500 MHz, DMSO-d6): δ 8.70 (s, 2H, Ha), 8.30 (d, J = 8.4 Hz, 2H, Hb), 7.98 (m, 4H, Hb,e), 7.67 (t, J = 8 Hz, 2H, Hc ), 7.58 (t, J = 8.0 Hz, 2H, Hd), 7.38 (merg, 3H, Hc',f), 6.98 (t, J = 8.0 Hz, 2H, Hd'), 6.65 (s, 1H, Hf' ), 6.59 (s, 2H, Ha'), 6.38 (d, J = 8Hz, 2H, He'), 5.72 (d, J = 14.28 Hz, 2H, CH2), 5.48 (d, J = 14.3 Hz, 2H, CH2), 5.45 (d, J = 14.3 Hz, 2H, CH2), 4.62 (d, J = 14.3 Hz, 2H, CH2), 2.37 (s, 6H, CH3), 2.22 (s, 3H, CH3), 1.74 (s, 3H, CH3), and 1.1 (s, 6H, CH3). ATR-IR (cm−1): 2018 (CO), 1913 (CO) and 1855 (CO).



Scheme 1. Synthesis of L1 and L2.




Scheme 2. Self-assembly of metallocycles C1 and C2.

Example 3
Infra-red spectroscopy analysis of metallocycles C1 and C2:
[0050] ATR-IR spectra were recorded on a Nicolet iS5 ATR-spectrometer.
[0051] The IR spectra of the metallocycles C1 and C2 displayed three intense bands in the range of 2020-1855 cm−1, characteristic of the fac-[Re(CO)3] core in an asymmetric environment (Figure 1).

Example 4
NMR analysis of metallocycles C1 and C2:
[0052] NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer.
[0053] Both the complexes C1 and C2 and ligand L1 were characterized by 1H-NMR spectra in DMSO-d6 (Figure 2, 4, 5). Metallocycle C1 and ligand L1 were also characterized using 19F-NMR spectra (Figure 3). Both the complexes displayed well-resolved splitting patterns in comparison to their corresponding free ligands. Two sets of well-resolved chemical resonances with equal intensity were observed for the benzimidazolyl and the central mesitylene spacer. The spectra indicated that the metallocyclic structure was retained in the solution.

Example 5
X-ray Crystallography of metallocycles C1 and C2
[0054] Single crystal X-ray structural study of metallocycles C1 and C2 were performed on a Bruker D8 QUEST diffractometer [λ(Mo Kα) = 0.71073 A] diffractometer. The molecular structures were refined using the SHELXL-2018/3 program (within the WinGX program package) and solved by direct methods using SHELXS-97 (Sheldrick 2008)). Refinement of all non-hydrogen atoms was done anisotropically.
[0055] The molecular structures of metallocycles C1 and C2 were clearly established through single-crystal X-ray diffraction analysis (Figure 6). Both the metallocycles exhibit similar structure, comprising of two fac-[Re(CO)3]+ cores, a bidentate sulfate anionic ligand that bridges the two rhenium centers, and two ditopic N-donor ligands (L1 or L2), resulting in a cyclic cage-like structure. Each rhenium center is coordinated by three terminal carbonyl groups, two nitrogen atoms from the two benzimidazolyl groups of L1 or L2, and one oxygen atom from SO42−, leading to a distorted octahedral geometry. The crystal structures of both metallocycles are stabilized by a range of intermolecular non-covalent interactions, including C-H···O, C-H···π, and lone pair···π interactions.

Example 6
Cell Viability Study of metallocycles C1 and C2
Materials and Methods
[0056] Dulbecco's Modified Eagle Medium (DMEM), trypsin−EDTA, Dimethyl sulfoxide (DMSO), Phosphate buffer (pH 7.4), Tris-HCl buffer (pH 7.4), and US-origin fetal bovine serum (FBS) were purchased from HiMedia Chemicals, Mumbai, India. Syringe filters (0.2 μm) were acquired from Sartorius (Carrigtwohill, Ireland). Milli-Q water was used from the Millipore, Billerica, MA system.

Cell Lines and Maintenance
[0057] Human cervical cancer cell line (HeLa) and murine melanoma cell line (B16) were obtained from the National Center for Cell Science (NCCS), Pune, India. The cell lines were cultured and maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) FBS, 1% L-glutamine, and 100 U/streptomycin/penicillin at 37 °C in a humidified atmosphere with 5% CO2.

Cell Viability Study
[0058] The cell viability of complex C1, C2, and ligands L1, L2 was determined using MTT assay in HeLa, B16, L929, and C2C12 cell lines. For this, 96-well plates were seeded with 7×103 cells/well, each with suitable growth media for the ambient environment. Different concentrations of complex C1, C2, ligands, and cisplatin in fresh media were added to the well plates and incubated for 24 hours. Further, the MTT substrate (5 mg/mL) was added and incubated for 3 hours. The supernatant from the MTT reagent was carefully discarded, and the resulting blue formazan crystals were dissolved by adding 1% DMSO to the wells. The absorbance of the dissolved formazan crystals was measured at 570 nm using the microplate reader (BIORAD Microplate Reader).
[0059] The anti-cancer activity of ligands L1 and L2 and the corresponding Re(I) complexes C1 and C2 were studied in cervical cancer cells (HeLa) and skin cancer cells (B16) using MTT assay (Figure 7). The MTT assay is a preliminary cell viability study used to measure cellular metabolic activity to assess the number of living cells/ viable cells upon treatment. This assay is based on the reduction of the yellow MTT substrate to dark blue formazan when incubated with viable cells. This assay determined the IC50 values (the concentration at which 50% of the cell growth is inhibited) after 24 h of incubation with the complexes. The IC50 values are tabulated in Table 1.
[0060] In this study, untreated cells were used as a negative control, while cisplatin was employed as a positive control. C1 complex with fluorine substitution exhibited significantly higher cytotoxic activity with IC50 values of 12.5 μM and 25 μM as compared to non-fluoro substituted C2 complex, which displayed IC50 of 25 μM and 45 μM for both the cell line. These values were comparable to that of cisplatin in both the studied cancer cell lines. On the other hand, ligands L1 and L2 were inactive against both cancer cell lines with IC50 of >50 μM each. This indicated a remarkable cytotoxic effect inducing cell death in the HeLa cancer cell line at much lower concentrations of C1 in comparison to cisplatin.
[0061] Further, an MTT assay was performed on L929 and C2C12 cell lines to evaluate the compatibility of the synthesized ligands and complexes. The results exhibited that the synthesized ligands and complexes are compatible with the normal cell lines, as shown in Figure 8.

Table 1. IC50 Values of Complexes C1-C2 determined using MTT assay towards Cervical Cancer (HeLa) and skin cancer (B16) cell line and comparison with cisplatin

IC50 (µM)
Complex Cervical Cancer Cells
(HeLa) Skin Cancer Cells
(B16)
C1 12.5 ± 4.92 µM 25 ± 2.97 µM
C2 25 ± 4.54 µM 45 ± 3.26 µM
L1 > 50 µM > 50 µM
L2 > 50 µM > 50 µM
Cisplatin 25 ± 3.34 µM 50 ± 2.57 µM










Example 7
Live-Dead Assay
[0062] Live-dead assay was performed on HeLa cells (7×103 cells/well), which were seeded and cultured for 24 hours in a 96-well plate for adhesion of the cells. The cells were treated with cisplatin (positive control) and complex C1 at its IC50 value. Further, the cells were incubated in a CO2 incubator for 24 hours. The live and dead cells were visualized using fluorescein diacetate (green fluorescence for live cell staining) and propidium iodide (red fluorescence for dead cell staining). FDA and PI stains were utilized at doses of 20 μM concentrations each. Finally, the images of live and dead cells were photographed using a fluorescent microscope.
[0063] After treatment of HeLa cells with C1 and cisplatin, live and dead cells were visualized using the live-dead cell imaging technique (Figure 9). The cells were exposed to the IC50 concentration. The live and dead cells were stained with propidium iodide (PI) and fluorescein diacetate (FDA). Cells treated with C1 exhibited considerable cell death compared to those treated with cisplatin. This qualitative analysis revealed that the synthesized complexes exhibit anti-cancer properties and could be utilized as anti-carcinogenic agents.

Example 8
ROS Generation Analysis
[0064] ROS generation of the complex C1 was investigated on HeLa cell lines by seeding 1×104 cells per well in 96-well cell culture plates and incubated for 24 hours. The cells were treated with the experimental groups (complex C1 and cisplatin). DCFDA of 10 μM concentration was added to the wells and incubated for 5 minutes. The Zoe microscope was used to visualize the cells. The absorbance was measured at 485 nm (excitation) and 535 nm (emission) by a microplate reader.
[0065] ROS are the common byproducts of cellular metabolism, and their generation occurs mainly in the mitochondria. However, the excess production interferes with the integrity of the mitochondrial membrane, leading to cell death. The mitochondrial stress by complex C1 was estimated using the DCFDA (2',7'-dichlorodihydrofluorescein diacetate) analysis (Figure 10). HeLa cells were treated with complex C1 and cisplatin to investigate the ROS generation during irradiation. Significant green fluorescence was observed in the complex C1-treated cells. Quantitative analysis using DCFDA showed elevated intracellular ROS levels following treatment with complex C1 compared to the control group. The findings indicated that complex C1 exhibited a photodynamic effect demonstrated by ROS production. Complex C1 is, therefore, ideal for combinatorial cancer treatment.

Example 9
JC1 Staining Assay
[0066] HeLa cells (5×104 cells per well) were cultured in 24-well plates. The cells were incubated for 24 hours and then treated with complex C1 and cisplatin with further incubation of an additional 6 hours. Fresh JC-1 (1μg mL−1) dye-containing medium was added by replacing the previous medium and incubated for another 20 minutes. Cells were washed with 1×PBS, and images were photographed with the ZOE™ fluorescence cell imager, using red and green filters. The mitochondrial membrane potential of cells was studied using green and red fluorescence filters, wherein the green fluorescence indicates the deficient mitochondrial potential, and red fluorescence indicates active mitochondrial membrane potential.
[0067] JC-1 staining assay was conducted to assess the physiological conditions of mitochondria (Figure 11). JC-1 is a fluorescent dye that, when added to the living cells (negative control), forms intense red fluorescent J-aggregates (emission maximum at ~590 nm). Upon the addition of complex C1, which induces cell death, membrane potential is lost, and membrane permeability is increased. Consequently, JC-1 does not form J aggregate with low ΔΨM; thus, it retains its monomeric form (emission maximum at ~529 nm) and displays intense green fluorescence. HeLa cells treated with cisplatin (positive control) also display green fluorescence, indicating the formation of J-monomers. This result showed that C1 induced mitochondrial membrane damage and thereby reduced the membrane potential.

Example 10
Et-Br Displacement Assay
[0068] For this assay, CT-DNA (100 μM of base pairs equivalent to 0.064 mg/ml) was pre-incubated with Ethidium Bromide (EtBr) (2 nM) for 1 hour at ambient temperature. Complex C1 (12.5 μM) and cisplatin (25 μM) (positive control) were dispersed in 10 mM Tris-HCl buffer of pH 7.4 and 1% DMSO, respectively. The samples were incubated for 30 minutes at room temperature. Complex C1 and cisplatin's primary stock were prepared using 100% DMSO and diluted with Tris HCl buffer to 1% DMSO level each. After 60 minutes of incubation, a fluorescent plate reader (excitation wavelength of 480 nm and an emission wavelength of 530-650 nm) was used to measure the readings.
[0069] Ethidium bromide (Et-Br) is an intercalating agent that exhibits high fluorescence intensity upon binding with CT-DNA. For the assay, complex C1 was treated with Et-Br intercalated CT-DNA. The maximum reduction in the fluorescence intensity was seen in C1 as compared to cisplatin (Figure 12). The results demonstrate that compound C1 has the ability to interact with CT-DNA by displacing the intercalated ethidium bromide from it. During the assay, CT-DNA served as the negative control, while Cisplatin was used as the positive control.

Example 11
Clonogenic Assay
[0070] Colony formation analysis was carried out using HeLa cells. The cells were cultured in the 96-well plate at 37 °C for 24 hours in a CO2 incubator for this experiment. Following a 24-hour incubation period, each group (control, complex C1, and cisplatin) was assigned four wells for the treatment. The cells were trypsinized on the 4th day of the incubation and were cultured on a 35 mm petri dish to investigate the regeneration of the remnant cells. The plates were washed with 1×PBS and were maintained in fresh media for 10 days in a CO2 incubator. After that, the developed colonies were thoroughly washed with PBS, fixed with a fixing agent, and visualized by staining with a 0.5 % crystal violet solution.
[0071] A colony formation assay was conducted to comprehend the re-emergence of trace cells post-laser irradiation in HeLa cells. Residual complex C1 and cisplatin-treated cells (12.5 μM) were trypsinized and seeded onto 35 mm petri plates on the 4th day. The remnant cells were cultured at 37 °C (5% CO2) for 7 days. Approximately 97.66 ± 1.69% of the cells in the untreated group and about 75± 1.69 % in Cisplatin treated group had re-grown. On the contrary, a small percentage of colonies developed, making up less than 44.95 ± 2.95% of the cells exposed to complex C1 upon quantitative analysis (Figure 13). Complex C1 controlled the cell development because of its inhibitory activity and reduced the probability of tumor recurrence. It increased cell death and suppressed colony formation.

Example 12
Apoptosis Detection
[0072] Cell death by apoptosis in HeLa cancer cells was evaluated by AO-EtBr staining. The cells were grown at 4×104 cells/well in 6-well plates and treated with complex C1 and cisplatin for 48 hours. Cells were collected and stained with an AO-EtBr dye mix (1:1 v/v from 100 μg/mL in PBS), and a fluorescence microscope was used for further examination.
[0073] The presence of apoptotic cells was studied in the HeLa cell line after treatment with C1 using acridine orange/ethidium bromide (AO/EB) and dual fluorescent dye staining assay (Figure 14). Untreated cells were used as the negative control, while Cisplatin was used as a positive control. AO stains the live cells exhibiting green fluorescence, and Ethidium bromide stains dead cells displaying red fluorescence. AO-EtBr staining showed yellowish-orange fluorescence after 24 hours of treatment, indicating early apoptotic cells. After 48 hours, both early and late apoptotic cells with nuclear shrinkage were observed. In all cases, unaffected cells displayed green fluorescence.









SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION
[0074] The present disclosure relates to a rhenium(I) metallocycle of Formula (I)


(I)
wherein R is H or F.

[0075] Such a rhenium(I) metallocycle of Formula (I) wherein the metallocycle is selected from the group consisting of:

C1

C2

[0076] The present disclosure also relates to a process of preparing a metallocycle of formula (I), comprising the steps of:

(I)

wherein R is H or F;

a) mixing dirhenium decacarbonyl (Re2(CO)10), sodium bisulphite, ligand of formula (II), in a solvent in a sealed Teflon-lined stainless-steel bomb; and

(II)

wherein R is H or F.

b) heating the mixture at a temperature range of 100 °C to 200°C to obtain the metallocycle of Formula (I).
[0077] Such a process is disclosed, wherein an amount of the Re2(CO)10 is in range of 50-100 mg
[0078] Such a process is disclosed, wherein an amount of the ligand is in range of 50mg-1g
[0079] Such a process is disclosed, wherein the ligand of formula (II) is selected from the group consisting of
(L1), and

(L2)
[0080] Such a process is disclosed, wherein the ligand of formula (II) is prepared by the process comprising the steps of:
a) mixing and stirring a compound of formula (L), Sodium hydride, and a solvent for 2 to 4 h at room temperature;


c) adding and stirring 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene for 36 to 52 h; and
b) evaporating the solvent and quenching by adding water to obtain the ligand of formula (II)
[0081] Such a process is disclosed, wherein a concentration of 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene is in range of 50mg-1g
[0082] Such a process is disclosed, wherein the solvent is selected from the group consisting of tetrahydrofuran, acetone, toluene, or a combination thereof.
[0083] Such a process is disclosed, wherein the heating is carried out for 36 hours to 52 hours.
, Claims:We Claim:
1. A rhenium(I) metallocycle of Formula (I)


(I)
wherein R is H or F.

2. The rhenium(I) metallocycle of Formula (I) as claimed in claim 1, wherein the metallocycle is selected from the group consisting of:

C1

C2

3. A process of preparing a metallocycle of formula (I), comprising the steps of:

(I)

wherein R is H or F

c) mixing dirhenium decacarbonyl (Re2(CO)10), sodium bisulphite, ligand of formula (II), in a solvent in a sealed Teflon-lined stainless-steel bomb; and

(II)

wherein R is H or F.

d) heating the mixture at a temperature range of 100 °C to 200°C to obtain the metallocycle of Formula (I).

4. The process as claimed in claim 3, wherein an amount of the Re2(CO)10 is in range of 50-100 mg

5. The process as claimed in claim 3, wherein an amount of the ligand is in range of 50mg-1g

6. The process as claimed in claim 3, wherein the ligand of formula (II) is selected from the group consisting of

(L1), and

(L2)

7. The process as claimed in claim 3, wherein the ligand of formula (II) is prepared by the process comprising the steps of:

a) mixing and stirring a compound of formula (L), Sodium hydride, and a solvent for 2 to 4 h at room temperature;


b) adding and stirring 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene for 36 to 52 h;
c) evaporating the solvent and quenching by adding water to obtain the ligand of formula (II)

8. The process as claimed in claim 7, wherein a concentration of the 1,3-bis(bromomethyl)-2,4,6-trimethylbenzene is in the range of 50mg -1g













9. The process as claimed in any one of claims 3 and 7, wherein the solvent is selected from the group consisting of tetrahydrofuran, acetone, toluene, or a combination thereof.

10. The process as claimed in claim 3, wherein the heating is carried out for 36 hours to 52 hours.

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