Description:FIELD OF THE INVENTION
[0001] The present disclosure relates generally to anti-cancer compounds. More specifically, the disclosure is directed to 2-amino-4H-pyran coupled imidazo[1,2-a]pyridine compounds of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof. The present disclosure also provides pharmaceutical compositions comprising the compounds and method of synthesis of the compounds.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Lung cancer is the most fatal and common malignancy worldwide irrespective of men and women. It is expected to cause about 2.45 million deaths globally by 2030. Based on the cellular origin, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases of lung cancer and of which, lung adenocarcinoma is more prevalent than squamous cell carcinoma and large cell carcinoma. Lung adenocarcinoma, a sub-type of NSCLC, is highly metastatic and mainly responsible for cancer-related mortality. The treatment options for lung cancer patients typically consist of surgery, radiation, and chemotherapy. Due to the poor prognostic nature, chemotherapy remains at the cornerstone of NSCLC treatment. Although chemotherapy has many drawbacks especially for drug resistance and non-specific cell toxicity, in absence of selective drug candidates, patients still get treated with chemotherapeutic drugs, like docetaxel, doxorubicin and cisplatin, that showed severe side effects.
[0004] There is a need in the art to develop novel compounds for selective and targeted therapies, for both cancer cell proliferation and tumor metastasis, against lung cancer treatment, specifically NSCLC.

OBJECTS OF THE INVENTION
[0005] An object of the present disclosure is to provide compounds for anti-cancer activity.
[0006] Another object of the present disclosure is to provide compounds that selectively inhibit tumor cell growth and proliferation.
[0007] Another object of the present disclosure is to provide a method of synthesis of the anti-cancer compounds.

SUMMARY OF THE INVENTION
[0008] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0009] Aspects of the present disclosure relate to novel compounds that have 2-amino-4H-pyran coupled to imidazo[1,2-a]pyridine.
[0010] In an aspect, the present disclosure provides an anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

Formula I
wherein R1 may be selected from (C6-C14)aryl or (C5-C10)heterocyclyl;
wherein R1 may be unsubstituted or substituted with one or more of halogen, nitro, (C1-C6)alkyl, amino, -NR2R3, -CN, (C1-C6)alkoxy, -SO2, -COOH, hydroxy, or -COOR2; and
wherein R2 and R3¬ may be independently selected from (C1-C6)alkyl.
[0011] In an embodiment, R1 may be substituted with one or more of F, Cl, Br, nitro, -CN, ethyl, methyl, trifluoromethyl, trichloromethyl, tribromomethyl, methoxy, ethoxy, -N(CH3)2, -N(C2H5)2, -COOH, -COOC2H5, or –COOCH3.
[0012] In a preferred embodiment, the compound of Formula I may be selected from
2-Amino-4-phenyl-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(p-tolyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-methoxyphenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-(dimethylamino)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-chlorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-fluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2,4-difluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(3-bromophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-cyanophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-nitrophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
Methyl 4-(2-amino-3-cyano-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridin-4-yl)benzoate;
2-Amino-4-(4-(trifluoromethyl)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(pyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2-chloropyridin-4-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(5-bromopyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0013] In another aspect, the present disclosure relates to a pharmaceutical composition comprising an anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof and one or more pharmaceutically acceptable excipient(s),

Formula (I)
wherein R1 is as defined earlier. In an embodiment of the present disclosure, the pharmaceutical composition of compound of Formula I may be in the form of a tablet, a pill, a dispersible film, an elixir, a capsule, a spray, an aerosol, a solution, a gel, a suspension, nanoparticles, microemulsions, microparticles, a patch, an oil, or a powder.
[0014] In an aspect, the present disclosure provides a method of synthesizing a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein the method comprises the steps of general scheme I comprising: (a) reacting 2-aminopyridine (1) with ethyl 2-bromoacetate to give 2-amino-1-(2-ethoxy-2-oxoethyl)pyridin-1-ium (2) and cyclizing it to give imidazo[1,2-a]pyridin-2-ol (3); and (b) reacting imidazo[1,2-a]pyridin-2-ol (3) of step (a) with malononitrile (4) and an aldehyde of Formula 5 to give the compound of Formula I

Scheme I
[0015] In another aspect, the present disclosure provides a medicament comprising a pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0016] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0018] Figure 1 provides the impact of compounds IMPA -2,-5,-6,-8, and -12, as per an embodiment of the present disclosure, on cell viability in A549 lung adenocarcinoma cell lines. (1A) Cell viability(%) of A549 cells after treatment with different concentrations of IMPA -2,-5,-6,-8, and -12 at 24h; (1B) Cell viability(%) of BEAS2B and mouse fibroblast L929 at concentrations of IC50 values of IMPA-2, -5, -6, -8, and -12.; (1C) SEM images of representative morphological characteristics of A549 cells after treatment with the compounds at 24h; (1D) Clonogenic assay of A549 cells; and (1E) Quantitative result of the clonogenic assay in A549 cells treated with IMPA-2,-5,-6,-8, and -12 in 24h.
[0019] Figure 2 provides the cell migration and invasion properties in A549 lung adenocarcinoma cell lines after treatment with compounds IMPA -2,-5,-6,-8, and -12, as per an embodiment of the present disclosure. (2A) Representative pictures of wound healing scratch assays of untreated A549 cells, and cells treated with IMPA-2, -5, -6, -8, and -12 at 0h, 4h, 8h, 12h and 24h; (2B) Quantification data of wound scratch assay; (2C) Demonstrative images of untreated cells, and IMPA -2,-5,-6,-8 and -12 treated cells by fluorescent microscopy at 24h for examination of migration abilities by transwell assay;(2D) Quantification data of cell migration assay; (2E) Demonstrative images of untreated cells, and IMPA -2,-5,-6,-8 and -12 for examination of invasion properties by transwell assay in A549 cells at 24h; and (2F) Quantitative analysis of invasion assay study in untreated and IMPA treated A549 cells.
[0020] Figure 3 provides the study of the effect of compounds IMPA -2,-5,-6,-8, and -12, as per an embodiment of the present disclosure, on the apoptotic signalling pathway in A549 lung adenocarcinoma cell lines by activating the cleaved caspase 3, 9 and compromising mitochondrial membrane potentiality. (3A) Flow cytometry analysis of apoptosis assay by Annexin V-FITC/PI in A549 cells after treatment with the compounds at 24h; (3B) Quantification data of apoptosis assay; (3C) Fluorescence microscopy images of mitochondrial membrane potential assay by JC-1 dye; (3D) Quantification study ROS activity by DCFDA reagent (hydrogen peroxide probe); (3E) Gene expression analysis of pro-apoptotic Bax, Bak1; (3F) Anti-apoptotic Bcl2 in untreated and compound treated A549 cells at 24h; (3G) Immunoblotting of cleaved caspase 3, and 9 protein expression of untreated and compound treated A549 cells at 24h; (3H) Relative protein expression of cleaved caspase 3, and 9 by western blot in NAC treatment followed by compound treated cells; and (3I) Quantification data of apoptosis assay in NAC treatment.
[0021] Figure 4 demonstrates that compounds IMPA -2,-5,-6,-8, and -12, as per an embodiment of the present disclosure, arrested the cell cycle by activating the tumor suppressor proteins in A549 lung adenocarcinoma cell lines. (4A) Flow cytometric analysis of cell cycle distribution by flow histogram; (4B) Quantification data of cell cycle distribution. (4C) Relative protein expression of pP38 by western blot at 24h; (4D) Expression profile of p53 gene in untreated control and compound treated A549 cells at 24h; (4E) Expression profile of p16, p21, and p27 gene in untreated and compound treated A549 cells at 24h; and (4F) Expression profile of p53 by qPCR of NAC treated compound treated cells.
[0022] Figure 5 provides representative data of compounds, IMPA -2,-5,-6,-8, and -12, as per an embodiment of the present disclosure, preventing the growth of 3D lung tumor spheroids. (5A) Phase contract images of multicellular tumor spheroids (A549, MRC-5 and THP-1) treated with the compounds for 24 h and 48 h respectively; (5B) Representative images of Calcein AM (green) and propidium iodide (red) staining of multicellular tumor spheroids treated with compounds for 24 h and 48 h respectively; (5C) Representative images of flow cytometric cell viability assay (Annexin-v and PI) on multicellular tumor spheroids treated with indicated compounds for 24 h and 48 h respectively; (5D) Graph representing the percentage of live cells in spheroids treated with indicated compounds for 24 h; and (5E) Graph representing the percentage of early apoptotic, late apoptotic and dead cells in 48 hours in cells treated with the compounds of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0023] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0024] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0025] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0026] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0027] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0028] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0029] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0030] Also, use of "(s)" as part of a term, includes reference to the term singly or in plurality, for example, the term pharmaceutically acceptable salt(s) indicates a single salt or more than one salt of the compound of Formula I.
[0031] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0032] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0033] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0034] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0035] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0036] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0037] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0038] The term "or", as used herein, is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
[0039] The term, "halogen" as used herein refers to chlorine, fluorine, bromine or iodine atom.
[0040] The term, “(C1-C6)alkyl”, as used herein, refers to the radical of saturated aliphatic groups, including straight or branched-chain alkyl groups having six or fewer carbon atoms in its backbone, for instance, C1-C6 for straight chain and C3-C6 for branched chain. As used herein, (C1-C6)alkyl refers to an alkyl group having from 1 to 6 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl and 3-methylbutyl.
[0041] Furthermore, unless stated otherwise, the alkyl group can be unsubstituted or substituted with one or more substituents, for example, from one to four substituents, independently selected from the group consisting of halogen, hydroxy, cyano, nitro and amino. Examples of substituted alkyl include, but are not limited to hydroxymethyl, 2-chlorobutyl, trifluoromethyl and aminoethyl.
[0042] The term,“(C1-C6)alkoxy" refers to a (C1-C6)alkyl having an oxygen radical attached thereto. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy. Furthermore, unless stated otherwise, the alkoxy groups can be unsubstituted or substituted with one or more groups. A substituted alkoxy refers to a (C1-C6)alkoxy substituted with one or more groups, particularly one to four groups independently selected from the groups indicated above as the substituents for the alkyl group.
[0043] The term "(C6-C14)aryl" or "aryl" as used herein refers to monocyclic, bicyclic, or tricyclic hydrocarbon groups having 6 to 14 ring carbon atoms, wherein at least one carbocyclic ring is having a p electron system. Examples of (C6-C14) aryl ring systems include, but are not limited to, phenyl and naphthyl.
[0044] The term, “(C5-10)heterocyclyl”, as used herein refers to a 5- to 10-membered, saturated, partially unsaturated or unsaturated monocyclic or bicyclic ring system containing 1 to 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur. Saturated heterocyclic ring systems do not contain any double bond, whereas partially unsaturated heterocyclic ring systems contain at least one double bond, and unsaturated heterocyclic ring systems form an aromatic system containing heteroatom(s). The oxidized form of the ring nitrogen and sulfur atom contained in the heterocyclyl to provide the corresponding N-oxide, S-oxide or S,S-dioxide is also encompassed in the scope of the present disclosure. Representative examples of heterocyclyls include, but are not limited to, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, dihydropyran, tetrahydropyran, thio-dihydropyran, thio-tetrahydropyran, piperidine, piperazine, morpholine, 1,3-oxazinane, 1,3-thiazinane, 4,5,6-tetrahydropyrimidine, 2,3-dihydrofuran, dihydrothiene, dihydropyridine, tetrahydropyridine, isoxazolidine, pyrazolidine, furan, pyrrole, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, benzofuran, indole, benzoxazole, benzothiazole, isoxazole, triazine, purine, pyridine, pyrazine, quinoline, isoquinoline, phenazine, oxadiazole, pteridine, pyridazine, quinazoline, pyrimidine, isothiazole, benzopyrazine and tetrazole.
[0045] The term, "therapeutically effective amount" as used herein refers to an amount of a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof or a composition comprising a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, effective in producing the desired therapeutic response in a particular subject suffering from a disease or disorder.
[0046] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia', including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0047] The terms, “treatment", "treat" and "therapy" and the like as used herein refer to alleviate, slow the progression, attenuation, prophylaxis or as such treat the existing diseases or condition (e.g. lung cancer proliferation). Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0048] Aspects of the present disclosure provide compounds that target both cancer cell proliferation and tumor metastasis. The compounds have potent anti-proliferative activity in lung adenocarcinoma cells which induces apoptotic cell death and cell cycle arrest.
[0049] In an embodiment, the present disclosure provides an anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

Formula I
wherein R1 may be selected from (C6-C14)aryl or (C5-C10)heterocyclyl;
wherein R1 may be unsubstituted or substituted with one or more of halogen, nitro, (C1-C6)alkyl, amino, -NR2R3, -CN, (C1-C6)alkoxy, -SO2, -COOH, hydroxy, or -COOR2; and
wherein R2 and R3¬ may be independently selected from (C1-C6)alkyl.
[0050] In an embodiment, the (C1-C6)alkyl group may be unsubstituted or substituted with one or more substituents, preferably from one to four substituents, independently selected from the group consisting of halogen, hydroxy, -CN, nitro and amino.
[0051] In an embodiment, R1 may be substituted with one or more of F, Cl, Br, nitro, -CN, ethyl, methyl, trifluoromethyl, trichloromethyl, tribromomethyl, methoxy, ethoxy, -N(CH3)2, -N(C2H5)2, -COOH, -COOC2H5, or –COOCH3.
[0052] In an embodiment, (C6-C14)aryl may be selected from phenyl, or naphthyl; wherein (C6-C14)aryl may be substituted with one or more of Cl, F, Br, nitro, -CN, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, -N(CH3)2, or -COOCH¬3.
[0053] In an embodiment, (C5-C10) heterocyclyl may be pyridine; wherein (C5-C10) heterocyclyl may be substituted or unsubstituted with one or more of Cl, or Br.
[0054] In a preferred embodiment, the compound of Formula I may be selected from
2-Amino-4-phenyl-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(p-tolyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-methoxyphenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-(dimethylamino)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-chlorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-fluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2,4-difluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(3-bromophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-cyanophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-nitrophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
Methyl 4-(2-amino-3-cyano-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridin-4-yl)benzoate;
2-Amino-4-(4-(trifluoromethyl)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(pyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2-chloropyridin-4-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(5-bromopyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0055] In an embodiment, the compound of Formula I may be selected from
2-Amino-4-(p-tolyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-chlorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-fluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(3-bromophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile; or
2-Amino-4-(4-(trifluoromethyl)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile.
[0056] In another embodiment, the compound of Formula I can be converted into a pharmaceutically acceptable salt. The pharmaceutical acceptable salts of the compound of Formula I according to the disclosure are prepared in a manner known to one skilled in the art. Pharmaceutically acceptable salts of the compound of the present disclosure include but are not limited to, an acid salt of a compound of the present disclosure containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.
[0057] In yet another embodiment, the pharmaceutically acceptable salts of the compound of the present disclosure containing acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.
[0058] In an embodiment, the solvates include hydrates such as monohydrate, dihydrate, sesquihydrate, tetrahydrate, or combinations thereof. The terms tautomers and stereisomers have the meaning as well-defined in the art.
[0059] The compounds of the present disclosure are effective anti-cancer compounds. Specifically, the compounds are effective against tumor metastasis or proliferation of lung cancer and more preferably for non-small cell lung cancer (NSCLC).
[0060] Without being bound to theory, it is believed that the compounds of Formula I significantly increase reactive oxygen species (ROS)-mediated mitochondrial pathway of apoptosis in lung cancer cells through the impairment of mitochondrial membrane potential, increased pro-apoptotic BAX and BAK1 expressions and decreased anti-apoptotic BCL2 expression, along with the induction of caspase-9/3 activation. Increased ROS production by these compounds also promotes p53 mediated A549 lung cell cycle arrest through the activation of p38 MAPK. The compounds show significant reduction of tumor growth and inhibition of cancer cells migration, invasion and colony formation.
[0061] The compounds demonstrate decreased cell viability for lung adenocarcinoma cells with high specificity. The compounds are capable of inhibiting NSCLC cells wound healing, invasion and metastasis abilities. They also repress NSCLC growth or cause cell arrest by blocking G1/S transition, or the G2/M phase.
[0062] In another embodiment, the present disclosure relates to a pharmaceutical composition comprising an anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof and one or more pharmaceutically acceptable excipient(s),

Formula (I)
wherein R1 is as defined earlier.
[0063] The present disclosure also relates to a process of preparation of the composition, which includes bringing a compound of Formula I, into a suitable administration form using the pharmaceutically acceptable excipient. The pharmaceutical compositions containing the compound of Formula (I) according to the disclosure are prepared in a manner known to one skilled in the art. In an embodiment of the present disclosure, the pharmaceutical composition of compound of Formula I may be in the form of a tablet, a pill, a dispersible film, an elixir, a capsule, a spray, an aerosol, a solution, a gel, a suspension, nanoparticles, microemulsions, microparticles, a patch, an oil, or a powder.
[0064] In an embodiment, the pharmaceutically acceptable excipient may be selected from carrier, solvent, binder, filler, solubilizers, surfactants, anti-oxidants, disintegrant, flavoring agent, coloring agent, preservatives, lubricant, or combinations thereof. However, a person of skill in the art would appreciate that any other excipient(s) may be employed without deviating from the spirit and scope of the invention
[0065] In an embodiment, the excipient may be selected depending on the type of composition for example, the composition may be a tablet, a pill or a film comprising the compound along with excipients including magnesium stearate, lactose, talc, corn starch, starch, cellulose, gum arabica, sodium glyconate, or combinations thereof; the composition may be a capsule comprising gelatin, waxes, fats or combinations thereof; or the composition may be a solution or elixir comprising a solvent such as water, sodium chloride solution, glycerol, ethanol, or sugar solution.
[0066] In an embodiment, the composition may be administered orally, nasally, intravenously, parenterally, intraperitoneally, rectally, buccally, transdermally, subcutaneously, or intramuscularly depending on the form of the composition. For example, a spray or aerosol may be taken nasally while a pill or solution or elixir may be taken orally.
[0067] In another embodiment, the pharmaceutical compositions normally contain about 1% to 99%, for example, about 5% to 70%, or from about 10% to about 30% by weight of the compound of Formula (I), a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof. The amount of the compound of Formula (I) in the pharmaceutical composition normally is from about 5 to 500 mg or may be lower than or higher than the lower and the upper limit respectively. The dose of the compound of Formula I, which is to be administered, may be decided by a skilled physician considering the weight, gender, age, previous medical records and severity of current medical condition of the subject.
[0068] In certain embodiments, the composition may also comprise in addition to a compound of Formula I, one or more other therapeutically or prophylactically active agents. Alternatively, the compound of Formula I may be co-administered along with the other therapeutically or prophylactically active agent, as either a single combination dosage form or as multiple, separate dosage forms, administration of the compound of the present disclosure first, followed by the other therapeutic agent or treatment and administration of the other therapeutic agent or treatment first, followed by the compound of present disclosure. Further, therapeutic agents are administered either simultaneously or sequentially. The other therapeutic agent may be any agent that is known in the art to treat, prevent, or reduce the symptoms of a disease or disorder, preferably for cancer.
[0069] In an embodiment, the present disclosure provides a method of synthesizing a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein the method comprises the steps as shown in general scheme I.

Scheme I
[0070] In an embodiment, the present disclosure provides a method of synthesizing a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein the method comprises the steps of: (a) reacting 2-aminopyridine (1) with ethyl 2-bromoacetate to give 2-amino-1-(2-ethoxy-2-oxoethyl)pyridin-1-ium (2) and cyclizing it to give imidazo[1,2-a]pyridin-2-ol (3); and (b) reacting imidazo[1,2-a]pyridin-2-ol (3) of step (a) with malononitrile (4) and an aldehyde of Formula 5 to give the compound of Formula I.
[0071] In an embodiment, the cyclization of compound of Formula 2 may be performed in a base and a solvent. The base may be selected from sodium ethoxide, sodium methoxide, sodium hydride, potassium tertiary butoxide, LiHMDS, cesium carbonate, or combinations thereof. The solvent used in step (a) may be selected from ethanol, isopropanol, methanol, tetra hydro furan, or combinations thereof.
[0072] In another embodiment, the step (b) is a pot-reaction that may be performed in a solvent selected from ethanol, water, isopropanol, methanol, tetra hydro furan with water or combinations thereof. In a preferred embodiment, the step may be performed in a solvent comprising ethanol and water in a ratio of about 1:1.
[0073] The process of the present disclosure gives compounds in high yields and with high purity. In some embodiments, the process may yield over 85% of the compound of Formula I.
[0074] In an embodiment, the present disclosure relates to use of a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof for treatment, alleviation or amelioration of cancer.
[0075] In another embodiment, the present disclosure provides a method of treatment of cancer by administering a therapeutically effective amount of a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0076] In a preferred embodiment, the cancer may be lung cancer. In a more preferred embodiment, the cancer may be non-small cell lung cancer.
[0077] In another embodiment, the present disclosure provides a medicament comprising a pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0078] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0079] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
[0080] MATERIALS AND METHODS: All the required chemicals were purchased from Sigma-Aldrich, Spectrochem Pvt. Ltd. and Combi Blocks, USA. Reactions were monitored by silica gel coated aluminium plates and visualized by UV light. Spectra of 1H NMR and 13C NMR were recorded on Avance (400 & 300 MHz), Bruker instruments. Spectral values were assigned in d and TMS as the internal standard. Mass was analyzed in the ESI+APCI instrument and IR data was recorded on FT-IR instrument recorded in cm-1 for major peaks. The purity of compounds was determined by HPLC (Agilent Technologies 1200).
[0081] All tissue culture materials were obtained from Gibco, Grand Island, NY. Phospho-p38 MAPK (Thr-180/Tyr-182; D3F9; Cat. No. #4511), cleaved Caspase 3 (Asp-175, Cat. No. #9664S) antibodies were purchased from Cell Signaling Technology, Danvers, MA; and ß-actin monoclonal antibody (AC-15) (Cat. No. #AM4302) from Invitrogen, Grand Island, NY. Caspase 9 (Cat. No. #BB-AB0245), antibody was procured from the BioBharati Life Science, India. ClarityTM Western ECL Substrate (Cat. No. #170-5060), N-Acetyl-L-cysteine (NAC) (Cat. No. #A0905) was procured from TCI, and iScript Reverse Transcription Supermix (Cat. No. #170-8841) from Bio-Rad Laboratories, Hercules, CA. Halt Protease and Phosphatase Inhibitor Cocktail (Cat. No. #78440) was purchased from Thermo-Scientific, Grand Island, NY. Different gene-specific primers were procured from Integrated DNA Technologies, India. All other chemicals and reagents used were purchased from Sigma, St. Louis MO, USA.
[0082] Cell lines and treatment conditions: The human lung adenocarcinoma A549, human peripheral blood monocyte THP-1and mouse fibroblast L929 cells were procured from the National Centre for Cell Science (NCCS), Pune, India and cultured in RPMI-1640(Cat. No. #12-115F, Lonza, USA). Human lung epithelial cells BEAS-2B and human lung fibroblast MRC-5 were obtained from NCCS, India and maintained in LHC-9 (Cat. No. #12680013, Thermo Fischer, USA), and EMEM (Cat. No. #12-125Q, Lonza, USA), respectively. All media were supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA) and penicillin (100 IU/ml)-streptomycin (100 ng/ml) (Invitrogen, USA) solution. The cells were cultured at 37°C in a 90% humidified incubator with 5% CO2 environment. Cells when attained 80% confluency were sub-cultured in fresh media.
[0083] Compounds synthesized in the examples (IMPA-2,-5,-6,-8,-12) and curcumin were dissolved in DMSO (HiMedia, India) to prepare a stock solution at a concentration of 1 mg/ml and stored in -20°C. The working solutions of these compounds were freshly prepared in the culture medium before use. The cells were seeded in a 6-well plate with 3.0X105 cells/well and allowed to adhere for 24h. Then, the cells were incubated with varied concentration of the compounds or curcumin for different periods. Control and treated cells images were captured using a light microscope (Evos XL core, USA). For NAC treatment, adherent cells were treated with 5mM as a working concentration of NAC in serum-free media 1h before the IMPA treatment. Upon termination of incubations, cells were washed twice with ice-cold Dulbecco’s phosphate-buffered saline (DPBS) and either harvested with trypsin–EDTA (Gibco, UK) solution or incubated with different chemicals for further studies. Harvested cells were centrifuged for 5 min for 2000 rpm to collect the cell pellets which were used to perform various assays.
[0084] Statistical analysis: In this study, all data were obtained from at least three separate experiments and standard deviation (S.D.) values were determined accordingly. The values were presented as the mean ± S.D. Student's t-tests were employed for statistical analysis using SigmaPlot 10.0 software. The mean fluorescence intensity was used for flow cytometry analysis. A level of p< 0.05 was considered statistically significant. Statistically significant results were shown as follows: ns: not significant, #p<0.05,*p<0.01 and **p<0.001
[0085] Example 1 : Synthesis of the compounds
[0086] 15 compounds of Formula I named IMPA-1-IMPA-15 were synthesized by the general scheme II.

Scheme II
[0087] Step a) Synthesis of 2-amino-1-(2-ethoxy-2-oxoethyl)pyridin-1-ium (2)
[0088] In a 100 mL multi neck RBF pyridine-2-amine (1 gm) was charged at room temperature. Then the ethyl 2-bromoacetate (5 vol.) was added slowly over 5 min and allowed to stir for 16 h at room temperature. MTBE was added and filtered the precipitated solid and washed twice with MTBE and dried to obtain 2-amino-1-(2-ethoxy-2-oxoethyl)pyridine-1-ium (2) as brown colour solid with a quantitative yield which was used for next step without any further purification.
[0089] Synthesis of imidazo[1,2-a]pyridin-2-ol(3)
[0090] To a stirred solution of 2-amino-1-(2-ethoxy-2-oxoethyl)pyridine-1-ium (2, 1 mM) in ethanol was added sodium ethoxide solution in ethanol (2 mM) drop wise over 15 min at room temperature. The resulting reaction mixture was stirred for 3 h at rt, the progress of the reaction monitored by TLC. After completion of starting material neutralized the reaction with 1N HCl solution and extracted with dichloromethane. Combined organic layers were dried over Na2SO4 and filtered, concentrated under reduced pressure to obtain imidazo[1,2-a]pyridin-2-ol (3) as light brown solid with 80% yield.
[0091] Step b) Synthesis of compound of Formula I: To a stirred solution of malononitrile (4, 1 mM) in an equity ratio of ethanol and water, was added corresponding aldehyde [(5 a-o, 1 mM] at rt and allowed to stir for 10 mins followed by addition of imidazo[1,2-a]pyridin-2-ol (3, 1 mM). The reaction mixture was heated to 100° C and maintained for 16 h. After the completion of the reaction, cooled the reaction mixture to room temperature and filtered the solid and washed with ethanol to yield compounds of Formula I, namely IMPA-1 – IMPA - 15. Table No. 1 below provides the characterization data and structure of the prepared compounds.
Table No. 1: Characterization data of compounds IMPA-1 to IMPA-15
Compound Characterization data

2-Amino-4-phenyl-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-1) Yield: 74%; off-white solid; mp: 241-245 °C; IR (KBr, cm-1) 3150, 2196, 1660, 1412, 1036, 738; 1H-NMR (400 MHz, DMSO-d6):d 7.68 (d, J = 6.8 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.36–7.33 (m, 2H), 7.29–7.23 (m, 4H), 7.12 (s, 2H), 6.89–6.85 (m, 1H), 5.18 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.54, 149.43, 140.89, 140.04, 128.85, 127.62, 127.54, 124.82, 123.94, 120.13, 116.11, 112.65, 100.71, 57.85, 36.89; LC-MS (APCI + ESI) m/z: 289 [M + H] +; Anal. Calcd for C17H12N4O: C, 70.82; H, 4.20; N, 19.43. Found: C, 70.98; H, 4.21; N, 19.49.

2-Amino-4-(p-tolyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-2) Yield: 69%; off-white solid; mp: 246-250 °C; IR (KBr, cm-1) 3149, 2193, 1657, 1404, 1035, 736; 1H-NMR (400 MHz, DMSO-d6):d 7.68 (d, J = 6.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.15–7.10 (m, 6H), 6.87 (t, J = 6.8 Hz, 1H), 5.13 (s, 1H), 2.26 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d160.46, 149.37, 139.99, 137.87, 136.70, 129.41, 127.52, 124.75, 123.96, 120.14, 116.08, 112.62, 100.81, 58.00, 36.54, 20.60; LC-MS (APCI + ESI) m/z: 303 [M + H] +; Anal. Calcd for C18H14N4O: C, 71.51; H, 4.67; N, 18.53. Found: C, 71.69; H, 4.69; N, 18.61.

2-Amino-4-(4-methoxyphenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-3) Yield: 72%; off-white solid; mp: 223-226 °C; IR (KBr, cm-1) 3171, 2196, 1659, 1403, 1036, 734; 1H-NMR (400 MHz, DMSO-d6):d 7.67 (d, J = 6.8 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 7.08 (s, 2H), 6.90–6.86 (m, 3H), 5.12 (s, 1H), 3.72 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d160.38, 158.52, 149.33, 139.98, 132.74, 128.73, 124.73, 123.95, 120.18, 116.07, 114.17, 112.60, 100.92, 58.20, 54.99, 36.14; LC-MS (APCI + ESI) m/z: 319 [M + H] +; Anal. Calcd for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: C, 68.02; H, 4.44; N, 17.65.

2-Amino-4-(4-(dimethylamino)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-4) Yield: 71%; off-white solid; mp: 230-233 °C; IR (KBr, cm-1) 3169, 2194, 1657, 1406, 1036, 741; 1H-NMR (400 MHz, DMSO-d6):d 7.67 (d, J = 6.8 Hz, 1H), 7.53 (d, J = 9.2 Hz, 1H), 7.25 (t, J = 7.2 Hz, 1H), 7.05–7.01 (m, 4H), 6.87 (t, J = 6.8 Hz, 1H), 6.66 (d, J = 8.4 Hz, 2H), 5.03 (s, 1H), 2.86 (s, 6H); 13C-NMR (100 MHz, DMSO-d6): d160.24, 149.66, 149.24, 139.87, 128.17, 127.96, 124.58, 124.00, 120.30, 116.02, 112.47, 101.25, 58.57, 36.09; LC-MS (APCI + ESI) m/z: 332 [M + H] +; Anal. Calcd for C19H17N5O: C, 68.87; H, 5.17; N, 21.13. Found: C, 68.99; H, 5.18; N, 21.18.

2-Amino-4-(4-chlorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-5) Yield: 76%; off-white solid; mp: 248-251 °C; IR (KBr, cm-1) 3140, 2195, 1659, 1428, 1037, 738; 1H-NMR (400 MHz, DMSO-d6):d 7.70 (d, J = 6.8 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.42–7.40 (m, 2H), 7.31–7.26 (m, 3H), 7.12 (s, 2H), 6.88 (td, J = 6.8, 0.8 Hz, 1H), 5.22 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.59, 149.49, 140.14, 139.90, 132.08, 129.53, 128.86, 124.98, 123.96, 120.00, 116.15, 112.79, 100.25, 57.43, 36.25; LC-MS (APCI + ESI) m/z: 323 [M + H] +; Anal. Calcd for C17H11ClN4O: C, 63.26; H, 3.44; N, 17.36. Found: C, 63.40; H, 3.45; N, 17.41.

2-Amino-4-(4-fluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-6) Yield: 78%; off-white solid; mp: 236-239 °C; IR (KBr, cm-1) 3141, 2198, 1659, 1410, 1040, 737; 1H-NMR (400 MHz, DMSO-d6):d 7.69 (d, J = 6.8 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.30–7.26 (m, 3H), 7.19–7.16 (m, 4H), 6.89 (t, J = 6.8 Hz, 1H), 5.22 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.52, 149.43, 140.10, 137.09, 137.06, 129.65, 129.56, 124.92, 123.94, 120.05, 116.14, 115.75, 115.53, 112.73, 100.52, 57.75, 36.13; LC-MS (APCI + ESI) m/z: 307 [M + H] +; Anal. Calcd for C17H11FN4O: C, 66.66; H, 3.62; N, 18.29. Found: C, 66.76; H, 3.63; N, 18.35

2-Amino-4-(2,4-difluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-7) Yield: 80%; off-white solid; mp: 219-222 °C; IR (KBr, cm-1) 3138, 2192, 1660, 1412, 1039, 740; 1H-NMR (400 MHz, DMSO-d6):d 7.67 (d, J = 6.8 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.31–7.21 (m, 5H), 7.05 (t, J = 8.4 Hz, 1H), 6.92 (t, J = 6.8 Hz, 1H), 5.45 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d161.04, 149.72, 140.09, 131.17, 131.12, 124.92, 123.70, 123.61, 123.46, 119.89, 116.20, 112.96, 112.26, 112.04, 99.18, 55.97, 30.92; LC-MS (APCI + ESI) m/z: 325 [M + H] +; Anal. Calcd for C17H10F2N4O: C, 62.96; H, 3.11; N, 17.28. Found: C, 63.10; H, 3.12; N, 17.34.

2-Amino-4-(3-bromophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-8) Yield: 79%; off-white solid; mp: 242-245 °C; IR (KBr, cm-1) 3152, 2193, 1658, 1404, 1037, 739; 1H-NMR (400 MHz, DMSO-d6):d 7.74 (d, J = 6.8 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H), 7.49–7.47 (m, 2H), 7.33–7.28 (m, 2H), 7.23–7.21 (m, 3H), 6.92 (td, J = 6.8, 1.2 Hz, 1H), 5.22 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.68, 149.54, 143.76, 140.18, 131.13, 130.56, 130.32, 126.73, 125.07, 123.96, 122.08, 119.96, 116.19, 112.87, 100.08, 57.31, 36.46; LC-MS (APCI + ESI) m/z: 367 [M + H] +; Anal. Calcd for C17H11BrN4O: C, 55.61; H, 3.02; N, 15.26. Found: C, 55.73; H, 3.02; N, 15.30.

2-Amino-4-(4-cyanophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-9) Yield: 82%; off-white solid; mp: 247-250 °C; IR (KBr, cm-1) 3123, 2197, 1661, 1405, 1040, 750; 1H-NMR (400 MHz, DMSO-d6):d 7.82 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 6.4 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.32–7.26 (m, 3H), 6.89 (t, J = 6.8 Hz, 1H), 5.33 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.80, 149.66, 146.46, 140.27, 132.91, 128.74, 125.16, 123.96, 119.86, 118.53, 116.22, 112.91, 110.44, 99.73, 56.80, 36.79; LC-MS (APCI + ESI) m/z: 314 [M + H] +; Anal. Calcd for C18H11N5O: C, 69.00; H, 3.54; N, 22.35. Found: C, 69.15; H, 3.55; N, 22.42.

2-Amino-4-(4-nitrophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-10) Yield: 85%; light yellow solid; mp: 243-246 °C; IR (KBr, cm-1) 3126, 2198, 1662, 1405, 1041, 721; 1H-NMR (400 MHz, DMSO-d6):d 8.21 (d, J = 8.4 Hz, 2H), 7.73 (d,J = 6.8 Hz, 1H), 7.58–7.53 (m, 3H), 7.32–7.28 (m, 3H), 6.89 (t, J = 6.8 Hz, 1H), 5.41 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.84, 149.65, 148.45, 146.92, 140.33, 129.07, 125.24, 124.17, 124.03, 119.85, 116.25, 112.96, 99.71, 56.66, 36.58; LC-MS (APCI + ESI) m/z: 334 [M + H] +; Anal. Calcd for C17H11N5O3: C, 61.26; H, 3.33; N, 21.01. Found: C, 61.40; H, 3.34; N, 21.08.

Methyl 4-(2-amino-3-cyano-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridin-4-yl)benzoate (IMPA-11) Yield: 83%; off-white solid; mp: 245-248 °C; IR (KBr, cm-1) 3136, 2193, 1658, 1403, 1038, 728; 1H-NMR (400 MHz, DMSO-d6):d 7.94 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 6.8 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.30–7.26 (m, 1H), 7.23 (s, 2H), 6.87 (td, J = 6.8, 0.8 Hz, 1H), 5.30 (s, 1H), 3.83 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d165.86, 160.71, 149.54, 146.22, 140.19, 129.83, 128.93, 128.12, 125.04, 123.94, 119.92, 116.18, 112.81, 100.10, 57.12, 52.09, 36.78; LC-MS (APCI + ESI) m/z: 347 [M + H] +; Anal. Calcd for C19H14N4O3: C, 65.89; H, 4.07; N, 16.18. Found: C, 65.98; H, 4.09; N, 16.22.

2-Amino-4-(4-(trifluoromethyl)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-12) Yield: 81%; off-white solid; mp: 258-260 °C; IR (KBr, cm-1) 3136, 2198, 1661, 1405, 1068, 731; 1H-NMR (400 MHz, DMSO-d6):d 7.73–7.71 (m, 3H), 7.57 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 7.6 Hz, 2H), 7.31–7.24 (m, 3H), 6.90 (t, J = 7.2 Hz, 1H), 5.34 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.73, 149.62, 145.62, 140.24, 128.54, 125.86, 125.82, 125.10, 123.96, 122.76, 119.93, 116.20, 112.90, 99.97, 57.09, 36.60; LC-MS (APCI + ESI) m/z: 357 [M + H] +; Anal. Calcd for C18H11F3N4O: C, 60.68; H, 3.11; N, 15.72. Found: C, 60.81; H, 3.12; N, 15.76.

2-Amino-4-(pyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-13) Yield: 79%; off-white solid; mp: 234-237 °C; IR (KBr, cm-1) 3156, 2190, 1656, 1402, 1034, 741; 1H-NMR (400 MHz, DMSO-d6):d 8.57 (d, J = 1.6 Hz, 1H), 8.49 (dd, J = 1.2, 4.8 Hz, 1H), 7.75 (d, J = 6.8 Hz, 1H), 7.58–7.56 (m, 2H), 7.37–7.28 (m, 2H), 7.25 (s, 2H), 6.90 (t, J = 6.8 Hz, 1H), 5.28 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.75, 149.66, 148.99, 148.95, 140.22, 136.29, 135.27, 125.08, 124.15, 123.93, 119.97, 116.21, 112.87, 99.73, 57.05, 34.45; LC-MS (APCI + ESI) m/z: 290 [M + H] +; Anal. Calcd for C16H11N5O: C, 66.43; H, 3.83; N, 24.21. Found: C, 66.55; H, 3.84; N, 24.26.

2-Amino-4-(2-chloropyridin-4-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-14) Yield: 80%; off-white solid; mp: 235-238 °C; IR (KBr, cm-1) 3099, 2191, 1656, 1408, 1040, 737; 1H-NMR (400 MHz, DMSO-d6):d 8.37 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 6.8 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.49 (s, 1H), 7.35–7.30 (m, 3H), 7.24 (d, J = 4.4 Hz, 1H), 6.94 (t, J = 6.8 Hz, 1H), 5.29 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d161.04, 153.63, 150.85, 150.69, 149.80, 140.41, 125.36, 124.07, 123.23, 122.20, 119.71, 116.27, 113.09, 98.77, 55.84, 35.92; LC-MS (APCI + ESI) m/z: 324 [M + H] +; Anal. Calcd for C16H10ClN5O: C, 59.36; H, 3.11; N, 21.63. Found: C, 59.51; H, 3.13; N, 21.71.

2-Amino-4-(5-bromopyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile (IMPA-15) Yield: 83%; off-white solid; mp: 253-256 °C; IR (KBr, cm-1) 3137, 2192, 1654, 1406, 1034, 742; 1H-NMR (400 MHz, DMSO-d6):d 8.65 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 7.91 (t, J = 2.0 Hz, 1H), 7.81 (d, J = 6.8 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.34–7.30 (m, 3H), 6.93 (td, J = 6.8, 0.8 Hz, 1H), 5.31 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): d160.89, 149.79, 149.73, 147.64, 140.33, 138.61, 137.76, 125.25, 123.98, 120.65, 119.87, 116.26, 113.02, 99.09, 56.49, 34.15; LC-MS (APCI + ESI) m/z: 368 [M + H] +; Anal. Calcd for C16H10BrN5O: C, 52.19; H, 2.74; N, 19.02. Found: C, 52.29; H, 2.75; N, 19.08.

[0092] Example 2: Biological Evaluation
2.1 Anti-proliferative activity
[0093] To examine the cytotoxic effect of the compounds on human lung adenocarcinoma cells, A549 cells were treated with the fifteen newly synthesized compounds IMPA 1-15 at varied concentrations for 24h and the cell viability was analyzed by MTT assay using Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, protocol adopted by Mossmann et al., J. Immunol. Methods. 1983, 65, (1-2), 55-63. Treatment of A549 cells with these compounds significantly decreased cell viability in a dose-dependent manner; however, the cytotoxic effect of five compounds (IMPA-2,-5,-6,-8, and -12) was more profound compared to other compounds as indicated by the IC50 values of IMPA-2, -5, -6, -8, and -12 which were 40.1 ± 6.6 µM, 47.8 ± 9.1 µM, 48.7 ± 12.7 µM, 44 ± 6.8 µM, and 47.9 ± 11.6 µM, respectively.
[0094] Figure 1A provides the cell viability (%) of A549 cells with variation in concentration for the compounds IMPA-2,-5,-6,-8, and -12 at 24h. The effect of these five compounds was then investigated on the viability of normal human lung epithelial cells BEAS-2B and mouse fibroblast L929 cells, but no significant cell mortality was observed at concentrations of IC50values of five compounds (refer Figure 1B) indicating their cytotoxic specificity.
[0095] Based on these results, IMPA-2,-5,-6,-8, and -12 were considered for further studies. Cellular morphological changes are often considered markers associated with cell pathology. The morphological differences of A549 cancer cells in response to these compounds were determined. The micrographs indicated markedly altered epithelial morphology with decreased cell-cell contacts in A549 treated cells with the compounds as compared to control cells. Scanning electron microscopy (SEM) images corroborated this observation with the appearance of membrane blebbing and formation of apoptotic bodies in IMPA-2,-5,-6,-8, and -12 treated A549 cells (Figure 1C). To further confirm the anti-proliferative activity of these compounds on A549 cells, a clonogenic assay was performed. In contrast, to control cells, IMPA-2,-5,-6,-8, and -12 treatments significantly suppressed cancer cell growth as indicated by decreased colony numbers (Figures 1D and 1E). All these results suggested that the compounds of the present disclosure, particularly IMPA-2,-5,-6,-8, and -12 had promising antitumor activity against human lung adenocarcinoma cells.
2.2 Anti-metastatic activity
[0096] NSCLC is highly metastatic in nature and is associated with higher metastasis-related death approximately 90% of all lung cancer mortality. The effect of compounds IMPA-2,-5,-6,-8, and -12 on migratory and invasive capabilities of A549 cells were studied and thus performed wound healing, trans-well migration and invasion assays.
[0097] 2.2.1 Wound healing assay: A549 cells (1X105 cells/well) were seeded in 12-well cell culture plates and cultured until they reached 90% confluency. Cells were then serum-starved for 12 h and treated with mitomycin C (1 µg/ml) for 1 h to stop cell proliferation. A straight scratch area was created to simulate wound formation in each well using a sterile micropipette tip and washed once to remove non-adherent cells. Cells were then treated without or with the compounds (IMPA-2, 5, 6, 8 and 12) individually. Cells migrations on the wound surface were observed under a microscope and images were captured at various time points (0h, 4h, 8h, 12h and 24h). The percentage of wound closure was measured from 3 random fields for each well by calculating the width of the wound remaining compared to the initial wound width area (refer Figures 2A and 2B).
[0098] 2.2.2 Trans-well cell migration and invasion assays: Assays were performed using trans-well inserts containing 8.0 µm pores (HiMedia, India). The serum-starved A549 cells (5X104cells/well) were incubated without or with the compounds in a serum-free medium for 24 h. On termination of incubations, cells were trypsinised and re-suspended in serum-free media. For migration assay, cells were directly placed in the upper chamber of trans-well inserts; whereas, for invasion assay, cells were placed in the upper chamber of Matrigel-coated trans-well inserts. The lower chamber was filled with 10% FBS containing media in both cases and then incubated for 24 h. The upper surface of the membrane was gently scrubbed with a cotton swab, and the cells that migrated or invaded to the lower membrane surface were fixed with 2.5% glutaraldehyde for 10 min and stained with 0.5% crystal violet solution for 2 h. The bright-field images of migrated and invaded cells were captured by a microscope (Nikon, Japan) at a magnification of 200x (refer Figure 2C and 2E). The migrated and invaded cell numbers also counted for at least 5 random fields for each membrane (refer Figure 2D and 2F).
[0099] Treatment of A549 cells with IMPA-2,-5,-6,-8, or -12 notably inhibited wound healing rate (Figures 2A,B) and cellular migration (Figures 2C,D) as compared to control untreated cells. A significant reduction of invasive cell numbers when A549 cells were treated with IMPA-2,-5,-6,-8, and -12 in comparison to control cells was also noted (Figures 2E,F). These results indicate that the compounds can counter the NSCLC invasion and metastasis abilities.
[00100] 2.3 Induction of apoptotic activity
[00101] To determine whether the inhibition of A549 cells proliferation by the compounds is due to the induction of apoptosis, apoptotic markers by flow cytometry were investigated. Apoptosis assessment was conducted using flow cytometry to determine the phosphatidylserine exposed apoptotic cells by Annexin V–FITC and propidium iodide (PI) double staining (FITC-Annexin V apoptosis detection kit, BD Biosciences) following the manufacturer's protocol used by Ravi Hingorani et al., BD Bioscience Detection of Apoptosis Using the BD Annexin V FITC Assay on the BD FACSVerse™ System BD Biosciences 2011. Dot-plot graphs were used to illustrate the viable cells (the lower left quadrant), early-phase apoptotic cells (the lower right quadrant), late-phase apoptotic or dead cells (the upper right quadrant), and the necrotic cells (the upper left quadrant).
[00102] Flow cytometric analysis of FITC-Annexin V/ Propidium Iodide (PI) revealed that treatment of A549 cells with IMPA-2,-5,-6,-8 and -12 for 24h significantly decreased the percentage of live cells along with the induction of early and late apoptotic cells as compared to control cells (Figures 3A, B).
[00103] To investigate further apoptotic cell death by the compounds, mitochondrial transmembrane potential (??m) as loss of ??m was measured which is a common and early feature of the intrinsic pathway of cellular apoptosis protocol adopted from Lakhani, S.A. et al., Science, 2006, 311(5762), 847-851. The JC-1 staining was performed to assess the loss ??m by fluorescence microscopy in A549 cells in response to compound treatments. JC-1 aggregates in the mitochondrial matrix and emits red fluorescence when the membrane potential is high; compared to mitochondria with lower membrane potential which emanate green fluorescence. Treatment with IMPA-2,-5,-6,-8 and -12 notably increased green fluorescence and reduced red fluorescence in A549 cells compared to untreated control cell (Figure 3C). This result revealed that compounds compromise mitochondrial membrane potential in A549 cells.
[00104] It has been reported that increased production of reactive oxygen species (ROS) lead to the mitochondrial membrane depolarization and mitochondrial damage which play a pivotal role in contributing apoptotic cell death at the levels beyond the cellular antioxidant defence mechanisms (F. Sivandzade, A. Bhalerao, L. Cucullo, Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe, Bio-Protocol. 9 (2019)). The intracellular ROS levels were studied by measuring the intensity of fluorescence generated from DCFDA through the cellular redox reactions. Detection of intracellular reactive oxygen species (ROS) is performed using DCFDA (#D6883, Sigma) fluorescent probe following the previously described method adopted from Chompoosor, A et al.,Small, 2010, 6(20), 2246-2249. Treatment of A549 cells with IMPA-2,-5,-6,-8 and -12 significantly increased the fluorescence intensity suggesting substantial production of intracellular ROS compared to control cells (Figure 3D). To determine the possible reasons of ROS mediated inhibition of ??m, the gene expression profile of Bax and Bak (pro-apoptotic markers) and Bcl2 (anti-apoptotic marker) was studied in IMPA-2,-5,-6,-8 and -12 treated A549 cells which are involved in the regulation of mitochondrial membrane permeability. Gene expression was analyzed by two-step qRT–PCR. Total RNA was extracted from the cells of different incubations using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instruction. Wagner, et al., Monitoring gene expression: quantitative real-time rt-PCR. Lipoproteins and Cardiovascular Disease. Humana Press, Totowa, NJ, 2013. The results demonstrated that incubations of these compounds significantly increase Bax and Bak1 gene expression with a concomitant decrease of Bcl2 (Figure 3E, 3F) augmenting the ratio of pro-apoptotic/anti-apoptotic gene expression. Induction of Bax/Bcl2 ratio directs the release of mitochondrial cytochrome c that facilitates intrinsic apoptotic pathway of caspase activation which subsequently degrades its cellular targets favoring A549 cancer cell apoptosis.
[00105] The caspase activation and its downstream target in IMPA treated A549 cells were examined. IMPA-2,-5,-6,-8 and -12 treatments notably increased the activated cleaved caspase 9 and cleaved caspase 3 levels in A549 cells (Figure 3G), however, pre-treatment of N-acetyl-L-cysteine (NAC) markedly prevents IMPAs effected cleavages of caspase-9 and caspase-3 along with a significant down-regulation of pro-apoptotic genes in A549 cells (Figures 3G-H). Taken together, these results implied that the compounds induced ROS production implements apoptotic cell death of A549 lung cancer cells. Figure 3I provides the relative protein expression of cleaved caspase 3, and 9 by western blot in NAC treatment followed by compound treated cells.
[00106] 2.4 Inhibition of cell cycle progression
[00107] To investigate the effect of the compounds on cell cycle progression in A549 lung cancer cells, cell cycle distribution by flow cytometry was analyzed using BD CycletestTMPlus DNA kit, protocol adopted from Gray, et al., Techniques in cell cycle analysis. Springer Science & Business Media, 1987. Cell populations in G0/G1 phase were markedly increased with a concomitant decrease of similar proportions of S phase populations in IMPA-2,-5,-6 and -12 treated A549 cells. Intriguingly, IMPA-8 did not increase the G0/G1 cell populations, instead, it enhanced G2/M phase cell populations (Figure 4A, B). This result confers that while IMPA-2,-5,-6 and -12 incubations repressed NSCLC growth through blocking G1/S transition, the IMPA-8 targets cell cycle arrest of NSCLC at the G2/M phase.
[00108] Since p38 MAPK signalling regulates both G1/S as well as G2/M cell cycle checkpoints in response to cellular stress such as ROS and DNA damage, p38 MAPK activation was checked by western blot analysis. Significant activation of p38 MAPK was noticed in IMPA-2,-5,-6,-8 and -12 treated A549 cells (Figure 4C). Activated p38 is known to associate with the stability and activation of p53, a tumor suppressor that regulates cell cycle checkpoints. Incubation of A549 cells with IMPA-2,-5,-6,-8 and -12 markedly increased p53 (Figure 4D) and its downstream p16INK4A, p21Cip1 and p27Kip1 (Figure 4E) gene expression and thus promoting cell cycle arrest by targeting CDK inhibition. Pre-treatment with NAC significantly waived the compound’s effect on p53 expression in A549 cells (Figure 4F). These findings confirm the mechanistic insights of ROS mediated A549 cell cycle arrest by the compounds.
[00109] 2.5 Inhibition of proliferation of 3D multicellular spheroid
[00110] 3D multi-cellular tumor spheroids can reflect in-vivo features of a tumor microenvironment better than 2D monolayers, therefore, the effect of different compounds on the 3D multi-cellular tumor spheroid model was analyzed. 3D multi-cellular tumor spheroids A549, MRC-5 and were generated by following the hanging drop plate method adopted from Ware, et al., Tissue Engineering Part C: Methods, 2016, 22 (4), 312-321. Phase-contrast imaging demonstrated a promising effect of the compounds on lung tumor spheroids size and nature. Spheroids were sensitive to the compounds as they showed a reduction in spheroids size and diameter when treated for 24h and a marked enhancement of spheroids disintegration at 48 h (Figure 5A).
[00111] To examine the viability of cells in multi-cellular (A549, MRC-5 and THP-1) tumor spheroids treated without or with different compounds for 24h and 48h followed by the determination of viability and cytotoxicity with the live/dead assay of 3D-multicellular spheroid protocol adopted from Patrick et al., Journal of neuroscience methods. 1997, 71(2), 205-213. Live/dead assay indicates a striking reduction of viable cells with the notable appearance of dead cells in IMPA-2, -5, -6, -8 and -12 treated tumor spheroids at 24 h and 48 h compared to the controls (Figure 5B). To demonstrate the effect of these compounds on tumor cell death is due to apoptosis, FITC-Annexin V/Propidium Iodide (PI) staining was performed by flow cytometry. As shown in Figure 5C, D; the compounds’ treatments significantly decreased the percentage of live cells with a concomitant increase of early and late apoptotic cells in tumor spheroids compared to the untreated controls both at 24 h and 48 h. Results indicate that the compounds effectively reduced lung cancer cells growth and viability in 3D multi-cellular lung tumor spheroids.
[00112] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

We Claims:

1. An anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

Formula I
wherein R1 is selected from (C6-C14)aryl or (C5-C10)heterocyclyl;
wherein R1 is unsubstituted or substituted with one or more of halogen, nitro, (C1-C6)alkyl, amino, -NR2R3, -CN, (C1-C6)alkoxy, -SO2, -COOH, hydroxy, or -COOR2; and
wherein R2 and R3¬ is independently selected from (C1-C6)alkyl.
2. The compound as claimed in claim 1, wherein R1 is substituted with one or more of F, Cl, Br, nitro, -CN, ethyl, methyl, trifluoromethyl, trichloromethyl, tribromoethyl, methoxy, ethoxy, -N(CH3)2, -N(C2H5)2, -COOH, -COOC2H5, or –COOCH3.
3. The compound as claimed in claim 1, wherein the (C1-C6)alkyl group is unsubstituted or substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, -CN, nitro and amino.
4. The compound as claimed in claim 1, wherein the (C6-C14)aryl is selected from phenyl, or naphthyl; and wherein the (C6-C14)aryl is unsubstituted or substituted with one or more of Cl, F, Br, nitro, -CN, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, -N(CH3)2, or -COOCH3.
5. The compound as claimed in claim 1, wherein the (C5-C10) heterocyclyl is pyridine; and wherein (C5-C10) heterocyclyl is substituted or unsubstituted with one or more of Cl, or Br.
6. The compound as claimed in claim 1, wherein the compound is selected from:
2-Amino-4-phenyl-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(p-tolyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-methoxyphenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-(dimethylamino)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-chlorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-fluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2,4-difluorophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(3-bromophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-cyanophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(4-nitrophenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
Methyl 4-(2-amino-3-cyano-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridin-4-yl)benzoate;
2-Amino-4-(4-(trifluoromethyl)phenyl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(pyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(2-chloropyridin-4-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
2-Amino-4-(5-bromopyridin-3-yl)-4H-pyrano[2',3':4,5]imidazo[1,2-a]pyridine-3-carbonitrile;
a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
7. A pharmaceutical composition comprising an anti-cancer pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof and one or more pharmaceutically acceptable excipient(s).
8. A method of synthesizing a compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein the method comprises the steps of:
(a) reacting 2-aminopyridine (1) with ethyl 2-bromoacetate to give 2-amino-1-(2-ethoxy-2-oxoethyl)pyridin-1-ium (2) and cyclizing it to give imidazo[1,2-a]pyridin-2-ol (3); and (b) reacting imidazo[1,2-a]pyridin-2-ol (3) of step (a) with malononitrile (4) and an aldehyde of Formula 5 to give the compound of Formula I.