Description:FIELD OF INVENTION
[0001] The invention relates to novel fused heterocyclic compounds. Particularly, the present invention relates to incorporation of Quinoline Bridged Hydroxamate-based anti-cancer agent and its acceptable salts and method for the preparation of such compounds for preventing the diseases associated with abnormal cell growth.
BACKGROUND OF THE INVENTION
[0002] Deoxyribonucleic acid (DNA) Topoisomerases are proteins found in all forms of life. In a typical DNA double helical structure, the length of the strands, along with their nature to intertwine, leads to various topological problems. The DNA double helix structure unwinds before it transcribes into a protein. The function of Topoisomerase is to help in such transcription. The abnormal increase in cell growth (leading to cancers, tumours etc.) lead to cascading effect on human body as various enzymes and proteins are involved in the process. This makes single drug targeted delivery systems not very effective during the advanced stage of such diseases. Thus, several combination therapies are currently available to overcome the disadvantage. These combination therapies especially include epigenetic therapies like those involving DNA Methyltransferases, Histone Demethylases, Reactive Oxygen Species (ROS)-generating agents like Adaphostin, β-Phenylethyl isothiocyanate, proteosome inhibitors like Bortezomib, Marizomib, Carfilzomib and DNA damaging agents like Radiation, Tubulin, Temozolomide etc. The combination therapy involves the mechanism of directly blocking several oncogenic signaling pathways to create a synergestic anti-tumour effect. These combination therapies are disadvantageous as these involve issues related to patient compliance, toxicity and pharmacokinetics.

[0003] Another method adapted for a more effective treatment for cancers has been the use of hybrid drugs which is usually a two drug cocktail. In this method, two drugs are usually incorporated in a single molecule to target the abnormally growing cells. But, dose –dependent toxicities and issues related to patient resistance are still a concern.

[0004] The patent application CA2893339A1 discloses a group of Aryl heteroaryl fused lactams. The application further discloses signal transduction inhibitors like multiple targeted Kinase inhibitors. The cited patent application But, the invention does not disclose any specific kinase inhibitor but multiple kinase inhibitors. There is no evidence of in-vivo testing of kinase inhibitors. The compound mentioned is Histone methyl Transferase but no such method of preparation is disclosed. Further, the compounds in the invention are mutant forms of EZH2. Synergistic dual drug inhibition of cancer cells is not explicitly disclosed.

Prototype and an example of the pharmaceutically accepted compound in the application
[0005] The patent application EP2678016B1 discloses pharmaceutically acceptable heterocyclic compounds and said compound may selectively inhibit one or more members type I or class I phosphatidylinositol 3-kinases (PI3-kinase). The major drawback of this application is that: There is no specific focused target. The application targets both kinases and/or histone di acetylases. Kinases are located in the cytoplasm thus the target is not specific as they are not found precisely in nucleus of the cell for a more target specific result. Further, the application does not claim any specific compound against the activity of kinases. The microsomal assay is disclosed [refer example 23of the prior art] where human microsomes from the liver are taken for invitro analysis. The application does not disclose any account of the data from the assay.

Prototype and an example of the pharmaceutically accepted compound in the cited application
[0006] The disclosed prior arts have their scope extended beyond anti-cancer activity like: Epilepsy, neurodegenerative diseases like: Alzheimer's disease, Parkinson’s disease and the like.

[0007] Thus an approach involving hybrid drugs have been proposed off-late. Histone deacetylase (HDAC) inhibitors have turned to be one of the emerging class of compounds successfully being incorporated in anti-cancer therapy. Histone deacetylase is an enzyme linked to altered expression and mutation of genes, thus leading to aberration in transcription. This in turn effects cellular functions including cell apoptosis thus leading to unchecked cellular growth.

[0008] Therefore, keeping in view of the problems associated with the state of the art, there is a need for dual, target specific inhibitors to prevent diseases related to abnormal cell growth.

OBJECTIVES OF THE INVENTION
[0009] The primary objective of the present invention is to provide a Quinoline Bridged Hydroxamate-Based Dual Inhibitors of Human Topoisomerase and Histone Deacetylase and their resultant anticancer activity.

[00010] Another objective of the invention is to provide a method for treating and preventing the abnormal cell growth using the Quinoline Bridged Hydroxamate-based Dual Inhibitors of Human Topoisomerases and Histone Deacetylase.

[00011] Yet another objective of the invention is to provide fused and/or ring expanded heterocyclic compound.

[00012] Another objective of the present invention is to provide a synergistic effect generated by the dual inhibitors of Human Topoisomerases and Histone Deacetylase on the cell.

[00013] Yet another objective of the invention is to provide acceptable salts of Quinoline Bridged Hydroxamate-Based Dual Inhibitors of Human Topoisomerases and Histone Deacetylase.

[00014] Other objective and advantages of the present invention will become apparent from the following description taken in connection with the accompanying drawings, wherein, by way of illustration and example, the aspects of the present invention are disclosed.

SUMMARY OF THE INVENTION
[00015] The present invention relates to a heterocyclic compound of formula 1 and its pharmaceutically acceptable salts and composition which is prophylactically and therapeutically active. The heterocyclic compound is preferably a Quinoline bridged Hydroxamate-based dual inhibitor. The invention also includes the methods of preparation of said compound. The formula 1 is represented as

wherein, each of A and B are independently selected from a group of: a homocyclic and a heterocyclic ring systems; W is selected from a group of: alkyl, alkenyl, alkynyl, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl; X and Y are independently selected from an Oxygen (O), an imidogen (NH) and a Sulphur (S); Z is selected from a group of: alkyl, alkenyl, alkynyl, alkoxy, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl or differently selected from a group of: heterocyclyl, azanol, and azaalkyl; L is selected from a group of: alkyl, alkenyl, and alkynyl, wherein the value of n ranges from 0-12; and R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from: hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

BRIEF DESCRIPTION OF DRAWINGS
[00016] Figure 1A-1C is a representation of a Spectral Data of N-hydroxy-7-((2-phenylquinolin-6-yl)oxy)heptanamide (3B1).

[00017] Figure 2A-2C is a representation of a Spectral Data of N-hydroxy-7-(4-(quinolin-2-yl)phenoxy)heptanamide (3B2).

[00018] Figure 3A-3C is a representation of a Spectral Data of N-hydroxy-6-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B3).

[00019] Figure 4A-4C is a representation of a Spectral Data of N-hydroxy-7-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B4).

[00020] Figure 5A-5D is a representation of a Spectral Data of N-hydroxy-6-((2-(3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)hexanamide (3B5).

[00021] Figure 6A-6C is a representation of a Spectral Data of N-hydroxy-7-((2- (3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)heptanamide (3B6).

[00022] Figure 7A-7C is a representation of a Spectral Data of N-hydroxy-7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B7).

[00023] Figure 8A-8Bis a representation of a Spectral Data of N-hydroxy-6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B8).

[00024] Figure 9A is a pictographic representation a decatenation assay to study the inhibitory effect of synthetics in comparison to etoposide kDNA.

[00025] Figure 9B is a graphical representation of the quantification of the decatenated product formed after the decatenation assay and of the investigated compounds.

[00026] Figure 9C is a pictographic representation of an intercalation assay conducted to study the inhibitory effect of synthetics.

[00027] Figure 9D is a pictographic representation of topol relaxation assay conducted to study the inhibitory effect of synthetics.

[00028] Figure 10A-10B are representations of readings related to change developed in a 3D culture model using A549 cancer cells upon treatment with investigational compounds.

[00029] Figure 11A-11B are graphical representations of the redox studies using H2DCFDA and Cell Rox red based fluorescent dyes.

[00030] Figure 12A is a graphical representations of PI Vs Annexin V assay to study the mode of cell death.

[00031] Figure 12 B is a representation of a Cell cycle analysis using propidium iodide.

[00032] Figure 12C is a representation of a Mitophagy experiment to investigate the potency of the synthetics in facilitating the process of mitophagy as detected by Green dye.

[00033] Figure 12D is a representation of an autophagy as detected using (MAK138 Sigma-Aldrich Autophagy Assay Kit).

[00034] Figure 13A is a representation of western blot analysis to detect the effect of synthetic on oncogenic proteins (PCNA, CDK-4, CyclinD1, p53, p21).

[00035] Figure 13B is a graphical representation of qPCR analysis of investigational compounds on miRNA let7a.

[00036] Figure 14A is a photographic representation of Effect of 3B3, Etoposide and SAHA treatment on the tumour size in A549-induced lung cancer Xenograft.

[00037] Figure 14B-14C are tumour volume curve and Kaplan-Meier survival curve respectively.

[00038] Figure 15 is a pictographic representation of the effect of 3B3, Etoposide and SAHA treatment on the tumour size in A-549-induced lung cancer xenograft.

[00039] Figure 16A-16C are graphical representations of effect of 3B3 Etoposide and SAHA treatment on average tumour weight, a percentage of tumour growth inhibition and percentage of body weight change.
DETAILED DESCRIPTION OF THE INVENTION
[00040] The present invention will be better understood after reading the following detailed description of the presently preferred aspects thereof, in which the features, other aspects and advantages of the invention shall be more apparent from certain exemplary embodiments of the invention.

[00041] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of invention. In addition, descriptions of well-known terms and functions and constructions are omitted for clarity and conciseness.

[00042] Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

[00043] The terms and words used in the following description are not limited to the bibliographical meanings, but, are merely used to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustrative purpose only and not for the purpose of limiting the invention.

[00044] It is to be understood that the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[00045] It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
[00046] Accordingly, present invention discloses a fused heterocyclic compound, preferably a Qunilone bridged Hydroxamate-based dual inhibitors, which inhibit the abnormal cell growth owing to the synergistic effects of the dual inhibitors. The resultant novel compound and its acceptable salts obtained are suitable for use as a drug/pharmaceutical agents with reduced dose dependent toxicities. The invention also provides methods for treating or preventing the diseases associated with abnormal cell growth not limiting to cancer, tumour and the like.

[00047] An exemplary embodiment of the present invention relates to a fused heterocyclic compound, preferably a Qunilone bridged Hydroxamate-based dual inhibitors (Represented as Formula 1) or its pharmaceutically acceptable salt,

i.

wherein,
• Each of A and B are independently selected from a group of, not limiting to, a homocyclic and a heterocyclic ring systems.
• W is selected from a group, not limiting to, an alkyl, an alkenyl, an alkynyl, an aralkyl, a heteroarylalkyl, a heterocyclylalkyl, an aryl, a cycloalkyl, a heteroaryl or a heterocyclyl.
• X and Y are independently selected from, not limiting to, an Oxygen (O), an imidogen (NH) and a Sulphur (S).
• Z is selected from a group, not limiting to, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aralkyl, a heteroarylalkyl, a heterocyclylalkyl, an aryl, a cycloalkyl, a heteroaryl or a group, not limiting to, a heterocyclyl, an azanol, and an azaalkyl.
• L is selected from a group, not limiting to, an alkyl, an alkenyl, and an alkynyl, where n ranges from 0-12.
• R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from, not limiting to, a hydrogen, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aralkyl, a heteroarylalkyl, a heterocyclylalkyl, an aryl, a cycloalkyl, a heteroaryl, and a heterocyclyl.

[00048] Further, the alkyl group unless otherwise specified, refers to a monoradical branched or a monoradical unbranched saturated hydrocarbon comprising of, not limiting to 1 carbon to about 20 carbon atoms. The alkyl group can be further exemplified by groups, not limiting to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-hexyl, n-decyl, tetradecyl, and the like. The exemplified alkyl groups may be further substituted with one or more substituents from, not limiting to, alkenyl, alkynyl, alkoxy, cycloalkyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, oxo, thiocarbonyl, carboxy, arylthio, thiol, alkylthio, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl); alternatively, -NRxRy (wherein Rx and Ry are hydrogen; alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl). (_ENREF_1_ENREF_1)

[00049] The above substituents may be further substituted by at least 1 to 3 substituents chosen from, but not limited to, alkyl, carboxy, aminocarbonyl, hydroxy, alkoxy, halogen, -CF3, amino, substituted amino, cyano, and –S(O)tRa(t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl). An alkyl group as defined above may further be interrupted by 1-5 atoms or groups independently chosen from oxygen, sulfur and –NRb- (where Rb can be, but not limited to, hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, and aryl). Unless otherwise constrained by the definition, all substituents may optionally be further substituted by at least 1 to 3 substituents chosen from alkyl, carboxy, aminocarbonyl, hydroxy, alkoxy, halogen, -CF3, amino, substituted amino, cyano, and –S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl); alternatively an alkyl group as defined above that has both substituents as defined above and is also interrupted by 1-5 atoms or groups as defined above.
[00050] Further, unless otherwise specified, the group alkenyl refers to a monoradical of a branched unsaturated hydrocarbon or unbranched unsaturated hydrocarbon comprising of, not limiting to 1 carbon to about 20 carbon atoms with either cis geometry or Trans geometry. Preferred alkenyl groups include, but not limiting to, ethenyl or vinyl (CH=CH2), 1-propylene or allyl (-CH2CH=CH2), or iso-propylene (-C(CH3)=CH2), and a bicyclo[2.2.1]heptene. Alternatively, if the alkenyl group is attached to a heteroatom, it is not feasible for the double bond to be attached to the alpha position to the heteroatom.

[00051] The alkenyl group may be further configured to be substituted with one or more substituents, not limiting to, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, oxo, thiocarbonyl, carboxy, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), heterocyclyl or heteroaryl). Unless otherwise constrained by the definition, all substituents may be optionally further substituted by at least 1-3 substituents, which may not be limited to, alkyl, carboxy, aminocarbonyl, hydroxy, alkoxy, halogen, -CF3, amino, substituted amino, cyano, or –S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl).

[00052] The group “alkynyl,” unless otherwise specified, refers to a monoradical of an unsaturated hydrocarbon, preferably comprising of 2 to 20 carbon atoms. Preferred alkynyl groups include, but not limited to, ethynyl (-CºCH), or propargyl (alternatively propynyl, -CH2CºCH), and the like. Alternatively, if the alkynyl group is attached to a heteroatom, it is not feasible for the triple bond to be attached to the alpha position to the heteroatom. The alkynyl group may be further substituted with one or more substituents, such as alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, oxo, thiocarbonyl, carboxy, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, or -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl). Unless otherwise constrained by the definition, all substituents may be optionally further substituted by 1-3 substituents, which can be alkyl, carboxy, aminocarbonyl, hydroxy, alkoxy, halogen,-CF3, amino, substituted amino, cyano, or –S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl).

[00053] The “cycloalkyl” group, unless otherwise specified, refers to a saturated cyclic alkyl groups or an unsaturated cyclic alkyl groups comprising of 3-30 carbon atoms having at least a single cyclic ring or multiple condensed rings and the like and said cycloalkyl group may contain an optional olefinic bond. The single ring structures in the cycloalkyl group may not be limited to, a cyclopropyl, a cyclobutyl, a cyclopentyl, a cyclooctyl, a cyclopropylene, a cyclobutylene and the like. Aalternatively, the multiple ring structures, in the cycloalkyl group may not be limited to, adamantanyl, and bicyclo [2.2.1]heptane, alternatively the cyclic alkyl groups may be fused by an aryl group, for example, indane and the like. The cycloalkyl may be further substituted with one or more substituents not limiting to, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, oxo, thiocarbonyl, carboxy, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), heterocyclyl or heteroaryl), heteroaryl or heterocyclyl. Unless otherwise constrained by the definition, all substituents may be optionally further substituted by 1 to 3 substituents, which may not be limited to, alkyl, carboxy, aminocarbonyl, hydroxy, alkoxy, halogen, -CF3, -NH2, substituted amino, cyano, or –S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl) groups.

[00054] The term “alkoxy” denotes the group O-alkyl, wherein the scope of the alkyl group is the same as defined above.

[00055] The term “aralkyl” refers to alkyl-aryl linked through alkyl portion (wherein the scope of the aralkyl group is the same as defined above) and the alkyl portion contains carbon atoms from at least 1-6.

[00056] The group “aryl,” unless otherwise specified, refers to phenyl or naphthyl ring, and the like. The aryl group may be optionally substituted with 1 to 3 substituents selected from the group consisting of, but not limiting to, a halogen (such as F, Cl, Br, I), hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, aryloxy, benzyloxy, -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), cyano, nitro, ester, carboxy, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, acyl and -(CH2)0-3C(=O)NRxRy (wherein Rx and Ry are same as defined earlier).

[00057] The “carboxy” group unless otherwise specified, refers to –C(=O)ORc (wherein Rc is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl, heteroarylalkyl or heterocyclylalkyl).

[00058] The group “heteroaryl”, unless otherwise specified, refers to an aromatic ring structure comprising of 5-6 carbon atoms. Alternatively, the heteroaryl group may be a bicyclic aromatic group comprising of 8 to 10 carbon atoms, with one or more heteroatom(s) being independently selected from the group consisting of, but not limited to, N, O and S, optionally substituted with 1 to 3 substituent(s), such as but not limited to, the halogen (F, Cl, Br, I), hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), heterocyclyl or heteroaryl)), alkoxy, aralkyl, cyano, nitro, acyl or -C(=O)NRxRy (wherein Rx and Ry are the same as defined earlier). Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, triazinyl, furanyl, benzofuranyl, indolyl, benzothiazolyl, benzoxazolyl, and the like, including analogous oxygen, sulphur, and mixed hetero atom containing groups.

[00059] The group “heterocyclyl”, unless otherwise specified, refers non-aromatic monocyclic or polycyclic ring having 3 to 10 atoms, in which 1 to 3 carbon atoms in a ring are replaced by heteroatoms selected from the group consisting of O, S and N, and optionally are benzofused or substituted by fused heteroaryl of 5-6 ring members, wherein the substituents can be halogen (F, Cl, Br, I), hydroxy, alkyl, alkenyl, alkynyl, hydroxyalkyl, cycloalkyl, carboxy, aryl, alkoxy, aralkyl, heteroaryl, heterocyclyl, heteroarylalkyl, heterocyclylalkyl, oxo, alkoxyalkyl or -S(O)tRa (are substituted), cyano, nitro, -NH2, substituted amino, acyl or -C(=O)NRxRy (wherein Rx and Ry are the same as defined earlier). Examples of heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, azabicyclohexane dihydropyridinyl, piperidinyl, isoxazoline, piperazinyl, dihydrobenzofuryl, isoindole-dione, and dihydroindolyl.

[00060] “Heteroarylalkyl” group unless otherwise specified, refers to an alkyl-heteroaryl group, wherein the alkyl and heteroaryl portions are the same as defined earlier.

[00061] “Heterocyclylalkyl” group unless otherwise specified, refers to an alkyl-heterocyclyl group, wherein the alkyl and heterocyclyl portions of the group are the same as defined earlier.

[00062] The “acyl” group refers to -C(=O)Ra wherein Ra is the same as defined earlier.

[00063] The term “substituted amino,” unless otherwise specified, refers to a group –N(Rk)2 wherein each Rk can be hydrogen [provided that both Rk groups are not hydrogen (defined as “-NH2”)], alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclylalkyl, heteroarylalkyl, acyl, S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), heterocyclyl or heteroaryl)), -C(=O)NRxRy, -C(=O)ORc (wherein Rx, Ry and Rc are the same as defined earlier) or -NHC(=O)NRyRx (wherein Ry and Rx are the same as defined earlier).

[00064] Unless otherwise constrained by the definition, all substituents optionally may be further substituted by not limiting to, 1-3 substituents, which may not be limited to, alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, carboxy, hydroxy, alkoxy, halogen, -CF3, cyano, -C(=O)NRxRy,-O(C=O)NRxRy (wherein Rx and Ry are the same as defined earlier) or -S(O)tRa (t being an integer from 0-2 and Ra is alkyl; alkenyl; alkylene; cycloalkyl; aryl; heterocyclyl; heteroaryl; aralkyl; heteroarylalkyl; heterocyclylalkyl), heterocyclyl or heteroaryl)).

[00065] Another embodiment of the present invention is directed towards the pharmaceutically acceptable salt form of formula 1. The salt may contain an organic salt or an inorganic salt. The inorganic salts may be inclusive of, but not be limited to, aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium and zinc as elements.

[00066] The organic salt may be inclusive of, but not limited to, a primary, a secondary, or a tertiary-amines, a naturally substituted amines, a cyclic amines, and modified salts prepared through basic ion exchange resin and the like. More preferably, the organic salts can be selected from the group consisting of, but not limited to, arginine, betain, caffeine, colin, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholin, N-ethylpiperidine, N-methylglucamine, glucamine, glucosamine, histidine, hydrapamine, N-(2-hydroxyethyl)piperidine, N-(2-hydroxyethyl) pyrrolidine, isopropylamine, lysine, methylglucamine, morpholin, piperazine, piperidine, polyamine resin, procain, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[00067] As another embodiment the pharmaceutically acceptable salts, diagnostic agents, acceptable solvates, enantiomers, diasteromers or N-oxides may also be utilized for treatment of abnormal cell growth leading to various diseases like cancers, tumours and the like.

[00068] Scheme A depicts a method for preparation of a novel Quinoline bridged Hydroxamate-Based inhibitors.

wherein, 2A series (2A1, 2A9, 2A3, 2A7, 2A6) represent mixture representative 2-aryl quinolone for synthesis of respective ester intermediates (3A1, 3A9, 3A3, 3A7 and 3A6). These ester intermediates undergo hydroxyl amination to form compound of formula 1 and its salts (3B1-3B8).

[00069] The method for preparation of a novel Quinoline bridged Hydroxamate-Based inhibitors (formula 1) comprises of the following steps:
A. Representative procedure for the synthesis of ester intermediates; and
B. hydroxylamination

The stages of sub-step A comprise of the following steps:
a) stirring a 100mg and 0.48mmol of the mixture representative 2-aryl quinoline (2A) along with one equivalent of ethyl-7-bromoalkanoate and three equivalents of potassium carbonate in a solvent, preferably Dimethylformamide (DMF) (qs) at room temperature for 10 h to obtain a mixture;
b) extracting the mixture of step a) by the process of Thin Layer Chromatography (TLC) with Ethyl acetate (three times in a quantity of 15 ml each) as the solvent, where the organic layer obtained was concentrated using rotary evaporator; and
c) drying the organic layer obtained in step b to obtain an ester intermediate [3A (3A1-3A8)].

The stages of sub-step B (hydroxyl amination) comprise of the following steps:
a) stirring the ester intermediate 3A using freshly prepared methanolic hydroxylamine solution for approximately 3-4 h at a temperature ranging from 0 ˚C to room temperature;

b) subjecting the mixture of step a to the process of Thin Layer Chromatography involving the use of Petroleum ether and Ethyl acetate to monitor the completion of reaction where the ratio of petroleum ether: ethyl acetate ranges between 3: 2, and neutralizing the reaction mixture with acetic acid; and

c) filtering the precipitates formed in step b and drying the precipitate and purifying it using (Silica gel #60-120 (used as a solid support); and Ethyl acetate: pet ether at 9:1 ratio) to obtain substituted molecules/synthetics belonging to Formula 1 (3B1-3B8).

[00070] In an exemplary embodiment, the present invention provides the following Synthetics (3B1-3B8) as mentioned below:

1. N-hydroxy-7-((2-phenylquinolin-6-yl)oxy)heptanamide (referred to as 3B1)

2. N-hydroxy-7-(4-(quinolin-2-yl)phenoxy)heptanamide (referred to as 3B2)

3. N-hydroxy-6-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)hexanamide (referred to as 3B3)

4. N-hydroxy-7-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)heptanamide (referred to as 3B4)

5. N-hydroxy-6-((2-(3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)hexanamide (referred to as 3B5)

6. N-hydroxy-7-((2- (3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)heptanamide (referred to as 3B6)

7. N-hydroxy-7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanamide (referred to as 3B7)

8. N-hydroxy-6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanamide (referred to as 3B8)

EXPERIMENTAL APPROACH

ANALYTICAL DATA AND TABLES

[00071] The analytical data of ester intermediates is as follows:

1. Ethyl 6-(4-(pyrazolo [1,5-c]quinazolin-2-yl)phenoxy)hexanoate (3A1)

A white yellowish compound with a melting point ranging from 108-110 °C. Other features include:
IR (KBr, cm-1): 1723 (C=O), 1610 (C=N). 1H NMR (CDCl3, 400 MHz, δ with TMS = 0): 9.09 (1H, s), 8.04 (1H, m), 7.94 -7.69 (5H, m), 7.67-7.57 (2H, m), 7.18 (1H,s), 6.99 (2H, d, J = 6.92 Hz), 4.13 (2H, q, J = 7.14 Hz), 4.02 (2H, t, J = 6.44 Hz), 2.35 (2H, m), 1.8-1.76 (2H, m), 1.74-1.69 (3H, m), 1.57-1.51 (2H, m), 1.28-1.22 (3H, m); 13C NMR (100 MHz, CDCl3, TMS = 0): 173.69, 160.07, 155.84, 140.05, 139.82, 139.29, 129.77, 128.71, 128.05, 128.02, 124.59, 123.32, 119.96, 114.84, 94.93, 67.76, 60.31, 34.27, 28.95, 25.67, 24.74, 14.28. HRMS (TOF-ESI) Calculated for C24H27NO3, 377.1991 [M]+; observed 378.2060 [M + H]+.
A yield of 52%, was obtained.

4. Ethyl 7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanoate (3A7)

A white yellowish compound with a melting point ranging from 93-96 °C. Other features include:
IR (KBr, cm-1): 1735 (C=O), 1617 (C=N), 1471 (C=N), 1210 (C-O). 1H NMR (DMSO-d6, 400 MHz, δ with TMS = 0) 8.25 (1H, d, J = 8 Hz), 8.08 (1H, d, J = 8 Hz), 7.91 (1H, d, J = 8 Hz), 7.48 (2H, s), 7.35-7.32 (2H, m) 4.05-3.99 (4H, m), 3.86 (6H, s), 3.69 (3H s), 2.26 (2H, t, J = 8 Hz, ) 1.75-1.70 (2H, m), 1.53-1.50 (3H, m), 1.43-1.39 (2H, m), 1.32-1.29 (1H, m), 1.13 (3H, t, J = 8 Hz); 13C NMR (100 MHz, DMSO-d6) δ 173.40, 157.06, 153.81, 153.71, 143.80, 139.19, 136.23, 134.90, 130.99, 128.54, 122.94, 119.54, 106.76, 104.80, 68.30, 60.64, 60.17, 56.54, 40.45, 40.24, 28.70, 24.93, 14.64. HRMS (TOF-ESI) Calculated for C27H33NO6 467.2308 [M]+; observed 468.3043 [M + H]+.
A yield of 63% was obtained.
5. Ethyl 6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanoate (3A8)

A yellow compound with a melting point ranging from 89-91 °C. Other features include:
IR (KBr, cm-1): 1737 (C=O), 1626 (C=N), 1465 (C=N), 1208 (C-O). 1H NMR (400 MHz, DMSO-d6 δ with TMS = 0) 8.26 (1H, d, J = 8.7 Hz), 8.08 (1H, d, J = 8.7 Hz), 7.91 (1H, d, J = 8.8 Hz), 7.48 (2H, s), 7.31 (2H, s), 4.11 – 3.95 (3H, m), 3.87 (6H, s), 3.69 (3H, s), 2.25 (2H, t, J = 7.3 Hz), 1.55 – 1.28 (6H, m), 1.12 (3H, t, J = 7.1 Hz). 13C NMR (100 MHz, DMSO-d6) δ 173.37, 157.04, 153.82, 153.71, 143.80, 139.19, 136.24, 134.90, 131.00, 128.54, 122.93, 119.56, 106.79, 104.80, 68.24, 60.64, 60.20, 56.55, 33.99, 28.80, 25.61, 24.76, 14.65. HRMS (TOF-ESI) Calculated for C26H31NO6 453.2151 [M]+; observed 454.2214 [M + H]+.
A yield of 68% was obtained.

The analytical data of various synthetics of formula 1 (3B) are as follows:

1.N-hydroxy-7-((2-phenylquinolin-6-yl)oxy)heptanamide (3B1)

A pale orange solid with a melting point ranging from 132-134 °C.
Figure 1A-1C represent the Spectral Data of N-hydroxy-7-((2-phenylquinolin-6-yl)oxy)heptanamide (3B1).
IR (KBr, cm-1): 3319 (-NH stretch), 2928 (C-H stretch), 2855 (=C-H stretch), 1649 (-C=O), 1467 (-C-H bending). 1H NMR (CDCl3, 400 MHz, δ with TMS = 0): 10.49 (1H, s), 8.32 (2H, d, J = 4.8 Hz), 8.21 – 8.19 (1H, m), 8.06 (1H, d, J = 6.8 Hz ), 7.72 – 7.54 (4H, m), 7.30 (1H, d, J = 6.4 Hz), 4.28 (2H, s), 2.78 (1H, s), 2.30 (2H, s), 2.03 (2H, d, J = 4.8 Hz ), 1.87 (2H, t, J = 5.6 Hz), 1.64 – 1.04 (5H, m). 13C NMR (100 MHz, CDCl3 + d6-DMSO) δ: 175.08, 161.85, 159.31, 148.83, 144.32, 140.40, 135.63, 133.73, 133.55, 132.94, 131.89, 127.43, 123.82, 110.51, 72.87, 37.64, 33.70, 33.62, 30.51, 30.27. HRMS (TOF-ESI) Calcd for C22H24N2O3, 364.1787 [M]+; observed 365.1789 [M + H]+.
A yield of 42% was obtained.
2. N-hydroxy-7-(4-(quinolin-2-yl)phenoxy)heptanamide (3B2)

A light yellow solid with a melting point ranging from 131 – 133 °C.
Figure 2A-2C is a representation of a Spectral Data of N-hydroxy-7-(4-(quinolin-2-yl)phenoxy)heptanamide (3B2).
IR (KBr, cm-1): 3429 (-NH stretch), 3051 (C-H stretch), 2356 (=C-H stretch), 1627 (-C=O). 1H NMR (CDCl3, 400 MHz, δ with TMS = 0): 10.36 (1H, s), 8.66 (1H, s), 8.32-8.00 (3H, m), 7.98-7.88 (3H, m), 7.73-7.69 (1H, t, J = 14.04 Hz), 7.52 (2H, t, J = 7.36 Hz), 7.04 (2H, d, J = 8.08 Hz), 3.98 (2H, m), 2.26-2.13 (1H, m), 2.01-1.78 (2H, m), 1.68-1.58 (3H, m), 1,57-1.48 (2H, m). 13C NMR (100 MHz, CDCl3, TMS = 0): 169.29, 159.97, 155.76, 147.57, 136.57, 130.97, 129.38, 128.83, 128.43, 127.43, 126.49, 125.66, 117.99, 114.41, 67.43, 32.29, 28.56, 28.37, 25.24, 25.06. HRMS (TOF-ESI) Calcd for C22H24N2O3, 364.1787 [M]+; observed 365.1847 [M + H]+.
A yield of 47% was obtained.

3. N-hydroxy-6-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B3)

A cream colored solid with a melting point ranging from 142 – 144 °C.
Figure 3A-3C is a representation of a Spectral Data of N-hydroxy-6-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B3).
IR (KBr, cm-1): 3280 (-NH stretch), 3054 (C-H stretch), 2856 (=C-H stretch), 1672 (-C=O), 1244 (C-N stretch). 1H NMR (d6-DMSO, 400 MHz, δ with TMS = 0): 10.35 (1H, s), 8.23 – 8.13 (3H, dd, J = 8.18 Hz), 7.98 (1H, d, J = 8 Hz), 7.87 (1H, d, J = 8 Hz), 7.33 (2H, m), 7.03 (2H, d, J = 8 Hz), 4.02 (2H, m), 3.79 (3H, s), 1.95 (2H, t, J = 8 Hz), 1.74 (2H, m), 1.54 (2H, m), 1.40 (2H, d, J = 8 Hz). 13C NMR (100 MHz, d6- DMSO) δ: 169.51, 160.78, 156.84, 153.87, 143.95, 136.28, 131.82, 130.83, 128.74, 128.23, 122.84, 118.93, 114.68, 106.83, 68.29, 55.77, 32.75, 28.87, 25.73, 25.43. HRMS (TOF-ESI) Calculated for C22H24N2O4, 380.1736 [M]+; observed 381.2336 [M + H]+.
A Yield of 39% was obtained.
4. N-hydroxy-7-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B4)

A creamish solid with a melting point ranging between: 147 – 149 °C.
Figure 4A-4C is a representation of a Spectral Data of N-hydroxy-7-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B4).
IR (KBr, cm-1): 3426 (-NH stretch), 2855 (C-H stretch), 2356 (=C-H stretch), 1624 (-C=O), 1119 (C-N stretch). 1H NMR (d6-DMSO, 400 MHz, δ with TMS = 0): 10.37 (1H, s), 8.71 (1H, s), 8.27 – 8.19 (3H, m), 8.03 – 7.90 (2H, dd, J = 6.4 Hz), 7.92- 7.90 (2H, m), 7.07 (2H, d, J = 5.9 Hz), 4.08 (2H, s), 3.82 (3H, s), 1.96 – 1.21 (10H, m). 13C NMR (100 MHz, d6- DMSO) δ: 169.43, 160.64, 156.81, 153.69, 143.80, 136.10, 131.66, 130.78, 128.70, 128.05, 122.83, 118.89, 114.56, 106.74, 68.21, 55.72, 32.68, 28.98, 28.88, 25.80, 25.56. HRMS (TOF-ESI) Cald for C23H26N2O4, 394.1893 [M]+; observed 395.2512 [M + H]+.
A Yield of 47% was obtained.
5. N-hydroxy-6-((2-(3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)hexanamide (3B5)

A Yellow colored solid with a melting point ranging from: 151 – 153 °C.
Figure 5A-5D is a representation of a Spectral Data of N-hydroxy-6-((2-(3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)hexanamide (3B5).
IR (KBr, cm-1): 3260 (-NH stretch), 2931 (C-H stretch), 2851 (=C-H stretch), 1667 (-C=O), 1254 (C-N stretch). 1H NMR (CDCl3, 400 MHz, δ with TMS = 0): 10.13 (1H, s), 8.56 (1H, s), 8.05 – 8.00 (2H, m), 7.69 – 7.68 (1H, m), 7.33 (1H, s), 6.99 – 6.89 (2H, m), 4.02 – 3.96 (2H, m), 3.92 (3H, m), 1.82 – 1.82 (10H, m). 13C NMR (100 MHz, CDCl3,) δ: 173.85, 158.49, 157.38, 152.82, 147.73, 138.06, 135.74, 130.95, 128.36, 127.72, 123.21, 118.67, 110.99, 105.62, 88.64, 67.81, 56.72, 34.28, 28.92, 25.73, 24.36. HRMS (TOF-ESI) Calcd for C22H23IN2O4, 506.0703 [M]+; observed 507.0733 [M + H]+.
A Yield of 31% was obtained.
6. N-hydroxy-7-((2- (3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)heptanamide (3B6)

A Yellow colored solid with a melting point ranging from: 159 – 161 °C.
Figure 6A-6C is a representation of a Spectral Data of N-hydroxy-7-((2- (3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)heptanamide (3B6).
IR (KBr, cm-1): 3298 (-NH stretch), 2988 (C-H stretch), 2864 (=C-H stretch), 1629 (-C=O), 1219 (C-N stretch). 1H NMR (CDCl3, 400 MHz, δ with TMS = 0): 10.42 (1H, s), 8.70 (2H, s), 8.30 – 8.25 (2H, m), 8.09 – 7.57 (2H, m), 7.40 (2H, s), 4.11 (2H, s), 3.93 (3H, s), 1.99-1.35 (11H, m) 13C NMR (100 MHz, CDCl3+d6-DMSO) δ: 170.42, 158.49, 156.77, 151.93, 143.72, 137.78, 135.15, 133.74, 129.78, 128.34, 127.89, 122.40, 118.43, 110.48, 105.65, 86.61, 68.12, 56.17, 32.55, 28.78, 28.60, 25.76, 25.41.HRMS (TOF-ESI) Calculated for C23H25IN2O4, 520.0859 [M]+; observed 521.1664 [M + H]+.
A Yield of 35% was obtained.
7. N-hydroxy-7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B7)

An off yellow solid with a melting point ranging from: 167 – 169 °C.
Figure 7A-7C is a representation of a Spectral Data of N-hydroxy-7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B7).

IR (KBr, cm-1): 3317 (-NH stretch), 2969 (C-H stretch), 1637 (-C=O). 1H NMR (d6-DMSO, 400 MHz, δ with TMS = 0): 9.89 (1H, s), 8.26 (1H, d, J = 8 Hz), 8.09 (1H, d, J = 8 Hz), 7.91 (1H, d, J = 8 Hz), 7.48 (2H, s), 7.35 (1H, s), 4.07 -3.98 (4H, m), 3.87 (6H, s), 3.69 (3H, s), 2.30 – 2.22 (3H, m), 1.77 – 1.42 (7H, m). 13C NMR (100 MHz, d6- DMSO) δ: 173.37, 157.04, 153.87, 153.71, 143.80, 139.19, 136.37, 136.24, 134.90, 131.00, 128.54, 122.80, 119.56, 106.79, 104.80, 68.24, 60.20, 56.55, 33.99, 28.80, 25.61, 25.56, 24.76. HRMS (TOF-ESI) Calcd for C25H30N2O6, 454.2104 [M]+; observed 455.2214 [M + H]+.
A Yield of 29% was obtained.

8. N-hydroxy-6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B8)

A Yellow solid with a melting point ranging from: 163 – 165 °C.
Figure 8A-8B is a representation of a Spectral Data of N-hydroxy-6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B8).
IR (KBr, cm-1): 3304 (-NH stretch), 2935 (C-H stretch), 2256 (=C-H stretch), 1629 (-C=O), 1179 (C-N stretch). 1H NMR. 1H NMR (d6-DMSO, 400 MHz, δ with TMS = 0): 9.95 (1H, s), 8.25 (1H, d, J = 12 Hz), 8.08 (1H, d, J = 8 Hz), 7.91 (1H, d, J = 8 Hz), 7.48 (1H, s), 7.36 -7.33 (2H, m), 4.07 – 3.99 (4H, m), 3.97 (3H, s), 3.69 (3H, s), 2.27 – 2.23 (2H, m), 1.74 -1.33 (9H, m); 13C NMR (100 MHz, d6- DMSO) δ: 173.40, 157.06, 153.81, 153.71, 143.80, 139.19, 136.23, 134.90, 130.99, 128.54, 122.94, 119.54, 106.76, 104.80, 68.30, 60.17, 33.39, 28.70, 25.26, 24.93. HRMS (TOF-ESI) Calculated for C24H28N2O6, 440.1947 [M]+; observed 441.1336 [M + H]+.
A Yield of 33% was obtained.
[00072] Biological Investigation:

A biological investigation of a compound is performed to test the biological activity of the compound like that of anti-cancer properties. The biological investigation of the novel compounds was conducted to asses for the topology, Histone deacetylase 1 and antiproliferative potential of the novel compounds (3B1-3B8) against anticancer cell lines. The cell lines considered were not limiting to, Lung cell lines (A549, H1299); Breast cell lines (MD-MDA-231, MCF-7) and Colorectal cell line (HT-29). The investigation involved Topo I and Topo II assay that is:
• Decatenation assay- A process of separating the physical linkage involved in the chromosomes;
• Intercalation assay: Aa process to determine the intercalating capacity of the compound with the DNA; and
• Relaxation assay- The process involves relaxation of the coiled DNA by an enzyme.

[00073] The compounds, not limiting to a Camptothecin (CPT) and Etoposide were used as positive controls in these TopoI and TopoII assays, respectively. The antiproliferative potential of the synthetics (3B1-3B8) is illustrated in Figures 9A-9D. The figure 9A is a representation of the decatenation assay to study TopoIIα inhibitory effect of synthetics (100 µM conc.) in comparison to etoposide (100 µM) on catenated kDNA as substrate. The figure 9B is a graphical representation of relative decatenation, where the X -axis represents investigational compounds (3B1-3B8) and Y- axis is represented by the decatenation values of k-DNA. The figure 9C depicts the results of an intercalation assay, where the Investigation compounds (3B1-3B8) of about 100 µM were incubated with supercoiled DNA (mPUC19) with reference to EtBr (1µg/mL). Figure 9D is the representation of the relaxation assay (Topol) which was conducted by incubating supercoiled DNA (pBR322). Relaxation activity of supercoiled form was evaluated in reference to the CPT (conc.100 µM).

[00074] The method for topoisomerase assay comprised of the following steps:

a. Procuring of an assay kit containing h TopoIIα enzyme, etoposide, buffer, the proteinase K and SDS as per the requirement for the as a first step;
b. preparing 10 mM stock solution by reconstituting the lyophilized etopside powder with dimethyl-sulfoxide (DMSO);
c. Mixing two buffer solutions i.e. buffer A and containing 0.5 M Tris-HCl (pH-8), 1.5 M sodium chloride, 5mM dithiothreitol, 300 µg/mL bovine serum albumin and buffer B containing 20 mM of Adenosine Triphosphate (ATP) in water; and
d. Dissolving the Investigation Compounds (3B1-3B8) were in DMSO to form 10 mM stock solution.

The method for Decatenation assay using human TopoIIα comprised of the following steps:

a. In a first step, adding to a 20 µL reaction mixture substrate, a 150ng of kDNA, two buffer solutions [buffer A and Buffer B, wherein the buffer A comprises of 0.5 M Tris-HCl (pH-8), 1.5 M sodium chloride, 5mM dithiothreitol, 300 µg/mL bovine serum albumin and buffer B contains 20 mM ATP in water are the same as prepared for the topoisomerase enzyme not limiting to 6 units;
b. adding a double distilled water to make up the reaction volume;
c. incubating the resultant reaction mixture of step a and b at not limiting to, 37 ˚C for not limiting to 30 minutes;
d. adding a broad spectrum serine protease, not limiting to a Proteinase K (500 µM/mL) and 10% Sodium Dodecyl Sulphate (SDS) leading to discontinuation of the reaction;
e. digesting the resultant mixture at 37 ˚C for 30 min;
f.. mixing the samples with 10X loading dye and were to resolve on 1% agarose gel electrophoresis containing 0.5 µg/mL ethidium bromide in TAE buffer; and
visualizing the decatenated kDNA on the gel on an imager.

The method for DNA Intercalation assay comprised of the following steps:
a. Subjecting a 200 ng of a negatively supercoiled DNA (pUC19) to incubation with 100 µM investigational compounds (3B1-3B8). Alternatively, subjecting a 200 ng of a negatively supercoiled DNA (pUC19) to incubation with1 µg/mL ethidium bromide at 37 ˚C for 30 minutes to obtain a sample;
b. loading the obtained sample of step a on 1 % agarose (non-ethidium bromide) gel electrophoresis in TAE buffer (the buffer contains Tris base, acetic acid and EDTA); and
c. staining the gel further for 15-20 minutes with ethidium bromide; and
d. destaining the stained gel of step c for not limiting to, 20-30 minutes to observe the retardation; and recording the pictographic images of the retardation.

The method for Topo-I relaxation assay comprised of the following steps:
a. In a first step, adding a 200 ng of negatively supercoiled pBR322 to a reaction mixture, where the reaction mixture consisted of: 10X buffer (100 mM Tris-HCl (pH-7.9), 10 mM EDTA, 1.5 M sodium chloride, 1% BSA, 1 mM spermidine, 50% glycerol) and the like.
b. adding a 100 µM investigational compounds or CPT and Topo-I to the mixture obtained in step a;
c. subjecting the mixtures of step a and b to incubation at, not limiting to, at 37 ˚C for 30 min;
d. adding a proteinase K ( (500 µM/mL)) and 10% of Sodium dodecyl sulphate (SDS) were to the mixture obtained at step c and was incubated at, not limiting to 50 ˚C for 20 min; and
e. subjecting the digested samples of step d to gel electrophoresis on a 1% agarose gel.

Results of the above analysis:

• The four synthetics 3B3-3B6 emerged as dual inhibitors of TopoI and TopoII as indicated by decatenation and relaxation assays without intercalating with DNA.
• However, all the synthetics exhibited excellent growth inhibitory activity at low micromolar concentration after 48 h of treatment. The four synthetics 3B3-3B6 emerged as multi-inhibitor of TopoI -TopoII and HDAC1. These selected synthetics were taken further for the cytotoxicity studies in normal cells (HEK-293 and hPBMCs). These synthetics, when incubated with respective cell lines at concentration of 10 μM (beyond their established IC50) separately for 48 h, did not exhibit any significant cytotoxicity.

[00075] Additional controls vorinostat and Trichostatin were also used for the biological investigation of the synthetics B1-B8. All the compounds except 3B2 and 3B7 were found to be potent inhibitors of HDAC1 (IC50 ranging from 14.16 – 47.43 nM) as compared to positive controls vorinostat (IC50= 64.34 nM) and trichostatin A (IC50= 77.01 nM). However, all the compounds exhibited excellent growth inhibitory activity at low micromolar concentration after 48 h of treatment.

[00076] An analysis of the effect of synthetics (3B3-3B6) on 3D cell culture of A549 cell was done through an MTT assay technique, where the technique employs a colorimetric assay for assessing cell metabolic activity. After achieving the 3D growth (21 days), of cells treatment of the cell mass with synthesized compounds at sub IC50 dose was initiated. The treatment was given for 17 days with a change of media and drug on every third day. At the end of the experiment, the diameter of the sphere was compared.

The method for MTT based Antiproliferative Assay consisted of the following steps:

a. Seeding a predetermined amount of cells for experimentation individually in each of the well plates;
b.subjecting the cells in the cell plates to incubation;
c.discarding the media responsible for holding the cells and separating the cells;
d. washing the separated cells with 1X of 100 µL per well phosphate buffered saline;
e. dissolving a colorimetric dye (MTT dye) in PBS (5 mg/mL) to obtain a dissolved dye solution;
f. adding the dissolved dye solution obtained in step e to each well (10 μL);
g. incubating each of the dyed cell in a Carbon-di-oxide incubator for approximately 3 - 4 h for the formation of Formazan crystals; and
h. dissolving the Formazan crystals in a biological grade DMSO and reading the absorbance on a microplate reader at 570 nm; and
i. representing the results mean ± Standard Deviation.

Results of the above analysis:
The results indicated that there was a noticeable decrease in mass of the 3D sphere by 3B5 and 3B6 whereas 3B3 and 3B4 exhibited potent anticancer effect as depicted in figure 3A making the 3D sphere tattered off. Further, the observation also disclosed that the synthetics were also able to hinder the 3D mass development when treatment was made concomitantly within 72 h of culture initiation, thus proving antimetastatic nature of the synthetics as depicted in figure 3B.

The following table 1 discloses the Antiproliferative potential (IC50) of the synthetic (3B1-3B8) as determined using MTT assay and their HDAC1 inhibitory potential:
Code Inhibitory Potential (IC50 (μM) ± SD)a
Lung cancer Cell Breast Cancer Cell Colon cancer Cell
A549 H1299 MDA-MB-231 MCF-7 HT-29 HDAC 1
IC50 (nM)
3B1 3.95 ± 0.19 4.25 ± 0.19 8.93 ± 0.19 9.38 ± 0.16 8.34 ± 0.17 47.43 ± 0.28
3B2 <1 2.46 ± 0.15 3.81 ± 0.15 4.82 ± 0.23 3.45 ± 0.14 66.54 ± 0.34
3B3 < 1 1.68 ± 0.18 < 1 1.98 ± 0.28 5.76 ± 0.18 14.16 ± 0.18
3B4 1.60 ± 0.23 2.33 ± 0.15 < 1 1.44 ± 0.19 3.91 ± 0.11 28.05 ± 0.23
3B5 1.77 ± 0.13 1.98 ± 0.13 1.49 ± 0.22 2.87 ± 0.17 2.86 ± 0.09 33.51 ± 0.27
3B6 1.97 ± 0.19 1.63 ± 0.15 1.55 ± 0.26 3.02 ± 0.18 1.86 ± 0.16 36.19 ± 0.24
3B7 3.75 ± 0.21 4.81 ± 0.22 5.87 ± 0.21 4.66 ± 0.14 4.97 ± 0.23 69.85 ± 0.41
3B8 1.85 ± 0.17 3.23 ± 0.27 6.24 ± 0.18 5.22 ± 0.22 1.90 ± 0.19 44.15 ± 0.36
CPT 6.86 ± 0.15 5.22 ± 0.16 7.36 ± 0.12 5.67 ± 0.20 6.87 ± 0.20 -
Vb 7.76 ± 0.28 4.21 ± 0.19 3.83 ± 0.16 4.99 ± 0.16 5.79 ± 0.27 64.34 ± 0.25
Tc - - - - - 77.01 ± 0.29
aCompounds were tested in triplicate and the result is presented in IC50 ± SD. bVorinostat; cTrichostatin as controls.
[00077] A further investigation of the synthetics (3B1-3B8) involving redox studies using H2DCFDA (a cell permeate indicator) and Cell Rox red based fluorescent dyes was conducted. The results of the investigation is represented graphically in figures 11A-11D. Figure 11A represents the Flourescence intensity of H2DCFDA versus the Count (i.e total number of cells within a tissue). Figure 11 B represents a Cell Rox assay analysed via Flow cytometer. Figure 11C represents graphically the mitochondrial membrane potential (∆ψm) detected by JC-1 (dye) based assay. Figure 11D depicts the relative ratio of change in Red/Green (J-aggregates Vs J-monomers) in the investigational compounds. Experiments were performed using A549 cells and treatment with investigational compounds were made at sub IC50 concentration. The analysis was done using BD C6 Flow cytometer.

[00078] The method for Mitochondrial Permeability Assay consisted of the following Steps:

a. as a first step, incubating the investigational compounds with cancerous cells for not limiting to, 48 hours;
b.trypsinizing followed by centrifuging and followed by washing the incubated cells with PBS and re-suspending the cells in the same;
c.adding a dye, not limiting to JC-1 dye;
d.incubating the suspension for further 30 minutes; and
e. analysing the mitochondrial membrane potential of the incubated cells using a flow cytometer in both the flow regions.

The dyes used in the assay rely on their accumulation in mitochondria. In healthy cells, dye accumulates as aggregates (J-aggregates) showing red fluorescence, whereas in unhealthy cells dye accumulates in the cytosol (J-Monomers) giving green fluorescence.

[00079] Figures 12A-12D is a graphical representation of the readings of the PI (Propedium Iodide) vs Annexin V assay performed on the compounds 3B3-3B6 In order to determine the exact mode of cell death induced by compounds upon treatment the PI vs Annexin V assay was performed. The analyses revealed apoptosis (both early and late) mode of cell death (refer Figure 12A) with cycle arrest at G1 and G2/M (refer Figure 12B). Post the apotysis the damaged cells and mitochondria therein may generate free radicals leading to elevation of Reactive Oxygen Species (ROS) levels leading to cancer precipitation and also a probable drug resistance. An investigation of the compounds was done to analyse whether their potential in inducing mitophag through a mitotracker green dye assay (refer Figure 12C) using two compounds 3B3 and 3B6. The analyses revealed that 3B3 has the highest potential to induce mitophagy in comparison to vorinostat and CPT employed as positive controls. Further, the compounds 3B3 and 3B6 were analysed for their ability to induce autophagy. It was concluded that synthetic 3B3 and 3B6 have the potency to induce autophagy (refer Figure 12D). The results were significant as autophagy in a cancer cell is associated with elimination and reversing drug resistance.

[00080] The method for Reactive Oxygen Species (ROS) Assay involved the following steps:

a.In the first step, a group of selected investigational molecules for investigating their effect on ROS levels were subjected to addition with two redox dyes, not limiting to, H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) dye for H2O2 Detection; and Cell rox red for detecting a superoxide or nitrite peroxidase;
b. measuring the fluorescence intensity of the dyed investigational molecules using BD C6 flow cytometer;
c. growing the cells in 35mm dishes and maintaining them under standard conditions;
d.incubating the Investigational compounds at varying concentration for 48 h with cancer cells;
e.trypsinising the incubated cells and washed with 1X PBS and pelleting down t=said cells;
f. rewashing the cells with PBS and centrifuging them at 1200 rpm; and
g. staining the centrifuged cells with appropriate redox dye and keepin at dark for 15-30 min and subsequently subjecting the cells to analysis using flow cytometer.

The method for Annexin V vs PI assay involved the following steps:

a. harvesting treated cellular samples for staining and transferring the harvested cells in a tube (1-106 cells per tube);
b. centrifuging the transferred cells at 1200 rpm for 5 min and washed with 1X PBS.
c. staining the suspended cells and incubating the stained for 30 minutes at room temperature in dark; and
d. analysing the cells of step c, preferably using a flow cytometer.
During the event of apoptosis, phosphatidylserine are configured to translocate from inner cytoplasmic membrane to outside, which is targeted by Annexin V dye, signifying the apoptotic population. On the other hand, PI intercalates with DNA in damaged cell and signifies cell death via necrosis.

[00081] A further analysis of the compounds 3B3 and 3B6 for their effect on oncogenic proteins PCNA (Proliferating Cell Nuclear Antigen); CDK4, cyclin D1, p53 and p21) and miRNA (let7a) was performed. For immunoblotting compounds, 3B3 and 3B6 were given treatment to MDAMB-231 cells at a concentration of 1, 3, 5 and 10 µM.. A positive control not limiting to, CPT and vorinostat were treated at a concentration of 5 µM. The analysis as represented in Figure 13A (RT-qPCR v P21,P53, Cyclin D1, CDK4,PCNA and Actin) it was observed that, PCNA levels were noticibly reduced for compound 3B3 in a dose-dependent manner. Further, though 3B6 was able to lower PCNA level, it was also able to significantly reduce at the highest concentration used. The PCNA levels were upregulated in case of CPT treated cells lysates. This could be plausibly indicative of CPT resistance in anticancer therapy. Further, a treatment with compound 3B3 suggested a decrease in CDK-4 levels, which may also be correlated with high subG1 population along with G1 arrest. The compound 3B6 was found to be able to induce CDK-4 inhibition at the highest concentration used, whereas CPT and vorinostat were found to increase the levels of CDK-4 suggesting non-mitogenic mode of cell death by these compounds .Further, the level of p53 were found to be down-regulated initially at a concentration of 1 and 3 µM by compound 3B3, but was upregulated at a concentration of 5 and 10 µM. Compound 3B6 was found to upregulate the level of p53 significantly at a concentration of 1-5 µM which was decreased at 10 µM concentration. In contrast, p21 was found to be down-regulated for compound 3B3 at every concentration whereas, for compound 3B6, p21 level was found to be upregulated initially at a concentration of 1 µM, which was further downregulated at higher concentration. Thus the results indicated extensive DNA damage.

[00082] Further, an analysis on p21 (an inhibitor of cell cycle progression), suggested the downregulation of p21 upon treatment with synthetics irrespective of upregulation of p53. Thus, p21 was found to be a negative regulator of p53. Further, to evaluate the effect of synthetic 3B3 and 3B6, at mRNA level, a tumour suppressor miRNAs was selected. The analysis found that the compounds 3B3 and 3B6 increased the expression of said miRNAs (refer Figure 13B), thus confirming their tumour suppressant behaviour.

[00083] Microsomal studies of compounds 3B3-3B6 were conducted. Further, a Microsomal stability assay using Human and Mouse liver microsomes to assess in vitro intrinsic clearance of investigational compounds was conducted. Human liver microsomes (HLM) and Mouse liver microsomes (MLM) were employed to study % Remaining 30 min, half-life (t½) and intrinsic clearance (CLint) of synthetics using Verapamil (High Clint and less t½) and carbamazepine (Low Clint and High t½) as controls. The results suggested 3B5 is stable to metabolism by CYPs having a half-life of more than 60 min with low CLint. 3B3, 3B4 and 3B6 have moderate clearance value.

[00084] Result of the above analysis: The results revealed the triple inhibition of TopoI, II and HDAC1 by the synthetics 3B3 – 3B6. The molecules were able to exhibit potential anticancer effect via modulating key oncogenic targets.

Table 2 represents Microsomal stability assay using Human and Mouse liver microsomes to assess in vitro intrinsic clearance of investigational compounds.
Compound % Remaining 30 min t½ (minutes) CLint (µl/min/mg)
HLM MLM HLM MLM HLM MLM
3B3 34.6 31.9 19.6 18.2 70.8 76.1
3B4 44.1 43.3 25.4 24.8 54.6 55.8
3B5 77.07 77.39 73.96 76.92 18.74 18.02
3B6 37.78 38.83 20.23 20.97 68.52 66.10
Verapamil 5.52 2.86 7.2 5.9 193 237
Carbamazepine 87.30 61.43 153.1 42.7 9 32

[00085] In Vivo Analysis: The effect of in vitro to in vivo to explore antitumor efficacy of 3B3 was performed by development of a xenograft animal model for lung cancer using A549 cells. The results suggested that both SAHA and Etoposide each at 30 mg/kg was used as a positive controls. A High dose 3B3 i.e. 30mg/kg had significantly decreased the size of the tumours when compared to tumor control group [Refer Figure 14 (A-C)]. A pictographic representation of macroscopic images of sacrificed mice and surgically removed tumors are shown in Figure 14A. A tumor volume-time curve is represented in Figure 14B, where the high dose of 3B3 is found to be highly effective. A Kaplan-Meier survival curve is depicted in Figure 14C.

[00086] Figure 15 is a Histopathological analysis of tumours excised from tumor control (TC), 3B3 (LD) 3B3 (HD), Etoposide and SAHA treated animals. The arrows depict the interstitial spaces in tumour samples. No such significant spaces were found in tumours of untreated animals. The percent tumor growth inhibition was found to be highest and average tumor weights were found to be lowest as represented graphically in figure 16 in the 3B3-HD, Etoposide and SAHA groups than the other respective groups. % Change in body weight was assessed in all animals of each group to determine the toxicological effects of each treatment. In addition to this, the Kaplan meier survival analysis indicated that 3B3-HD; Etoposide and SAHA improved the survival of the mice.

Amongst, other biological works: a 3D culture assay was performed where the process involved the following steps:

a. As a first step, suspending A 4 X 105 cancer cells (A549 and MDAMB-231) in 1 mL of in DMEM media;
b. incubating the suspended cells of step a with 3D culture scaffold in 12 well plates where the well plates are supplemented with 10% of Foetal Bovine Serum (FBS);
c. changing the media every third day under standard culture conditions.
d. subjecting the cells to incubation to ensure the filling of the scaffold completely with a 3D cell mass;
e. obtaining a complete growth of the 3D-cell mass was obtained after 21 days;
f. treating the cells of step e for not limiting to, 17 days by changing of media and drug on every third day; and
g. comparing the diameter of the sphere of the cancerous cell with results existing in the prior art.

[00087] Cell cycle analysis is perfomed to determine how the progression of cell cycle is affected by investigational compounds cell cycle analysis. This analysis is based on the mechanism of ability of the propidium iodide as a dye to stain the cellular DNA in a stoichiometric manner, the amount of stain is directly proportional to the amount of DNA within the cell.
The method involved the steps of:
a. prepared cellular samples for staining by transferring 1 X 105 to 1 X 106 cells to each tube;
b. centrifuging the prepared cellular samples at 1200 rpm for 5 min and washing the cells with 1X PBS;
c. fixing the cells using chilled ethanol and incubating the fixed cells for 3 h at -20 °C;
d. centriguging the cells of step c at 2500 rpm and washingwith 1 X PBS.
e. Adding 50 µL of propidium iodide (along with 50 µL Ribonuclease A) to each well and incubating said cells for 30 minutes at room temperature in dark;
f. analysing the cells of step e .

[00088] Autophagy analysis was done in accordance with manufacturer protocol (MAK138 Sigma-Aldrich Autophagy Assay Kit) by following steps:
a. Treating the cancer cells at sub-IC50 concentration with investigational compounds;
b. Trypsinising the treated cells and suspending in 1X PBS;
c. Staining the suspended cells with autophagy dye, preferably 10mM, 1µL per 100 µL sample and keeping the sample in dark for 30 min and
d. Analysing the sample using the cytometer.

[00089] Western Blotting was perfomed involving the following steps:
a. As a first step, treating respective cancer cells with investigational compound for 48 hrs;
b. scrapping, washing and collecting the cells of step a and lysing said cells using RIPA buffer comprising of: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA which is accompanied with protease inhibitor cocktail (BRAND). Lysed protein samples were quantified using Bradford assay;
c. resolution of proteins was assisted using electrophoresis on 10% SDS-PAGE and transferred onto Nitrocellulose membranes using Biorad Gel assembly;
d. blocking the membranes in non-fat dry milk prepared in 50mM Tris-HCl (pH 7.4), containing 0.05% Tween 20 (TBST) for 2 hr.
e. washing the blocked membranes wth (1X PBS);
f. incubating the washed blocked membranes with respective primary antibody and then incubated with the respective secondary; and
g. performing immune detection using ECL and bands and subsequently visualizing and documenting the results of the treated cells using a gel documentation system.

h. In vivo studies were conducted to anlayse whether 3B3 can translate the antitumor efficacy in vivo. Verapamil Hydrochloride and carbamazepine were used as a positive control.

The method involved the following steps:
a. developing a xenograft model of lung cancer, by subcutaneous injection of A549 cells into the flank of nude mice and evaluating its efficacy by allowing tumours to grow to around 100 mm3 of the tumour volume; and
b. dividing the animals into four groups (n = 6) and subjecting the Control mice to a 0.9% saline solution and checking the tumour volume not limiting to, bi-weekly up to four weeks.

[00090] Microsomal Assay was performed using the following steps:
a. As a first step weighing the test compounds and dissolving in DMSO to make 10mM stock solutions;
b. preparing a100mM potassium phosphate buffer (pH 7.4) by combining potassium phosphate monobasic solution and potassium phosphate dibasic solution and pre-warming said solution at 37 °C for one hour;
c. incubating the compound of step b at a final concentration of 1 µM with human (hLM), mouse (mLM) or rat (rLM) liver microsomes (0.5 mg/mL);
d. initiating by addition of the NADPH (1mM) in phosphate buffer (pH 7.4) at 37 °C. and terminating the at 0 and 30 min by the addition of cold acetonitrile containing propranolol (50 ng/mL) as an internal standard, where each reaction was carried out in duplicate (n=2) ;
e. partitioning the reaction mixture by centrifugation at 15,000 rpm for 15 min and analysing the resulting supernatants

ADVANTAGES OF THE INVENTION

• The present invention provides a compound and its synthetics to generate a synergestic mechanism for the treatment of abnormal cell growth.
• The synthetics disclosed in the present invention are therapeutically or prophylactically effective.
• The present invention has pharmaceutically acceptable salts for mitigating the growth of cancer cells.
• The compound disclosed in the present invention has minimal drug resistance and toxicity issues.
• The compound is effective in treatment of later stage/advanced stage cancers owing to the synergistic effect it provides with numerous anti-cancer drugs.

, Claims:WE CLAIM:

1. A heterocyclic compound represented by Formula (1):

wherein,
each of A and B are independently selected from a group of: a homocyclic and a heterocyclic ring systems;
W is selected from a group of: alkyl, alkenyl, alkynyl, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl or heterocyclyl;
X and Y are independently selected from an Oxygen (O), an imidogen (NH) and a Sulphur (S);
Z is selected from a group of: alkyl, alkenyl, alkynyl, alkoxy, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl or differently selected from a group of: heterocyclyl, azanol, and azaalkyl;
L is selected from a group of: alkyl, alkenyl, and alkynyl, wherein the value of n ranges from 0-12; and
R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aralkyl, heteroarylalkyl, heterocyclylalkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.
2. The heterocyclic compound as claimed in claim 1, wherein the heterocyclic compound is selected from a group consisting of N-hydroxy-7-((2-phenylquinolin-6-yl)oxy)heptanamide (3B1), N-hydroxy-7-(4-(quinolin-2-yl)phenoxy)heptanamide (3B2), N-hydroxy-6-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B3), N-hydroxy-7-((2-(4-methoxyphenyl)quinolin-6-yl)oxy)heptanamide (3B4), N-hydroxy-6-((2-(3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)hexanamide (3B5), N-hydroxy-7-((2- (3-iodo-4-methoxyphenyl)quinolin-7-yl)oxy)heptanamide (3B6), N-hydroxy-7-((2- (3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)heptanamide(3B7) and N-hydroxy-6-((2-(3,4,5-trimethoxyphenyl)quinolin-6-yl)oxy)hexanamide (3B8).

3. The heterocyclic compound as claimed in claim 1, wherein the heterocyclic compound is Qunilone bridged Hydroxamate-based dual inhibitor.

4. The heterocyclic compound as claimed in claim 1, wherein an anti-tumorigenic activity is exhibited in malignant cells of animals in a dosage ranging from 15mg/kg to  30mg/kg when tested in vivo.

5. A process for preparing heterocyclic compound of Formula (1)

comprising the steps of:
a. mixing 0.48 mmol of 2-aryl quinolone of formula (2A)

Formula 2A

with one equivalent of ethyl-7-bromoalkanoate and three equivalents of potassium carbonate in a solvent with stirring at a temperature ranging between 0 ˚C to room temperature for a period of ranging between 10 to 18 h to obtain a mixture;
b. extracting the mixture obtained in step (a) in the presence of an organic solvent to obtain an ester intermediate of Formula 3A;

Formula 3A
c. stirring the ester intermediate of formula 3A in the presence of freshly prepared solution of reducing agent for a period of 3-4 h at a temperature ranging between 0 ˚C - room temperature to obtain a reaction mixture;
d. neutralizing the reaction mixture obtained in step (c) with mild acid to obtain precipitate of reaction mixture; and
e. filtering and purifying the precipitates obtained in step (d) to obtain the heterocyclic compound of Formula (1).

6. The process as claimed in claim 5, wherein the solvent in step (a) is selected from the group consisting of: dimethyl formamide (DMF), acetonitrile, methanol, methyl ethyl ketone, 1 butanol, t-butanol, tert-butyl methyl ether, triethylamine and toluene.

7. The process as claimed in claim 5, wherein the organic solvent in step (b) is Ethyl acetate.

8. The process as claimed in claim 5, wherein the reducing agent in step (c) is methanolic hydroxylamine.

9. The process as claimed in claim 5, wherein the extraction of mixture in step (a) is done using the chromatography technique selected from Thin layer chromatography (TLC) and Reversed-Phase High Performance Liquid Chromatography (RP-pHPLC).

10. The process as claimed in claim 5, wherein the mild acid is selected from a group non-toxic acid like acetic acid.

11. The process as claimed in claim 5, wherein the filtering and purification is done using the silica gel-based chromatography.

12. The process as claimed in claim 5, wherein the yield of the product is in the range of 30% to 55%.