CHEMOTHERAPEUTIC AGENT

Abstract

ABSTRACT A chemotherapeutic agents of a following structural formula; Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF. (Fig. 1)

Specification

Description:FORM 2

THE PATENTS ACT 1970
(39 OF 1970)
&
The Patent Rules, 2003

COMPLETE SPECIFICATION
(See section 10 and Rules 13)



TITLE OF THE INVENTION
"Chemotherapeutic Agent''



APPLICANTS:
(a) Aligarh Muslim University,
(b) An Indian Central University,
(c) Distt. Aligarh, Uttar Pradesh - 202002, INDIA




PREAMBLE TO THE DESCRIPTION
The following specification describes the invention and the manner in which it is to be performed.
FIELD OF INVENTION
The present invention relates to chemotherapeutic agent and a process for the preparation thereof. Specifically, this invention relates to anti-cancer chemotherapeutic agent for use during chemotherapy for the treatment of cancer. More specifically the present invention relates to a single X-ray structure of metal based Bilastine (BLA), phen (1,10-phenanthroline) and metal {Co(II), Cu(II) & Zn(II)} based chemotherapeutic agent for use for the treatment of cancer and a process for the preparation thereof.
BACKGROUND OF THE INVENTION
As we all know cancer is a serious disease and most of the people get afraid and hopeless if they come to know that they have a disease like cancer. It is observed that cancer cells present in the body start growing uncontrollably and do not respond to chemotherapeutic medicines used to treat the disease. In some case, the cancerous cells even migrate/spread from one part of the body to another neighboring or distant part of the body, though blood or lymph, resulting in secondary cancers called metastasis. It's been a big health problem worldwide to treat cancer caused by the cancer cells developed in the human body.
It is known that metals are important elements to all the living beings and it helps in many physiological functions like growth, making energy and building DNA. The metal plays a key role in enzymes and proteins needed to keep the human bodies healthy and protect the body from different kind of diseases. Also, it is noticed that if we have too little or too much metals in our body then it can lead to the issues like genetic diseases, arthritis, ulcers, and even cancer.
The metals are usually known as an anti-oxidant agent and for the chelation therapies and therefore have been explored in cancer research to inhibit angiogenesis and potentially slow down growth of cancer cells in the human body. It is also known in the prior art that metal-based compounds mainly hinge on the characteristics of ligands and donor atoms bonded to the metal ions making it less toxic. Also, the metal is available at lower cost and therefore trying to make cancer treatment better than other medications present in the market.
European patent application (EP2148675A1) discloses a polymeric anti-cancer medicine having a micelle structure performing diagnosis and treatment of cancer at the same time and comprising, a chain end functionalized polymer expressed as a following chemical formula - 1;
NNHHCCIH-C- OH
wherein, R is methyl, n-butyl, sec-butyl, tert-butyl or methoxy; and n is an integer of 10 to 500;
a contrast material; and a chemotherapeutic agent,
wherein the anti-cancer medicine is in a form of a nano-particle having a micelIe structure.
Also the method of preparing a polymeric anti-cancer medicine of the above chemical formula having a micelle structure comprises, dissolving a drug in dimethyl sulfoxide (DMSO) to prepare a DMSO solution of the drug; adding triethylamine to the solution obtained; dissolving a chain end functionalized polymer in the DMSO to prepare a DMSO solution of the polymer mixing the DMSO solution of the drug and the DMSO solution of the polymer; adding a contrast material to the solution obtained; and dialyzing and lyophilizing the solution obtained.
Chinese patent application (CN116239635A) discloses about the 2-amino-8-hydroxyquinoline nickel complex for resisting drug-resistant ovarian cancer is characterized by having a chemical structural formula as shown in the specification:

Also the process of preparing a 2-amino-8-hydroxyquinoline nickel complex of the above formula comprises, weighing 0.200mmol of 2-amino-8-hydroxyquinoline (H-AQ), 0.100mmol of NiCl2.6H2O and 0.100mmol of an auxiliary ligand, respectively, into a 15.0cm thick-wall resistant tube, adding 2.5mL of MeOH and 0.5mL of CH into 2 Cl 2 and 0.5mL of triethylamine, vacuumizing, sealing a pipe orifice, and carrying out coordination reaction for 3 days at 80 ? to obtain the catalyst.
Chinese patent application (CN105924390A) discloses a synthetic method of Yi Zhong Mei Tafeini comprising following steps;
Step 1, synthesizes compound 3 by Buchwald catalytic coupling method: with the fluoro-PAP of compound 2 and 3-as raw material, with toluene or DMF as solvent, at CuI, Cs2CO3, organic ligand 1,10-phenanthroline (Phen) or 3,4,7,8-tetramethyl- 1,10-phenanthroline (Me4Phen) under effect, 700C - 120 0C reacting generating compound 3;
Step 2, compound 3 and isocyanates carry out additive reaction synthesis compound 1: the chloro-3-trifluoromethylbenzene of compound 3 and 4- Based isocyanate is in DMF or dichloromethane, under triethylamine or N, N-diisopropyl ethyl amine (Hunig alkali) catalysis, in room temperature is reacted and to be obtained thick product, obtains compound 1 after refining.
Chinese patent (CN105873569B) discloses a nanoscale particle for co-delivery of a plurality of therapeutic agents, the nanoscale particle comprising: a core comprising a metal organic matrix material, wherein the metal organic matrix material comprises a metal bisphosphonate coordination polymer comprising a polyvalent metal ion and a bisphosphonate; and a plurality of therapeutic agents, wherein a plurality of the therapeutic agents comprises at least one non-nucleic acid chemotherapeutic agent and at least one nucleic acid therapeutic agent, wherein the nanoscale particle further comprises a lipid bilayer covering at least a portion of the outer surface of the metal-organic matrix material core, wherein the lipid bilayer comprises (i) a cationic lipid or (ii) a thiol-or dithiol-functionalized lipid, wherein at least one of the nucleic acids is electrostatically attached to the cationic lipid or covalently attached to the thiol-or dithiol-functionalized lipid; and wherein said bisphosphonate comprises said at least one non-nucleic acid chemotherapeutic agent, and wherein said at least one non-nucleic acid chemotherapeutic agent is cisplatin or oxaliplatin prodrug.
US patent (US10092567B2) discloses a method for treating breast cancer in a human patient in need thereof comprising, administering to the patient,
a) a therapeutically effective amount of a compound of formula:


or a pharmaceutically acceptable salt thereof; and
b) a therapeutically effective amount of paclitaxel, or a pharmaceutically acceptable salt thereof.
Korean Patent No. KR100704464B1 discloses a Copper aminoalkoxide compound represented by the following formula (1): Formula 1

Where m is an integer of 1-3 and R and R' are an alkyl group of C1-4.
US Patent No. US6703050B1 discloses a method of treating cancer in an animal, comprising administering to an animal with cancer a biologically effective amount of at least a first agent that binds copper and forms an agent-copper-protein complex.
Indian Patent No. 202213015027 discloses a Cu (II)-Schiff base Complex represented by the following formula:

wherein R1, R2, and R3 are selected from a group consisting of H, methyl, isopropyl, methoxy, and hydroxy.
There are disadvantages associated with known anti-cancer drugs. One of the disadvantages is the systemic toxicity of the known anticancer drug regime.
Another disadvantage associated with the known anti-cancer drugs in clinic is that the dose of the anticancer drug is high and known anticancer drugs are resistant to the treatment.
Yet another disadvantage associated with the known anti-cancer drug is that the manufacturing cost of these conventional anticancer drugs is not affordable.
Still another disadvantage associated with the known anti-cancer drug is that the conventional anti-cancer drug kills healthy cells along with the diseased cells.
OBJECTIVES OF THE INVENTION
Therefore, an object of the present invention is to provide chemotherapeutic agents for treating chronic disease, like cancers, and a process for the preparation thereof which obviates the disadvantages associated with the prior art.
Another object of the present invention is to provide chemotherapeutic agents comprising a unique architecture of biocompatible ligand framework around the core metal ion to yield a stable good product.
Yet another object of the present invention is to provide chemotherapeutic agents having reduced toxic effects in organs.
Still, another object of the present invention is to provide chemotherapeutic agents which is highly effective at very low doses against drug-resistant cancers.
A further object of the present invention is to provide chemotherapeutic agents which is capable to be prepared / produced easily at low cost.
Another advantage of the present invention is to provide chemotherapeutic agents which is safe to use and does not kill the healthy (normal) cells of the body, but creates an impact only on diseased / infected cells.
Still, another advantage of the present invention is to provide chemotherapeutic agents which has high therapeutic potency for cancer treatment.
SUMMARY OF THE INVENTION
According to the invention, there is provided a chemotherapeutic agent of the following structural formula;

Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF
In one embodiment the chemotherapeutic agent of the present invention is represented as follows;
[BLA (phen)2M(II)]+.X?, wherein M = Co (1), Cu (2), & Zn (3) and X? = NO3, ClO4.
Furter according to the present invention there is provided a process for the preparation of the chemotherapeutic agent comprising deprotonating 0.0461 - 0.465 gm (1mmol) methanolic solution of bilastine by 0.138 - 0.142 ml (1mmol) triethylamine and then reacting with (1mmol) methanolic solution of metal salts in a ratio of 1:1 under stirring for 2.45 to 3.15 hours, followed by addition of 0.196 - 200 % by weight methanolic solution of 1,10-phenanthroline and stirring the same for 3 - 4 hours until completion of reaction, filtering the reaction mixture and drying the same to obtain chemotherapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
A chemotherapeutic agent and a process for the preparation thereof, according to a preferred embodiment, is herein described and illustrated in the accompanying drawings and examples wherein;
Figure 1 - illustrates invented anticancer drug formulation,
Figure 2 - illustrates process flow diagram of the invented formulation,
Figure 3 - illustrates ORTEP view of the invented formulation,
Figure 4 - illustrates UV-vis absorption spectra of the invented formulation 1-3.
Figure 5-6 - illustrates X-band EPR spectrum of the invented formulation 1-2.
Figure 7-8 - illustrates H-NMR spectrum of the invented formulation 3.
Figure 9 - illustrates ESI-MS spectra of the invented formulation 1-3.

Figures 10 - illustrates absorption spectra of the invented formulation in presence of ct-DNA,
Figures 11 - illustrates emission spectra of the invented formulation, EB ct-DNA
Figures 12 - illustrates CD spectra in presence of ct-DNA
Figure 13-14 - illustrates survival of tumor cells as a function of different concentrations of the invented formulation determined by SRB assay after incubating for 48 h,
Figures 15-17 - illustrates molecular docked model of the invented formulation with ct-DNA (PDB ID: 1BNA).
DETAILED DESCRIPTION OF THE INVENTION
A chemotherapeutic agent and a process for the preparation thereof is herein described and illustrated with numerous specific details so as to provide a complete understanding of the invention. However, these specific details are exemplary details and should not be treated as the limitations to the scope of the invention. Throughout this specification the word "comprises" or variations such as "comprises or comprising", will be understood to imply the inclusions of a stated element, integer or step, or group of elements, integers or steps, but not the exclusions of any other element, integer or step or group of elements, integers or steps. Same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.
Referring to the drawings, particularly figure 1, Chemotherapeutic agent's formula is shown. The chemotherapeutic agent's formula, according to the present invention, is represented as follows;
[BLA(phen)2M(II)]+.X?, where M = Co (1), Cu (2), & Zn (3), and X? = NO3, ClO4
The structural formula of the invented chemotherapeutic agent as shown in figure is as follows;

Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF
IUPAC Nomenclature:
Complex (1) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)cobalt(II)]chlorate
Complex (2) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)copper(II)]aqua nitrate
Complex (3) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)zinc(II)]chlorate Dimethylformamide
The complex 1 & 2, both are EPR active while complex 2 is NMR active.
As shown in the above structural formula, the chemotherapeutic agent comprises six coordinated distorted octahedral geometry of complex around M(II) center. Zn, X? = ClO4 complex is a strong intercalating agent with ct-DNA capable of inducing cell death at low concentration. Here hexa coordinated distorted octahedral geometry of complex around metal center.
In one embodiment the chemotherapeutic agent comprises 0.0462 - 0.464 % by weight (1mmol) methanolic solution of bilastine, and 0.240 - 0.242 % by weight (1mmol) Cu(NO3)2.3H2O, 0.371 - 0.373 % weight (1mmol) Zn(ClO4)2.6H2O, and 0.364 - 0.366 % by weight (1mmol) Co(ClO4)2.6H2O, metal salts.
Referring to the drawings, particularly figure 2, a process flow is shown. The chemotherapeutic agent formula, according to the process of this invention, is prepared by reacting deprotonated methanolic solution of bilastine [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoic acid] with methanolic solution of metal salts of (Cu, Zn and Co) in a stoichiometric ration 1:1 under stirring for 2.45 to 3.15 hours, followed by addition of phen (1,10-phenanthroline) in the ration of 1:2 (mixture deprotonated bilastine and methanolic solution of metals, respectively) and the reaction mixture is further stirred for a period of 3.45 to 4.15 hours until the reaction is completed. The mixture is then filtered and the product is dried to obtain the chemotherapeutic agent / product. The recrystallized product is kept for further crystallization for few days until suitable crystals separated out for single X-ray diffraction.
In one embodiment methanolic solution of is prepared by dissolving (0.463gm) Bilastine in (20ml) mixture of MeOH (Methanol) and DMF (dimethyl formamide). The mixture of MeOH and DMF is prepared by mixing MeOH and DMF in the ratio of 19:1. The methanolic solution of bilastine, so obtained, is deprotonated by adding triethylamine (0.138 - 0.142 ml, 1 mmol) into it. (0.462 - 0.464g,1mmol) deprotonated methanolic solution of bilastine is then reacted with (0.462 - 0.464g,1mmol) methanolic solution of metal salts (Cu, Zn and Co) to obtain the chemotherapeutic agent / complex. The methanolic solution of metal salts is prepared by mixing (0.240 - 0.242g, 1mmol) Cu(NO3)2.3H2O, (0.371 - 0.373g, 1mmol) Zn(ClO4)2.6H2O, and (0.364 - 0.366g, 1mmol) Co(ClO4)2.6H2O.
Complex 1 [C52H52ClCoN7O7], MW: 982.39, Yield 85%, m.p.: 220 °C; Anal. calc. (%) C, 63.64; H, 5.34; N, 9.99. Found (%): C, 63.04; H, 5.02; N, 9.59. FT-IR (KBr, ?max/cm-1): 2958 ?(-CH), 2920, 2851 ?(-NH), 1625 ?(-C=O), 1515 ?(-C=N), 1105 ?(-ClO4 ionic), 510 ?(M-O), 424 ?(M-N); ESI-MS(m/z); 881 [C52H52CoN7O3]+, 882 [C52H52CoN7O3 + H]+; UV-vis (1x10-4M, DMSO, ?/nm): 267, 565. ?m: ?-1cm-2mol-1 = 82 (DMSO).
Complex 2 [C52H56CuN8O7], MW: 984.60, yield: 80%, m.p.: 210 °C; Anal. calc. (%) C, 63.43; H, 5.73; N, 11.38. Found (%): C, 63.20; H, 5.72; N, 10.89. FT-IR (KBr, ? max/cm-1): 2971 ?(-CH), 2928, 2850 ?(-NH), 1592 ?(-C=O), 1517 ?(-C=N), 1384 ?(-NO3 ionic), 486 ?(M-O), 429 ?(M-N); ESI-MS(m/z): 983 [C52H56CuN8O8-H]+, 982 [C52H56CuN8O8-2H]+ 965 [C52H53CuN8O8-H2O]+, 966 [C52H53CuN8O8 -H2O + H]+, 464 [C27H37N3O3]+ ;UV-vis (1x10-4M, DMSO, ?/nm): 270, 680. ?m: ?-1cm-2mol-1 = 42 (DMSO).
Complex 3 [C55H59ClZnN8O8], MW: 1060.94, yield: 75%, m.p.: 230 °C; Anal. calc. (%) C, 63.26; H, 5.61; N, 10.56. Found (%): C, 62.87; H, 5.65; N, 10.53. FT-IR (KBr, ?max/cm-1): 2958 ?(-CH), 2926, 2865 ?(-NH), 1624 ?(-C=O), 1517 ?(-C=N), 1103 ?(-ClO4 ionic), 510 ?(M-O), 423 ?(M-N) ; 1H-NMR (400 MHz, DMSO, d, ppm): 8.90(d,4H), 8.70(s,4H), 7.90(d,4H), 7.50(t,4H), 7.08-7.16(m,8H), 4.37(t,2H), 3.61(t,4H), 3.30(t,4H), 2.78(s,1H), 2.24 (t,4H), 1.36(s,6H), 0.95(t,4H), 0.79(t,3H). 13C-NMR (100 MHz, DMSO, d, ppm): 14.90, 26.8, 29, 38, 43.9, 45.7, 53, 61.2, 65.7, 68.36, 110.02, 118.45, 121.09, 121.52, 128.31, 127.3, 134.8, 139.89, 149.10, 142.23, 172.1. ESI-MS(m/z); 988[C52H55ClZnN7O7]+, 989 [C52H55ClZnN7O7 + H]+, 464 [C27H37N3O3]+ ; UV-vis (1x10-4M, DMSO, ?/nm): 265, 330. ?m: ?-1cm-2mol-1 = 65 (DMSO).
All the above complexes are soluble in dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF).
The chemotherapeutic agent and the process for the preparation therefore is now described with the help of the following examples.
EXAMPLE - 1
0.463 gm Bilastine was dissolved in 20ml mixture of MeOH (Methanol) and DMF (dimethyl formamide) to obtain methanolic solution of blastine. The methanolic solution of blastine was deprotonated by adding 0.139 ml triethylamine into it and reacting the same with methanolic solution of metals (Cu, Zn and Co) in a stoichiometric ration of 1:1, under stirring for 3 hours, followed by addition of phen (1,10-phenanthroline) in the ration of 1:2 (mixture deprotonated bilastine and methanolic solution of metals, respectively). The reaction mixture was further stirred for a period of 4 hours until the reaction is completed. The mixture was then filtered and dried to obtain the chemotherapeutic agent / product. The crystallized chemotherapeutic agent / product was kept for few days for further crystallization and suitable crystals of chemotherapeutic agent / product were separated out.
The chemotherapeutic agent/complex, so prepared, was subjected to different kinds of test and analysis to evaluate its properties and efficacy and the results of such test and analysis along with the corresponding properties are shown in different drawings. These tests and analyses may include spectroscopic studies (such as UV-Vis, IR, NMR), stability assessments, cytotoxicity tests, and crystallographic analysis, among others. Each drawing illustrates specific findings, such as structural characteristics, absorption spectra, or biological activity, providing a comprehensive overview of the complex's potential as a therapeutic agent.
Referring to figure 3, an ORTEP (Oak Ridge Thermal Ellipsoid Plot) view of the invented formulation is shown.
Referring to figure 4 and 5, X-band EPR (Electron Paramagnetic Resonance) spectrum of the invented formulation/complex 1 and 2 are shown.
Referring to figure 6, H-NMR (hydrogen-1 nuclear magnetic resonance, or proton nuclear magnetic resonance) spectrum of the invented formulation/complex 3 is shown.
The chemotherapeutic agent/complex, so prepared, were subjected to the single x-ray crustal studies and following results were found.
Single crystal X-ray studies
Single crystal X-ray data of the invented drug / complex was collected at 100(2) K on a Bruker APEX-II CCD diffractometer employing graphite monochromated MoKa radiation (l = 0.71073 Å). The linear absorption coefficients, the anomalous dispersion corrections and scattering factors for the atoms were referred from the international tables for X-ray crystallography. The structure was solved by Olex2 with the olex2.solve structure solution program using charge flipping and refined with the olex2. refinement package with Gauss-Newton minimization. All non-hydrogen atoms were refined anisotropically. A summary of the selected crystallographic refinement data is provided in the following table.
Table 1 Crystal structure refinement data of chemotherapeutic agents.
Complex 1 2 3
CCDC 2269180 2153926 2326732
Empirical formula C52H52ClCoN7O7 C52H56CuN8O8 C55H59ClZnN8O8
Temperature 293K 293K 293K
Crystal system monoclinic triclinic triclinic
a/Å 13.8983(10) 13.8962(4) 12.4495(2)
b/Å 13.9966(10) 14.5101(4) 14.2858(4)
c/Å 15.2105(10) 15.7711(4) 15.3421(2)
a/° 90 87.974(2) 89.321(2)
ß/° 81.790(3) 64.155(3) 78.885(1)
?/° 90 70.434(2) 81.020(2)
Volume 2743.5(4) 2673.63(15) 2644.14(9)
Space group P1n1 P i P i
Mr 982.39 984.60 1060.94
Dx, g cm-3 1.307 1.199 1.334
Z 2 2 2
Mu 0.473 0.463 0.577
F000 1122.0 1012.0 1114.0
h, k, lmax 18,18,19 18,18,20 14, 16, 18
Nerf 12602 12251 9300
Tmin,Tmax 0.803, 1.000 0.659, 1.000 0.734, 1.000
Data completeness 1.77/0.89 0.947 0.998
Theta(max) 27.501 27.478 24.999
R (reflections) 0.0578(7061) 0.0531(8132) 0.0726(6411)
wR2(reflections) 0.1611(11188) 0.1632(11597) 0.2254(9282)
S 1.002 1.096 1.082
Npar 661 619 664


In figure 1, The chemotherapeutic agents/complexs, so prepared, were subjected to in-vitro ct-DNA binding and cleavage studies and following results were found.
In vitro ct-DNA binding and cleavage studies
DNA/RNA binding experiments of the invented drug / complex were carried out in Tris-HCl buffer at pH 7.2, following the standard methods and practices adopted previously. While measuring absorption spectra an equal amount of ct-DNA was added to sample and reference solutions in order to eliminate the absorbance of ct-DNA itself, and Tris-HCl buffer was detracted via base line correction in figure 7.
The chemotherapeutic agent/complex, so prepared, were subjected to in silico docking studies and following results were found.
In silico docking studies
Molecular modeling and computational analysis were conducted using HEX 6.0 software. The crystal structure of B-DNA (PDB ID: BDNA) was obtained from the Protein Data Bank (http://www.rcsb.org/pdb). Before executing the docking experiments, the structure of the complex was converted into PDB format. Visualization of the results was carried out using PyMOL and the LigPlus graphics program, with the findings illustrated in Figures 12-14. These figures depict the binding interactions and conformational details of the complex with B-DNA, highlighting key residues and molecular interactions.
The chemotherapeutic agents/complexes, so prepared, were subjected to spectroscopic characterisation and following results were found.
Spectroscopic characterization of the invented drug / complex
IR spectra of Bilastine-(Phen)2-based drug candidates 1-3 provide valuable information about different vibration modes of their functional groups. The characteristic carbonyl band of bilastine carboxylic acid was observed at 1664 cm-1, while in complexes 1-3, asymmetric stretching of carboxylate ion ?(-COO-) appeared at 1626, 1592 and 1625 cm-1. Complexes 1-3 exhibited medium intensity bands at 2958-2851 cm-1 assigned to ?(-CH), symmetric and asymmetric stretching mode of vibrations. A strong band was observed at 1100 cm-1 and at 624 cm-1 in complexes 1 and 3, respectively indicative of the antisymmetric stretching and antisymmetric bending vibration of perchlorate ion. However, in complex 2, a sharp and strong band was observed at 1384 cm-1 which was attributed to nitrate as counter ion outside the coordination sphere. The far-infrared bands of complexes 1-3 were observed at 429-423 cm-1 and 587-574 cm-1, assigned to ?(M-N) and ?(M-O) vibrations, respectively.
The UV-visible absorption spectra of the free ligand and Bilastine-(Phen)2-based drug candidates 1-3 were obtained in DMSO. Two intense bands were observed at 260-280 nm and 320-340 nm, which were assigned to p-p* and n-p* intra-ligand transitions of bilastine and 1,10-phenanthroline ligands. The characteristic broad d-d bands were observed at 685 nm in Cu(II) complex 2 and at 565 nm in case of Co(II) complex 1, which is suggestive of an octahedral geometry of central metal ion in the complexes, well corroborated with their single crystal X-ray structure. Figure 4.
The chemotherapeutic agent/complex, so prepared, were subjected to EPR spectra and following results were found.
EPR spectra of 1 & 2 was performed in the solid state at room temperature with magnetic field 3000 G to find the geometry of complex around the metal. The EPR spectrum of paramagnetic high spin cobalt(II) complex 1 displayed g|| = 2.71 and g? = 1.93 values. EPR spectrum of complex 2 exhibited an isotropic signal with g¦ = 2.126 and g- = 2.073. Complex 2 followed the trend g¦ > g- > 2.0023 indicating that the Cu(II) complex has dx2-y2 ground state with tetragonally extended octahedron geometry around the metal center with d9 configuration in figure 5-6.
The 1H-NMR spectrum of complex 3 recorded in DMSO-d6 showed the absence of the acidic (-COOH) proton confirming the coordination of the ligand with the metal centre through -COO-. All the aromatic protons of bilastine and 1, 10-phenthroline appeared in the characteristic aromatic range 8.9-7.08 ppm. The aliphatic protons of bilastine fragments found in the range of 4.37-0.79 ppm (Fig. S4). Similarly, 13C-NMR spectra of complex 3, revealed that the considerable shift in the d values of carboxylic carbon from 177.70 to 172.1 ppm confirming the complexation of bilastine with metal via carboxylate group. The aromatic and aliphatic carbons were found in the range of 142.23-110.02 ppm and 68.36-14.90 ppm which are in good agreement with the reported literature values in figure 7-8.
The ESI-MS spectra of complex 1 exhibited prominent molecular peaks at m/z = 881 and 882 assigned to [C52H52CoN7O3]+, and [C52H52CoN7O3 + H]+. In complex 2, the molecular ion peak was observed at m/z = 983 for [C52H56CuN8O8-H]+, in addition, two fragmented peaks at m/z = 965 and 966 were also observed consistent with the molecular composition [C52H56CuN8O8 - H2O + H]+ and [C52H56CuN8O8 - H2O + 2H]+. The complex 3 exhibited the isotropic peaks at m/z = 988, 989 and 990 with molecular formulae [C52H52ClN7O7 Zn + H]+, [C52H52ClN7O7Zn + 2H]+ and [C52H52ClN7O7 Zn + 3H]+, Figure 9.
The chemotherapeutic agent/complex, so prepared, were subjected to single crustal X-ray diffraction studies and following results were found.
Single Crystal X-ray diffraction studies
Single-crystal X-ray diffraction studies of Bilastine-(Phen)2-based drug candidates 1-3 revealed that complex 1 crystallized in the monoclinic (P1n1), and complexes 2&3 crystalized in triclinic (Pi) crystal systems. The crystal lattice parameter of complexes were found to be a = 13.8983(10) Å, b = 13.9966(10) Å, c = 15.2105(10) Å, and a = 90, ß = 81.790(3), ? = 90 in 1, a = 13.8962(4) Å, b = 14.5101(4) Å, c = 15.7711(4) Å and a = 87.974(2), ß = 64.155(3), ? = 70.434(2) in 2, and a = 12.4495(2) Å, b = 14.2858(4) Å, c = 15.3421(2) Å, and a = 89.321(2), ß = 78.885(1), ? = 81.020(2) in 3, per unit, respectively. The central metal ion was found coordinated with carboxylate group of bilastine in all the complexes and remaining coordination sites were occupied by the nitrogen atoms of the two 1, 10-phenanthroline ligands. The crystallographic data revealed that the carboxylate group of bilastine was situated in equatorial position while nitrogen atoms occupied axial and basal position at octahedron complexes 1-3. In complex 1, the cobalt-oxygen (Co-O) and cobalt-nitrogen (Co-N) bond distances are Co(1)-O(4) = 2.150 Å, Co(1)-O(8) = 2.151 Å and Co(1)-N(5) = 2.115 Å, Co(1)-N(7) = 2.113 Å, Co(1)-N(9) = 2.132 Å, Co(1)-N(K) = 2.108 Å, while the bond angles were found to be O(4)-Co(1)-O(8) = 60.9, N(5)-Co(1)-N(9) = 78.8o and N(7)-Co(1)-N(K) = 78.1o. In complex 2, copper-oxygen (Cu-O) and copper -nitrogen (Cu-N) bond lengths were found to be Cu(1)-O(28) = 2.798 Å, Cu(1)-O(27) =1.961 Å and Cu(1)-N(45) = 2.000(2) Å, Cu(1)-N(59) = 2.002(3) Å, Cu(1)-N(49) = 2.064(2) Å and Cu(1)-N(63) = 2.199(2) Å, respectively, indicating slight distortion of the octahedral structure. The respective bond angles are also given in Table 1, and the ORPET view of complexes shown in Fig. 3. Bilastine-(Phen)2-based drug candidates 3 revealed that the zinc-oxygen (Zn-O) and zinc-nitrogen (Zn-N) bond distances were Zn(1)-O(3) = 2.100 Å, Zn(1)-O(4) = 2.289 Å and Zn(1)-N(5) = 2.136 Å, Zn(1)-N(6) = 2.144 Å, Zn(1)-N(A) = 2.127 Å, Zn(1)-N(C) = 2.128 Å and bond angles were O(3)-Zn(1)-O(4)= 59.2o , N(5)-Zn(1)-N(6)= 77.7o and N(A)-Zn(1)-N(C) = 78.0o, figure 3.
in vitro ct-DNA binding studies
UV-vis titration studies of Bilastine-(Phen)2- based drug candidates with ct-DNA
UV-visible spectroscopy is often employed to identify the binding mode of interaction of drug candidates with DNA. When a metal complex is titrated with ct-DNA, a shift (red shift or blue shift) and a hypochromic or hyperchromic is observed in the absorption spectra of complexes. To fixed concentration (10-4 M) of complexes 1-3, incremental addition of ct-DNA (0.1 x 10-4 M) in aqueous saline tris HCl buffer was performed that resulted in 'hypochromic' shift in all the complexes at the absorbance bands at 295 nm. This 'hypochromic' shift has been attributed to the intercalation binding mode of complexes 1-3, (Fig. 10), where the complexes due to the presence of aromatic bilastine and phen chromophores wedge-in between the nucleobases of DNA helix via p-p stacking interactions with the DNA base pairs. While free bilastine ligand (BLA) binds to ct-DNA via electrostatic binding mode of interaction as early reported. The intrinsic binding constant Kb values of free ligand bilastine and its complexes 1-3 were found to be 1.48 ± 0.10 x 104 M-1, and 2.15 ± 0.01 x 105 M-1, 3.80 ± 0.02 x 105 M-1, 3.50 ± 0.02 x 105 M-1 respectively, as quantified by Wolfe-Shimer equation, suggesting the higher binding propensity of 2 as compared to 1 and 3, figure 10.
Fluorescence studies of Bilastine-(Phen)2 based drug candidates with ct-DNA
Ethidium bromide (EB) displacement assay, which provides strong evidence for the competitive binding of drugs with ct-DNA, further validated the interactions of complexes 1-3 with ct-DNA. EB has a planar structure consisting of phenanthridium ring that fits into the double helical DNA and is stabilized by p-p stacking interactions with the base pair of the DNA. these interactions are known to stabilize the EB+DNA system. Although EB is a weak fluorescent molecule, it is found to emit strongly owing to its intercalation between the base pairs that occurs in presence of ct-DNA. However, after the addition of complexes to the EB+DNA system, the complexes compete with EB and EB is partially knocked out of the helix which caused a decrease fluorescence emission of the EB+DNA system. The decrease in emission intensity at 585 and 595 nm in the EB+DNA systems on addition of complexes 1-3, implicated the breakdown of the EB+DNA complex due to the insertion of a drug molecule36 as illustrated in Fig. 11. To ascertain emission quenching, the Stern-Volmer equation and Io/I vs [Q] graph were utilized to compute the Stern-Volmer quenching constant (Ksv) for all the complexes. Complex 2 demonstrated a notably higher Ksv value compared to complexes 1 and 3, with values of 0.7 x 104, 5.0 x 104, and 3.5 x 104, figure11.
Circular dichroic studies
Circular dichroism (CD) spectroscopy is highly sensitive and effective technique for investigating conformational changes pertaining to ct-DNA structure and its interaction with drug-complexes 1-3. Ct-DNA exhibits two consecutive bands, a positive band at 272 nm due to base stacking and a negative band at 240 nm attributed to right-handed helicity. the interaction of small molecules is significantly known to influence both these bands. The circular dichroic pictogram has been successfully used to predict the transformation in conformations of DNA in presence of 1-3, with a distinct CD spectrum exhibited by various DNA structures. In classical intercalation, CD band intensities increase due to base stacking interactions in the presence of an intercalator. Conversely, simple groove binding and electrostatic interactions leads to small, or no perturbations in DNA-CD bands, figure 9.
On addition of complexes 1-3 to ct-DNA, both positive and negative band intensities decreased with a slight red shift observed in the case of 2, indicative of p- p stacking interactions between DNA and complex 2. However, complex 1 and 3, induced a redshift of the positive band (272 nm) to 276 nm and the negative band (240 nm) to 243 nm in both cases. The CD spectra of ct-DNA with complexes are depicted in (Fig. 12), suggesting that the interaction of complexes 1-3 with ct-DNA occurs by strong intercalating mode of binding that causes bending, kinks, and damage to the secondary structure of DNA and changes the conformation of DNA, figure 12.
Cytotoxicity
The cytotoxicity activity of metal complex 2 was evaluated against five cancer cell lines, MCF-7 (breast cell), MDA-MB-231 (triple negative breast cancer cell), HeLa (cervix), MIA-PA-CA-2 (epithelial cell), and Hep-G2 (human hepatoma) by SRB assay about GI50 values. The GI50 is the concentration of the drug candidates that inhibits the cell growth by 50%. TGI, GI50 and LC50 values of complex 2 against five cell lines. Complex 2 has shown remarkably good activity with GI50 values <10 and displayed greater activity than the standard drug Adriamycin as well as cisplatin against MCF-7, MBA-MD-231, HeLa, Hep-G2, Mia-Pa-Ca-2 cancer cells. The coordination environment, like planar ring present around Cu(II) complex which provide the stability of complex could be responsible for the higher activity. All control cell lines exhibited fixed volume, shape, and structure but after treating with complex 2 and ADR, volume of cell decreased, and diversity of cell was reduced shown in (Fig. 13). Morphological studies of complex 2 on treatment with all tested cell lines was investigated and image shown in (Fig. 14). The corroborative results of cytotoxicity and the binding studies have confirmed the complex 2 is a potent cytotoxic agent against tested cell lines, with much superior anticancer efficacy against human pancreatic cancer cell line (Mia-Pa-CA-2) cancer cells, figure 13.
Molecular Docking
To further elucidate the observed spectroscopic binding results of complexes 1-3, molecular docking studies were carried out to explore the preferred binding sites, orientation, and binding affinity regarding the energy of complexes within the DNA biomolecules. The molecular docking approach plays a crucial role in discovering the drug-DNA interaction and mechanistic investigation by inserting a molecule in the binding site of a particular section of DNA noncovalently. It is well acknowledged that the binding affinity between the receptor (DNA) and "the ligand" (complexes molecule 1-3) increases as the binding free energy decreases. In this study, DNA duplex d{CGCGAATTCGCG}2 dodecamer sequence with PDB:1bna was used Hex 6.0 to dock with complexes 1-3 and was visualized in Pymol and ligplus to generate the 2D Ligplot, which help to understand the drug-DNA interactions more easily .The resulting docking model exhibited that complexes 1-3 fitted into DNA via intercalation mode and further showed binding by hydrogen bonding or other hydrophobic interaction as well Complex 1, da18(B), dc21(B), da6(A), da4(A), da5(A), dg22(B), dc9(A), dt19(B), da17(B), dt20(B), dc23(B), dt23(B), dt7(A), dt8(A) base connected through hydrophobic interaction with the complex atom C30, C38, C33, C24, C14, C47, C35, C36, C41, C49, C39 as shown in (Fig. 15). Hydrogen bonds were created between the nitrogen atom in the metal complex 2 and the oxygen atom of DT8(A) base (3.34 Å), as shown in the (Fig. 16), also there are hydrophobic interaction between da5(A), da6(A), dc21(B), dg4(A), dt7(A), dt20(B), da18(B), dc9(A), dt19(B), dg22(B) and C55, C62, C50, C56, C54, C11, C6, C4, C32, C60 atoms , respectively. Complex 3, from the 3D profile and 2D Ligplot revealed that the dg4(A), da5(A), da6(A), dg22(B), dc23(B), dg24(B) related to complex atoms C5, Cl6, Cl2, C10, C01, C14 as shown in (Fig. 17). The docked result revealed that the binding affinity was higher in the case of complex 2 than complexes, 1&3 with relative binding energy to -351.49, -381.5 and -320.19, KJ mol-1, respectively, figure 14-17.
Certain features of the invention have been described with reference to the example embodiments. However, the description is not intended to be construed in a limiting sense. Various modifications of the example embodiments as well as other embodiments of the invention, which are apparent to the persons skilled in the art to which the invention pertains, are deemed to lie within the scope of the invention.
, Claims:FORM 2

THE PATENTS ACT 1970
(39 OF 1970)
&
The Patent Rules, 2003

COMPLETE SPECIFICATION
(See section 10 and Rules 13)



TITLE OF THE INVENTION
"Chemotherapeutic Agent''



APPLICANTS:
(a) Aligarh Muslim University,
(b) An Indian Central University,
(c) Distt. Aligarh, Uttar Pradesh - 202002, INDIA




PREAMBLE TO THE DESCRIPTION
The following specification describes the invention and the manner in which it is to be performed.
FIELD OF INVENTION
The present invention relates to chemotherapeutic agent and a process for the preparation thereof. Specifically, this invention relates to anti-cancer chemotherapeutic agent for use during chemotherapy for the treatment of cancer. More specifically the present invention relates to a single X-ray structure of metal based Bilastine (BLA), phen (1,10-phenanthroline) and metal {Co(II), Cu(II) & Zn(II)} based chemotherapeutic agent for use for the treatment of cancer and a process for the preparation thereof.
BACKGROUND OF THE INVENTION
As we all know cancer is a serious disease and most of the people get afraid and hopeless if they come to know that they have a disease like cancer. It is observed that cancer cells present in the body start growing uncontrollably and do not respond to chemotherapeutic medicines used to treat the disease. In some case, the cancerous cells even migrate/spread from one part of the body to another neighboring or distant part of the body, though blood or lymph, resulting in secondary cancers called metastasis. It's been a big health problem worldwide to treat cancer caused by the cancer cells developed in the human body.
It is known that metals are important elements to all the living beings and it helps in many physiological functions like growth, making energy and building DNA. The metal plays a key role in enzymes and proteins needed to keep the human bodies healthy and protect the body from different kind of diseases. Also, it is noticed that if we have too little or too much metals in our body then it can lead to the issues like genetic diseases, arthritis, ulcers, and even cancer.
The metals are usually known as an anti-oxidant agent and for the chelation therapies and therefore have been explored in cancer research to inhibit angiogenesis and potentially slow down growth of cancer cells in the human body. It is also known in the prior art that metal-based compounds mainly hinge on the characteristics of ligands and donor atoms bonded to the metal ions making it less toxic. Also, the metal is available at lower cost and therefore trying to make cancer treatment better than other medications present in the market.
European patent application (EP2148675A1) discloses a polymeric anti-cancer medicine having a micelle structure performing diagnosis and treatment of cancer at the same time and comprising, a chain end functionalized polymer expressed as a following chemical formula - 1;
NNHHCCIH-C- OH
wherein, R is methyl, n-butyl, sec-butyl, tert-butyl or methoxy; and n is an integer of 10 to 500;
a contrast material; and a chemotherapeutic agent,
wherein the anti-cancer medicine is in a form of a nano-particle having a micelIe structure.
Also the method of preparing a polymeric anti-cancer medicine of the above chemical formula having a micelle structure comprises, dissolving a drug in dimethyl sulfoxide (DMSO) to prepare a DMSO solution of the drug; adding triethylamine to the solution obtained; dissolving a chain end functionalized polymer in the DMSO to prepare a DMSO solution of the polymer mixing the DMSO solution of the drug and the DMSO solution of the polymer; adding a contrast material to the solution obtained; and dialyzing and lyophilizing the solution obtained.
Chinese patent application (CN116239635A) discloses about the 2-amino-8-hydroxyquinoline nickel complex for resisting drug-resistant ovarian cancer is characterized by having a chemical structural formula as shown in the specification:

Also the process of preparing a 2-amino-8-hydroxyquinoline nickel complex of the above formula comprises, weighing 0.200mmol of 2-amino-8-hydroxyquinoline (H-AQ), 0.100mmol of NiCl2.6H2O and 0.100mmol of an auxiliary ligand, respectively, into a 15.0cm thick-wall resistant tube, adding 2.5mL of MeOH and 0.5mL of CH into 2 Cl 2 and 0.5mL of triethylamine, vacuumizing, sealing a pipe orifice, and carrying out coordination reaction for 3 days at 80 ? to obtain the catalyst.
Chinese patent application (CN105924390A) discloses a synthetic method of Yi Zhong Mei Tafeini comprising following steps;
Step 1, synthesizes compound 3 by Buchwald catalytic coupling method: with the fluoro-PAP of compound 2 and 3-as raw material, with toluene or DMF as solvent, at CuI, Cs2CO3, organic ligand 1,10-phenanthroline (Phen) or 3,4,7,8-tetramethyl- 1,10-phenanthroline (Me4Phen) under effect, 700C - 120 0C reacting generating compound 3;
Step 2, compound 3 and isocyanates carry out additive reaction synthesis compound 1: the chloro-3-trifluoromethylbenzene of compound 3 and 4- Based isocyanate is in DMF or dichloromethane, under triethylamine or N, N-diisopropyl ethyl amine (Hunig alkali) catalysis, in room temperature is reacted and to be obtained thick product, obtains compound 1 after refining.
Chinese patent (CN105873569B) discloses a nanoscale particle for co-delivery of a plurality of therapeutic agents, the nanoscale particle comprising: a core comprising a metal organic matrix material, wherein the metal organic matrix material comprises a metal bisphosphonate coordination polymer comprising a polyvalent metal ion and a bisphosphonate; and a plurality of therapeutic agents, wherein a plurality of the therapeutic agents comprises at least one non-nucleic acid chemotherapeutic agent and at least one nucleic acid therapeutic agent, wherein the nanoscale particle further comprises a lipid bilayer covering at least a portion of the outer surface of the metal-organic matrix material core, wherein the lipid bilayer comprises (i) a cationic lipid or (ii) a thiol-or dithiol-functionalized lipid, wherein at least one of the nucleic acids is electrostatically attached to the cationic lipid or covalently attached to the thiol-or dithiol-functionalized lipid; and wherein said bisphosphonate comprises said at least one non-nucleic acid chemotherapeutic agent, and wherein said at least one non-nucleic acid chemotherapeutic agent is cisplatin or oxaliplatin prodrug.
US patent (US10092567B2) discloses a method for treating breast cancer in a human patient in need thereof comprising, administering to the patient,
a) a therapeutically effective amount of a compound of formula:


or a pharmaceutically acceptable salt thereof; and
b) a therapeutically effective amount of paclitaxel, or a pharmaceutically acceptable salt thereof.
Korean Patent No. KR100704464B1 discloses a Copper aminoalkoxide compound represented by the following formula (1): Formula 1

Where m is an integer of 1-3 and R and R' are an alkyl group of C1-4.
US Patent No. US6703050B1 discloses a method of treating cancer in an animal, comprising administering to an animal with cancer a biologically effective amount of at least a first agent that binds copper and forms an agent-copper-protein complex.
Indian Patent No. 202213015027 discloses a Cu (II)-Schiff base Complex represented by the following formula:

wherein R1, R2, and R3 are selected from a group consisting of H, methyl, isopropyl, methoxy, and hydroxy.
There are disadvantages associated with known anti-cancer drugs. One of the disadvantages is the systemic toxicity of the known anticancer drug regime.
Another disadvantage associated with the known anti-cancer drugs in clinic is that the dose of the anticancer drug is high and known anticancer drugs are resistant to the treatment.
Yet another disadvantage associated with the known anti-cancer drug is that the manufacturing cost of these conventional anticancer drugs is not affordable.
Still another disadvantage associated with the known anti-cancer drug is that the conventional anti-cancer drug kills healthy cells along with the diseased cells.
OBJECTIVES OF THE INVENTION
Therefore, an object of the present invention is to provide chemotherapeutic agents for treating chronic disease, like cancers, and a process for the preparation thereof which obviates the disadvantages associated with the prior art.
Another object of the present invention is to provide chemotherapeutic agents comprising a unique architecture of biocompatible ligand framework around the core metal ion to yield a stable good product.
Yet another object of the present invention is to provide chemotherapeutic agents having reduced toxic effects in organs.
Still, another object of the present invention is to provide chemotherapeutic agents which is highly effective at very low doses against drug-resistant cancers.
A further object of the present invention is to provide chemotherapeutic agents which is capable to be prepared / produced easily at low cost.
Another advantage of the present invention is to provide chemotherapeutic agents which is safe to use and does not kill the healthy (normal) cells of the body, but creates an impact only on diseased / infected cells.
Still, another advantage of the present invention is to provide chemotherapeutic agents which has high therapeutic potency for cancer treatment.
SUMMARY OF THE INVENTION
According to the invention, there is provided a chemotherapeutic agent of the following structural formula;

Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF
In one embodiment the chemotherapeutic agent of the present invention is represented as follows;
[BLA (phen)2M(II)]+.X?, wherein M = Co (1), Cu (2), & Zn (3) and X? = NO3, ClO4.
Furter according to the present invention there is provided a process for the preparation of the chemotherapeutic agent comprising deprotonating 0.0461 - 0.465 gm (1mmol) methanolic solution of bilastine by 0.138 - 0.142 ml (1mmol) triethylamine and then reacting with (1mmol) methanolic solution of metal salts in a ratio of 1:1 under stirring for 2.45 to 3.15 hours, followed by addition of 0.196 - 200 % by weight methanolic solution of 1,10-phenanthroline and stirring the same for 3 - 4 hours until completion of reaction, filtering the reaction mixture and drying the same to obtain chemotherapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
A chemotherapeutic agent and a process for the preparation thereof, according to a preferred embodiment, is herein described and illustrated in the accompanying drawings and examples wherein;
Figure 1 - illustrates invented anticancer drug formulation,
Figure 2 - illustrates process flow diagram of the invented formulation,
Figure 3 - illustrates ORTEP view of the invented formulation,
Figure 4 - illustrates UV-vis absorption spectra of the invented formulation 1-3.
Figure 5-6 - illustrates X-band EPR spectrum of the invented formulation 1-2.
Figure 7-8 - illustrates H-NMR spectrum of the invented formulation 3.
Figure 9 - illustrates ESI-MS spectra of the invented formulation 1-3.

Figures 10 - illustrates absorption spectra of the invented formulation in presence of ct-DNA,
Figures 11 - illustrates emission spectra of the invented formulation, EB ct-DNA
Figures 12 - illustrates CD spectra in presence of ct-DNA
Figure 13-14 - illustrates survival of tumor cells as a function of different concentrations of the invented formulation determined by SRB assay after incubating for 48 h,
Figures 15-17 - illustrates molecular docked model of the invented formulation with ct-DNA (PDB ID: 1BNA).
DETAILED DESCRIPTION OF THE INVENTION
A chemotherapeutic agent and a process for the preparation thereof is herein described and illustrated with numerous specific details so as to provide a complete understanding of the invention. However, these specific details are exemplary details and should not be treated as the limitations to the scope of the invention. Throughout this specification the word "comprises" or variations such as "comprises or comprising", will be understood to imply the inclusions of a stated element, integer or step, or group of elements, integers or steps, but not the exclusions of any other element, integer or step or group of elements, integers or steps. Same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.
Referring to the drawings, particularly figure 1, Chemotherapeutic agent's formula is shown. The chemotherapeutic agent's formula, according to the present invention, is represented as follows;
[BLA(phen)2M(II)]+.X?, where M = Co (1), Cu (2), & Zn (3), and X? = NO3, ClO4
The structural formula of the invented chemotherapeutic agent as shown in figure is as follows;

Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF
IUPAC Nomenclature:
Complex (1) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)cobalt(II)]chlorate
Complex (2) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)copper(II)]aqua nitrate
Complex (3) - [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoato-bis(1,10-phenanthroline)zinc(II)]chlorate Dimethylformamide
The complex 1 & 2, both are EPR active while complex 2 is NMR active.
As shown in the above structural formula, the chemotherapeutic agent comprises six coordinated distorted octahedral geometry of complex around M(II) center. Zn, X? = ClO4 complex is a strong intercalating agent with ct-DNA capable of inducing cell death at low concentration. Here hexa coordinated distorted octahedral geometry of complex around metal center.
In one embodiment the chemotherapeutic agent comprises 0.0462 - 0.464 % by weight (1mmol) methanolic solution of bilastine, and 0.240 - 0.242 % by weight (1mmol) Cu(NO3)2.3H2O, 0.371 - 0.373 % weight (1mmol) Zn(ClO4)2.6H2O, and 0.364 - 0.366 % by weight (1mmol) Co(ClO4)2.6H2O, metal salts.
Referring to the drawings, particularly figure 2, a process flow is shown. The chemotherapeutic agent formula, according to the process of this invention, is prepared by reacting deprotonated methanolic solution of bilastine [2-(4-(2-(4-(1-2-ethoxyethyl)-1H-benzo[d]imidazole-2-yl)-piperidin-1-yl)-ethyl)-phenyl)-2-methylpropanoic acid] with methanolic solution of metal salts of (Cu, Zn and Co) in a stoichiometric ration 1:1 under stirring for 2.45 to 3.15 hours, followed by addition of phen (1,10-phenanthroline) in the ration of 1:2 (mixture deprotonated bilastine and methanolic solution of metals, respectively) and the reaction mixture is further stirred for a period of 3.45 to 4.15 hours until the reaction is completed. The mixture is then filtered and the product is dried to obtain the chemotherapeutic agent / product. The recrystallized product is kept for further crystallization for few days until suitable crystals separated out for single X-ray diffraction.
In one embodiment methanolic solution of is prepared by dissolving (0.463gm) Bilastine in (20ml) mixture of MeOH (Methanol) and DMF (dimethyl formamide). The mixture of MeOH and DMF is prepared by mixing MeOH and DMF in the ratio of 19:1. The methanolic solution of bilastine, so obtained, is deprotonated by adding triethylamine (0.138 - 0.142 ml, 1 mmol) into it. (0.462 - 0.464g,1mmol) deprotonated methanolic solution of bilastine is then reacted with (0.462 - 0.464g,1mmol) methanolic solution of metal salts (Cu, Zn and Co) to obtain the chemotherapeutic agent / complex. The methanolic solution of metal salts is prepared by mixing (0.240 - 0.242g, 1mmol) Cu(NO3)2.3H2O, (0.371 - 0.373g, 1mmol) Zn(ClO4)2.6H2O, and (0.364 - 0.366g, 1mmol) Co(ClO4)2.6H2O.
Complex 1 [C52H52ClCoN7O7], MW: 982.39, Yield 85%, m.p.: 220 °C; Anal. calc. (%) C, 63.64; H, 5.34; N, 9.99. Found (%): C, 63.04; H, 5.02; N, 9.59. FT-IR (KBr, ?max/cm-1): 2958 ?(-CH), 2920, 2851 ?(-NH), 1625 ?(-C=O), 1515 ?(-C=N), 1105 ?(-ClO4 ionic), 510 ?(M-O), 424 ?(M-N); ESI-MS(m/z); 881 [C52H52CoN7O3]+, 882 [C52H52CoN7O3 + H]+; UV-vis (1x10-4M, DMSO, ?/nm): 267, 565. ?m: ?-1cm-2mol-1 = 82 (DMSO).
Complex 2 [C52H56CuN8O7], MW: 984.60, yield: 80%, m.p.: 210 °C; Anal. calc. (%) C, 63.43; H, 5.73; N, 11.38. Found (%): C, 63.20; H, 5.72; N, 10.89. FT-IR (KBr, ? max/cm-1): 2971 ?(-CH), 2928, 2850 ?(-NH), 1592 ?(-C=O), 1517 ?(-C=N), 1384 ?(-NO3 ionic), 486 ?(M-O), 429 ?(M-N); ESI-MS(m/z): 983 [C52H56CuN8O8-H]+, 982 [C52H56CuN8O8-2H]+ 965 [C52H53CuN8O8-H2O]+, 966 [C52H53CuN8O8 -H2O + H]+, 464 [C27H37N3O3]+ ;UV-vis (1x10-4M, DMSO, ?/nm): 270, 680. ?m: ?-1cm-2mol-1 = 42 (DMSO).
Complex 3 [C55H59ClZnN8O8], MW: 1060.94, yield: 75%, m.p.: 230 °C; Anal. calc. (%) C, 63.26; H, 5.61; N, 10.56. Found (%): C, 62.87; H, 5.65; N, 10.53. FT-IR (KBr, ?max/cm-1): 2958 ?(-CH), 2926, 2865 ?(-NH), 1624 ?(-C=O), 1517 ?(-C=N), 1103 ?(-ClO4 ionic), 510 ?(M-O), 423 ?(M-N) ; 1H-NMR (400 MHz, DMSO, d, ppm): 8.90(d,4H), 8.70(s,4H), 7.90(d,4H), 7.50(t,4H), 7.08-7.16(m,8H), 4.37(t,2H), 3.61(t,4H), 3.30(t,4H), 2.78(s,1H), 2.24 (t,4H), 1.36(s,6H), 0.95(t,4H), 0.79(t,3H). 13C-NMR (100 MHz, DMSO, d, ppm): 14.90, 26.8, 29, 38, 43.9, 45.7, 53, 61.2, 65.7, 68.36, 110.02, 118.45, 121.09, 121.52, 128.31, 127.3, 134.8, 139.89, 149.10, 142.23, 172.1. ESI-MS(m/z); 988[C52H55ClZnN7O7]+, 989 [C52H55ClZnN7O7 + H]+, 464 [C27H37N3O3]+ ; UV-vis (1x10-4M, DMSO, ?/nm): 265, 330. ?m: ?-1cm-2mol-1 = 65 (DMSO).
All the above complexes are soluble in dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF).
The chemotherapeutic agent and the process for the preparation therefore is now described with the help of the following examples.
EXAMPLE - 1
0.463 gm Bilastine was dissolved in 20ml mixture of MeOH (Methanol) and DMF (dimethyl formamide) to obtain methanolic solution of blastine. The methanolic solution of blastine was deprotonated by adding 0.139 ml triethylamine into it and reacting the same with methanolic solution of metals (Cu, Zn and Co) in a stoichiometric ration of 1:1, under stirring for 3 hours, followed by addition of phen (1,10-phenanthroline) in the ration of 1:2 (mixture deprotonated bilastine and methanolic solution of metals, respectively). The reaction mixture was further stirred for a period of 4 hours until the reaction is completed. The mixture was then filtered and dried to obtain the chemotherapeutic agent / product. The crystallized chemotherapeutic agent / product was kept for few days for further crystallization and suitable crystals of chemotherapeutic agent / product were separated out.
The chemotherapeutic agent/complex, so prepared, was subjected to different kinds of test and analysis to evaluate its properties and efficacy and the results of such test and analysis along with the corresponding properties are shown in different drawings. These tests and analyses may include spectroscopic studies (such as UV-Vis, IR, NMR), stability assessments, cytotoxicity tests, and crystallographic analysis, among others. Each drawing illustrates specific findings, such as structural characteristics, absorption spectra, or biological activity, providing a comprehensive overview of the complex's potential as a therapeutic agent.
Referring to figure 3, an ORTEP (Oak Ridge Thermal Ellipsoid Plot) view of the invented formulation is shown.
Referring to figure 4 and 5, X-band EPR (Electron Paramagnetic Resonance) spectrum of the invented formulation/complex 1 and 2 are shown.
Referring to figure 6, H-NMR (hydrogen-1 nuclear magnetic resonance, or proton nuclear magnetic resonance) spectrum of the invented formulation/complex 3 is shown.
The chemotherapeutic agent/complex, so prepared, were subjected to the single x-ray crustal studies and following results were found.
Single crystal X-ray studies
Single crystal X-ray data of the invented drug / complex was collected at 100(2) K on a Bruker APEX-II CCD diffractometer employing graphite monochromated MoKa radiation (l = 0.71073 Å). The linear absorption coefficients, the anomalous dispersion corrections and scattering factors for the atoms were referred from the international tables for X-ray crystallography. The structure was solved by Olex2 with the olex2.solve structure solution program using charge flipping and refined with the olex2. refinement package with Gauss-Newton minimization. All non-hydrogen atoms were refined anisotropically. A summary of the selected crystallographic refinement data is provided in the following table.
Table 1 Crystal structure refinement data of chemotherapeutic agents.
Complex 1 2 3
CCDC 2269180 2153926 2326732
Empirical formula C52H52ClCoN7O7 C52H56CuN8O8 C55H59ClZnN8O8
Temperature 293K 293K 293K
Crystal system monoclinic triclinic triclinic
a/Å 13.8983(10) 13.8962(4) 12.4495(2)
b/Å 13.9966(10) 14.5101(4) 14.2858(4)
c/Å 15.2105(10) 15.7711(4) 15.3421(2)
a/° 90 87.974(2) 89.321(2)
ß/° 81.790(3) 64.155(3) 78.885(1)
?/° 90 70.434(2) 81.020(2)
Volume 2743.5(4) 2673.63(15) 2644.14(9)
Space group P1n1 P i P i
Mr 982.39 984.60 1060.94
Dx, g cm-3 1.307 1.199 1.334
Z 2 2 2
Mu 0.473 0.463 0.577
F000 1122.0 1012.0 1114.0
h, k, lmax 18,18,19 18,18,20 14, 16, 18
Nerf 12602 12251 9300
Tmin,Tmax 0.803, 1.000 0.659, 1.000 0.734, 1.000
Data completeness 1.77/0.89 0.947 0.998
Theta(max) 27.501 27.478 24.999
R (reflections) 0.0578(7061) 0.0531(8132) 0.0726(6411)
wR2(reflections) 0.1611(11188) 0.1632(11597) 0.2254(9282)
S 1.002 1.096 1.082
Npar 661 619 664


In figure 1, The chemotherapeutic agents/complexs, so prepared, were subjected to in-vitro ct-DNA binding and cleavage studies and following results were found.
In vitro ct-DNA binding and cleavage studies
DNA/RNA binding experiments of the invented drug / complex were carried out in Tris-HCl buffer at pH 7.2, following the standard methods and practices adopted previously. While measuring absorption spectra an equal amount of ct-DNA was added to sample and reference solutions in order to eliminate the absorbance of ct-DNA itself, and Tris-HCl buffer was detracted via base line correction in figure 7.
The chemotherapeutic agent/complex, so prepared, were subjected to in silico docking studies and following results were found.
In silico docking studies
Molecular modeling and computational analysis were conducted using HEX 6.0 software. The crystal structure of B-DNA (PDB ID: BDNA) was obtained from the Protein Data Bank (http://www.rcsb.org/pdb). Before executing the docking experiments, the structure of the complex was converted into PDB format. Visualization of the results was carried out using PyMOL and the LigPlus graphics program, with the findings illustrated in Figures 12-14. These figures depict the binding interactions and conformational details of the complex with B-DNA, highlighting key residues and molecular interactions.
The chemotherapeutic agents/complexes, so prepared, were subjected to spectroscopic characterisation and following results were found.
Spectroscopic characterization of the invented drug / complex
IR spectra of Bilastine-(Phen)2-based drug candidates 1-3 provide valuable information about different vibration modes of their functional groups. The characteristic carbonyl band of bilastine carboxylic acid was observed at 1664 cm-1, while in complexes 1-3, asymmetric stretching of carboxylate ion ?(-COO-) appeared at 1626, 1592 and 1625 cm-1. Complexes 1-3 exhibited medium intensity bands at 2958-2851 cm-1 assigned to ?(-CH), symmetric and asymmetric stretching mode of vibrations. A strong band was observed at 1100 cm-1 and at 624 cm-1 in complexes 1 and 3, respectively indicative of the antisymmetric stretching and antisymmetric bending vibration of perchlorate ion. However, in complex 2, a sharp and strong band was observed at 1384 cm-1 which was attributed to nitrate as counter ion outside the coordination sphere. The far-infrared bands of complexes 1-3 were observed at 429-423 cm-1 and 587-574 cm-1, assigned to ?(M-N) and ?(M-O) vibrations, respectively.
The UV-visible absorption spectra of the free ligand and Bilastine-(Phen)2-based drug candidates 1-3 were obtained in DMSO. Two intense bands were observed at 260-280 nm and 320-340 nm, which were assigned to p-p* and n-p* intra-ligand transitions of bilastine and 1,10-phenanthroline ligands. The characteristic broad d-d bands were observed at 685 nm in Cu(II) complex 2 and at 565 nm in case of Co(II) complex 1, which is suggestive of an octahedral geometry of central metal ion in the complexes, well corroborated with their single crystal X-ray structure. Figure 4.
The chemotherapeutic agent/complex, so prepared, were subjected to EPR spectra and following results were found.
EPR spectra of 1 & 2 was performed in the solid state at room temperature with magnetic field 3000 G to find the geometry of complex around the metal. The EPR spectrum of paramagnetic high spin cobalt(II) complex 1 displayed g|| = 2.71 and g? = 1.93 values. EPR spectrum of complex 2 exhibited an isotropic signal with g¦ = 2.126 and g- = 2.073. Complex 2 followed the trend g¦ > g- > 2.0023 indicating that the Cu(II) complex has dx2-y2 ground state with tetragonally extended octahedron geometry around the metal center with d9 configuration in figure 5-6.
The 1H-NMR spectrum of complex 3 recorded in DMSO-d6 showed the absence of the acidic (-COOH) proton confirming the coordination of the ligand with the metal centre through -COO-. All the aromatic protons of bilastine and 1, 10-phenthroline appeared in the characteristic aromatic range 8.9-7.08 ppm. The aliphatic protons of bilastine fragments found in the range of 4.37-0.79 ppm (Fig. S4). Similarly, 13C-NMR spectra of complex 3, revealed that the considerable shift in the d values of carboxylic carbon from 177.70 to 172.1 ppm confirming the complexation of bilastine with metal via carboxylate group. The aromatic and aliphatic carbons were found in the range of 142.23-110.02 ppm and 68.36-14.90 ppm which are in good agreement with the reported literature values in figure 7-8.
The ESI-MS spectra of complex 1 exhibited prominent molecular peaks at m/z = 881 and 882 assigned to [C52H52CoN7O3]+, and [C52H52CoN7O3 + H]+. In complex 2, the molecular ion peak was observed at m/z = 983 for [C52H56CuN8O8-H]+, in addition, two fragmented peaks at m/z = 965 and 966 were also observed consistent with the molecular composition [C52H56CuN8O8 - H2O + H]+ and [C52H56CuN8O8 - H2O + 2H]+. The complex 3 exhibited the isotropic peaks at m/z = 988, 989 and 990 with molecular formulae [C52H52ClN7O7 Zn + H]+, [C52H52ClN7O7Zn + 2H]+ and [C52H52ClN7O7 Zn + 3H]+, Figure 9.
The chemotherapeutic agent/complex, so prepared, were subjected to single crustal X-ray diffraction studies and following results were found.
Single Crystal X-ray diffraction studies
Single-crystal X-ray diffraction studies of Bilastine-(Phen)2-based drug candidates 1-3 revealed that complex 1 crystallized in the monoclinic (P1n1), and complexes 2&3 crystalized in triclinic (Pi) crystal systems. The crystal lattice parameter of complexes were found to be a = 13.8983(10) Å, b = 13.9966(10) Å, c = 15.2105(10) Å, and a = 90, ß = 81.790(3), ? = 90 in 1, a = 13.8962(4) Å, b = 14.5101(4) Å, c = 15.7711(4) Å and a = 87.974(2), ß = 64.155(3), ? = 70.434(2) in 2, and a = 12.4495(2) Å, b = 14.2858(4) Å, c = 15.3421(2) Å, and a = 89.321(2), ß = 78.885(1), ? = 81.020(2) in 3, per unit, respectively. The central metal ion was found coordinated with carboxylate group of bilastine in all the complexes and remaining coordination sites were occupied by the nitrogen atoms of the two 1, 10-phenanthroline ligands. The crystallographic data revealed that the carboxylate group of bilastine was situated in equatorial position while nitrogen atoms occupied axial and basal position at octahedron complexes 1-3. In complex 1, the cobalt-oxygen (Co-O) and cobalt-nitrogen (Co-N) bond distances are Co(1)-O(4) = 2.150 Å, Co(1)-O(8) = 2.151 Å and Co(1)-N(5) = 2.115 Å, Co(1)-N(7) = 2.113 Å, Co(1)-N(9) = 2.132 Å, Co(1)-N(K) = 2.108 Å, while the bond angles were found to be O(4)-Co(1)-O(8) = 60.9, N(5)-Co(1)-N(9) = 78.8o and N(7)-Co(1)-N(K) = 78.1o. In complex 2, copper-oxygen (Cu-O) and copper -nitrogen (Cu-N) bond lengths were found to be Cu(1)-O(28) = 2.798 Å, Cu(1)-O(27) =1.961 Å and Cu(1)-N(45) = 2.000(2) Å, Cu(1)-N(59) = 2.002(3) Å, Cu(1)-N(49) = 2.064(2) Å and Cu(1)-N(63) = 2.199(2) Å, respectively, indicating slight distortion of the octahedral structure. The respective bond angles are also given in Table 1, and the ORPET view of complexes shown in Fig. 3. Bilastine-(Phen)2-based drug candidates 3 revealed that the zinc-oxygen (Zn-O) and zinc-nitrogen (Zn-N) bond distances were Zn(1)-O(3) = 2.100 Å, Zn(1)-O(4) = 2.289 Å and Zn(1)-N(5) = 2.136 Å, Zn(1)-N(6) = 2.144 Å, Zn(1)-N(A) = 2.127 Å, Zn(1)-N(C) = 2.128 Å and bond angles were O(3)-Zn(1)-O(4)= 59.2o , N(5)-Zn(1)-N(6)= 77.7o and N(A)-Zn(1)-N(C) = 78.0o, figure 3.
in vitro ct-DNA binding studies
UV-vis titration studies of Bilastine-(Phen)2- based drug candidates with ct-DNA
UV-visible spectroscopy is often employed to identify the binding mode of interaction of drug candidates with DNA. When a metal complex is titrated with ct-DNA, a shift (red shift or blue shift) and a hypochromic or hyperchromic is observed in the absorption spectra of complexes. To fixed concentration (10-4 M) of complexes 1-3, incremental addition of ct-DNA (0.1 x 10-4 M) in aqueous saline tris HCl buffer was performed that resulted in 'hypochromic' shift in all the complexes at the absorbance bands at 295 nm. This 'hypochromic' shift has been attributed to the intercalation binding mode of complexes 1-3, (Fig. 10), where the complexes due to the presence of aromatic bilastine and phen chromophores wedge-in between the nucleobases of DNA helix via p-p stacking interactions with the DNA base pairs. While free bilastine ligand (BLA) binds to ct-DNA via electrostatic binding mode of interaction as early reported. The intrinsic binding constant Kb values of free ligand bilastine and its complexes 1-3 were found to be 1.48 ± 0.10 x 104 M-1, and 2.15 ± 0.01 x 105 M-1, 3.80 ± 0.02 x 105 M-1, 3.50 ± 0.02 x 105 M-1 respectively, as quantified by Wolfe-Shimer equation, suggesting the higher binding propensity of 2 as compared to 1 and 3, figure 10.
Fluorescence studies of Bilastine-(Phen)2 based drug candidates with ct-DNA
Ethidium bromide (EB) displacement assay, which provides strong evidence for the competitive binding of drugs with ct-DNA, further validated the interactions of complexes 1-3 with ct-DNA. EB has a planar structure consisting of phenanthridium ring that fits into the double helical DNA and is stabilized by p-p stacking interactions with the base pair of the DNA. these interactions are known to stabilize the EB+DNA system. Although EB is a weak fluorescent molecule, it is found to emit strongly owing to its intercalation between the base pairs that occurs in presence of ct-DNA. However, after the addition of complexes to the EB+DNA system, the complexes compete with EB and EB is partially knocked out of the helix which caused a decrease fluorescence emission of the EB+DNA system. The decrease in emission intensity at 585 and 595 nm in the EB+DNA systems on addition of complexes 1-3, implicated the breakdown of the EB+DNA complex due to the insertion of a drug molecule36 as illustrated in Fig. 11. To ascertain emission quenching, the Stern-Volmer equation and Io/I vs [Q] graph were utilized to compute the Stern-Volmer quenching constant (Ksv) for all the complexes. Complex 2 demonstrated a notably higher Ksv value compared to complexes 1 and 3, with values of 0.7 x 104, 5.0 x 104, and 3.5 x 104, figure11.
Circular dichroic studies
Circular dichroism (CD) spectroscopy is highly sensitive and effective technique for investigating conformational changes pertaining to ct-DNA structure and its interaction with drug-complexes 1-3. Ct-DNA exhibits two consecutive bands, a positive band at 272 nm due to base stacking and a negative band at 240 nm attributed to right-handed helicity. the interaction of small molecules is significantly known to influence both these bands. The circular dichroic pictogram has been successfully used to predict the transformation in conformations of DNA in presence of 1-3, with a distinct CD spectrum exhibited by various DNA structures. In classical intercalation, CD band intensities increase due to base stacking interactions in the presence of an intercalator. Conversely, simple groove binding and electrostatic interactions leads to small, or no perturbations in DNA-CD bands, figure 9.
On addition of complexes 1-3 to ct-DNA, both positive and negative band intensities decreased with a slight red shift observed in the case of 2, indicative of p- p stacking interactions between DNA and complex 2. However, complex 1 and 3, induced a redshift of the positive band (272 nm) to 276 nm and the negative band (240 nm) to 243 nm in both cases. The CD spectra of ct-DNA with complexes are depicted in (Fig. 12), suggesting that the interaction of complexes 1-3 with ct-DNA occurs by strong intercalating mode of binding that causes bending, kinks, and damage to the secondary structure of DNA and changes the conformation of DNA, figure 12.
Cytotoxicity
The cytotoxicity activity of metal complex 2 was evaluated against five cancer cell lines, MCF-7 (breast cell), MDA-MB-231 (triple negative breast cancer cell), HeLa (cervix), MIA-PA-CA-2 (epithelial cell), and Hep-G2 (human hepatoma) by SRB assay about GI50 values. The GI50 is the concentration of the drug candidates that inhibits the cell growth by 50%. TGI, GI50 and LC50 values of complex 2 against five cell lines. Complex 2 has shown remarkably good activity with GI50 values <10 and displayed greater activity than the standard drug Adriamycin as well as cisplatin against MCF-7, MBA-MD-231, HeLa, Hep-G2, Mia-Pa-Ca-2 cancer cells. The coordination environment, like planar ring present around Cu(II) complex which provide the stability of complex could be responsible for the higher activity. All control cell lines exhibited fixed volume, shape, and structure but after treating with complex 2 and ADR, volume of cell decreased, and diversity of cell was reduced shown in (Fig. 13). Morphological studies of complex 2 on treatment with all tested cell lines was investigated and image shown in (Fig. 14). The corroborative results of cytotoxicity and the binding studies have confirmed the complex 2 is a potent cytotoxic agent against tested cell lines, with much superior anticancer efficacy against human pancreatic cancer cell line (Mia-Pa-CA-2) cancer cells, figure 13.
Molecular Docking
To further elucidate the observed spectroscopic binding results of complexes 1-3, molecular docking studies were carried out to explore the preferred binding sites, orientation, and binding affinity regarding the energy of complexes within the DNA biomolecules. The molecular docking approach plays a crucial role in discovering the drug-DNA interaction and mechanistic investigation by inserting a molecule in the binding site of a particular section of DNA noncovalently. It is well acknowledged that the binding affinity between the receptor (DNA) and "the ligand" (complexes molecule 1-3) increases as the binding free energy decreases. In this study, DNA duplex d{CGCGAATTCGCG}2 dodecamer sequence with PDB:1bna was used Hex 6.0 to dock with complexes 1-3 and was visualized in Pymol and ligplus to generate the 2D Ligplot, which help to understand the drug-DNA interactions more easily .The resulting docking model exhibited that complexes 1-3 fitted into DNA via intercalation mode and further showed binding by hydrogen bonding or other hydrophobic interaction as well Complex 1, da18(B), dc21(B), da6(A), da4(A), da5(A), dg22(B), dc9(A), dt19(B), da17(B), dt20(B), dc23(B), dt23(B), dt7(A), dt8(A) base connected through hydrophobic interaction with the complex atom C30, C38, C33, C24, C14, C47, C35, C36, C41, C49, C39 as shown in (Fig. 15). Hydrogen bonds were created between the nitrogen atom in the metal complex 2 and the oxygen atom of DT8(A) base (3.34 Å), as shown in the (Fig. 16), also there are hydrophobic interaction between da5(A), da6(A), dc21(B), dg4(A), dt7(A), dt20(B), da18(B), dc9(A), dt19(B), dg22(B) and C55, C62, C50, C56, C54, C11, C6, C4, C32, C60 atoms , respectively. Complex 3, from the 3D profile and 2D Ligplot revealed that the dg4(A), da5(A), da6(A), dg22(B), dc23(B), dg24(B) related to complex atoms C5, Cl6, Cl2, C10, C01, C14 as shown in (Fig. 17). The docked result revealed that the binding affinity was higher in the case of complex 2 than complexes, 1&3 with relative binding energy to -351.49, -381.5 and -320.19, KJ mol-1, respectively, figure 14-17.
Certain features of the invention have been described with reference to the example embodiments. However, the description is not intended to be construed in a limiting sense. Various modifications of the example embodiments as well as other embodiments of the invention, which are apparent to the persons skilled in the art to which the invention pertains, are deemed to lie within the scope of the invention.


WE CLAIM
1. A chemotherapeutic agents of a following structural formula;

Wherein M = Co, Cu and Zn, X = NO3 and CIO4, Y = H2O and DMF

2. A process for preparing an chemotherapeutic agents formula comprising deprotonating 0.462 - 0.464 gm (1mmol) methanolic solution of bilastine by 0.138 - 0.142 ml (1mmol) triethylamine and then reacting with (1mmol) methanolic solution of metal salts in a ratio of 1:1 under stirring for 2.45 to 3.15 hours, followed by addition of 0.196 - 200 % by weight methanolic solution of 1,10-phenanthroline and stirring the same for 3 - 4 hours until completion of reaction, filtering the reaction mixture and drying the same to obtain chemotherapeutic agent.
3. A process for preparing a chemotherapeutic agent formulation as claimed in claim 2, wherein methanolic solution of bilastine is prepared by dissolving (0.463g) Bilastine in (20ml) mixture of MeOH (Methanol) and DMF (dimethyl formamide).
4. A process for preparing a chemotherapeutic agent formulation as claimed in claim 3, wherein mixture of Methanol and DMF is prepared by mixing Methanol with DMF in a ratio of 19:1.
5. A process for preparing a chemotherapeutic agent formulation as claimed in claim 2, wherein methanolic solution of metal salts is prepared by mixing (0.240 - 0.242g, 1mmol) Cu(NO3)2.3H2O, (0.371 - 0.373g, 1mmol) Zn(ClO4)2.6H2O, and (0.364 - 0.366g, 1mmol) Co(ClO4)2.6H2O.

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