The present disclosure relates generally to pharmaceutical compounds. More specifically, the disclosure is directed to anti-cancer pyrido[2,3-a]carbazole compounds of Formula I, their stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, or mixtures thereof and a pharmaceutical composition comprising them. The disclosure also provides a process of synthesizing 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] According to World Health Organization (WHO) reports, cancer is the second leading cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer. Deaths due to cancer are likely to rise to over 13.1 million by 2030. Therefore, anti-cancer drugs are a key field of research. Pyridocarbazoles are an interesting class of antitumor agents which bind intercalatively with DNA nucleobases via 71-71* stacking, but did not gain significant importance due to their toxicity to normal cells because of their biological features. Ellipticine and its synthetic derivatives based on the linear annulated carbazole (ellitpicine acetate, olivacince, datellitium, retellipticine, pazellipticine, etc) are reported to have cytotoxic and antitumor activities due to their planar and aromatic structural features which facilitate binding interaction with DNA intercalatively. However, there is a need to find compounds with improved aromaticity, planarity and hydrophobicity for better anti-cancer activity.
[0004] The inventors of the present disclosure provide compounds with higher aromaticity, planarity and hydrophobicity for improved anti-cancer activity.

OBJECTS OF THE INVENTION
[0005] An object of the present disclosure is to provide novel compounds that
exhibit improved anti-cancer activity.
[0006] An object of the present disclosure is to provide anti-cancer pyrido[2,3-
ajcarbazole compounds with improved aromaticity, planarity, and hydrophobicity.
[0007] Another object of the present disclosure is to provide anti-cancer
compounds that also possess anti-microbial activity.
[0008] Yet another object of the present disclosure is to provide a process of
synthesizing the anti-cancer compounds.
SUMMARY OF THE INVENTION
[0009] 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.
[0010] In an aspect, the present disclosure provides an anti-cancer pyrido[2,3-ajcarbazole compound of Formula I, its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof, or mixtures thereof,

O
Formula I wherein Ar may be C6-C14 aryl, or C5-C14 heterocyclyl; wherein Ar may be unsubstituted or substituted with one or more of halogen, C1-C6 alkyl, C1-C6

alkoxy, cyano, nitro, -NH2, -N(R1)2, -COOH, -COOR1, or -OH; and wherein R1
may be a C1-C6 alkyl.
[0011] In an embodiment, the Ar may be phenyl; wherein the phenyl may be
unsubstituted or substituted with one or more of CI, Br, methyl, methoxy, nitro, or
-N(CH3)2.
[0012] In an embodiment, the Ar may be unsubstituted furanyl, thiophenyl,
pyrrolyl, orpyridinyl.
[0013] In an embodiment, the compound may be:
8-methyl-2,4-diphenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Bromo-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(2-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-p-tolyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Methoxy-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-
a]carbazole;
8-Methyl-4-(4-nitro-phenyl)-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
Dimethyl-[4-(8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole-4-yl)-
phenyl]-amine;
4-Furan-2-yl-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-thiophene-2-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-(lH-pyrrolo-2-yl)-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-pyridin-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof,
or mixtures thereof.
[0014] In an aspect, the present disclosure provides a pharmaceutical composition
comprising a compound of Formula I, its stereoisomer, a tautomer, a solvate, a
pharmaceutically acceptable salt thereof, or mixtures thereof; and one or more
pharmaceutically acceptable excipient(s)

H,C

Formula I wherein Ar is as defined earlier.
[0015] In an aspect, the present disclosure provides a process of synthesizing compounds of Formula I using a one-pot synthesis, wherein the process comprises the step of reacting a 8-methyl-l,2,3,4-tetrahydro-9H-carbazole-l-one (1) with N-phenacylpyridinium bromide (2), aldehyde (3) and ammonium acetate (4) in presence of an organic solvent followed by microwaving to yield a compound of Formula I as shown in general Scheme I.

Fonnula I

CH3COO-XHj+(4)
+ Ar-CHO ^

H,C

Scheme I
where Ar is as defined previously.
[0016] In an embodiment, the organic solvent may be acetic acid.
[0017] 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
[0018] 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.
[0019] Figure 1 provides Electronic spectra of compounds (A) 5a, (B) 5b,(C) 5c, (D) 5d, (E) 5e, (F) 5f, (G) 5g, (H) 5h, (I) 5i, (J)5j, (K)5k and (L) 51, wherein the compounds are as per an embodiment of the present disclosure, in Tris-HCl buffer upon addition of ct-DNA. Arrow shows the absorption intensities decrease upon increasing DNA concentration.
[0020] Figure 2 provides plot between [DNA] and [DNA]/[£a-£b]X 10"8 for compounds (A) 5a, (B) 5b,(C) 5c, (D) 5d, (E) 5e, (F) 5f, (G) 5g, (H) 5h, (I) 5i, (J)5j, (K)5k and (L) 51, wherein the compounds are as per an embodiment of the present disclosure.
[0021] Figure 3 provides effect of increasing amounts of compounds (5a-51), as per an embodiment of the present disclosure, on the relative viscosity r|/ r|o of ct-DNA.
[0022] Figure 4 provides Cyclic voltammogram of compound 5g, as per an embodiment of the present disclosure, in the (a) absence and (b) presence of ct-DNA with scan rate of 10 mV/s.
[0023] Figure 5 provides photographic images showing morphological changes in cancer cells (MCF-7) induced by varying concentrations: (a) 0.1 uM; (b)l uM; (c) 10 uM and (d) 50 uM of compound 5g as per an embodiment of the present disclosure.
[0024] Figure 6 provides photographic images showing morphological changes in cancer cells (HeLa) induced by varying concentrations: (a)0.1 uM; (b)l uM; (c) 10 uM and (d) 50 uM of compound 5g as per an embodiment of the present disclosure.
[0025] Figure 7 provides plot of percentage (%) inhibition in growth of cancer cell with various concentrations of the compounds (5b, 5c, 5d, 5f, 5g, 5i, 5j, 5k,

and 51), as per an embodiment of the present disclosure, against MCF-7 cancer cell line.
[0026] Figure 8 provides plot of the percentage (%) inhibition in growth of cancer cell with various concentrations of the compounds (5b, 5c, 5d, 5f, 5g, 5i, 5j, 5k, and 51), as per an embodiment of the present disclosure, against HeLa cancer cell line.
DETAILED DESCRIPTION OF THE INVENTION
[0027] 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.
[0028] 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. [0029] 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 features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. [0030] 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 considering 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. [0031] 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.
[0032] 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. [0033] 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."
[0034] 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. [0035] 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. [0036] 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.
[0037] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of 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.
[0038] 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. [0039] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. [0040] 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.
[0041] The term "or", as used herein, is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

[0042] 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. [0043] 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. [0044] 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.
[0045] The term 'C6-C14 aryl' or "aryl" as used herein refers to monocyclic or bicyclic hydrocarbon groups having 6 to 14 ring carbon atoms, wherein at least one carbocyclic ring is having a 71 electron system. Examples of (C6-C14) aryl ring systems include, but are not limited to, phenyl and naphthyl. Unless indicated otherwise, aryl group can be unsubstituted or substituted with one or more substituents, for example 1 -4 substituents independently selected from the group consisting of halogen, C1-C6 alkyl, hydroxy, cyano, nitro, -COOH, amino and Ci-C6 alkoxy.
[0046] The term, 'C5-C14 heterocyclyl', as used herein refers to a 5- to 14-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 contains 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 invention. 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 andtetrazole. Unless stated otherwise, C5-C10 heterocyclyl can be unsubstituted or substituted with one or more substituents, for example, substituents independently selected from the group consisting of oxo, halogen, hydroxy, cyano, nitro,amine, Ci-Ce alkyl and COOH.
[0047] The term, "halogen" as used herein refers to chlorine, fluorine, bromine or iodine atom.
[0048] The term, "therapeutically effective amount" as used herein refers to an amount of a compound of Formula (I) or a composition comprising a compound of Formula (I) effective in producing the desired therapeutic response in a particular patient (subject) suffering from a disease or disorder. [0049] The term "pharmaceutically acceptable excipient(s)" as used herein refers to a diluent, binder, disintegrant, glidant, lubricant, coating material or the like, which is non-toxic, and inert, which does not have undesirable effects on a subject to whom it is administered and is suitable for delivering a therapeutically active agent to the target site without affecting the therapeutic activity of the said agent.

[0050] 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. [0051] 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, i.e., cancer. Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0052] Aspects of the present disclosure provide a series of pyrido[2,3-a]carbazole compounds with anti-cancer and anti-microbial activity. The disclosure also provides a process of their synthesis by microwave one-pot condensation.
[0053] In an embodiment, the present disclosure provides an anti-cancer pyrido[2,3-a]carbazole compound of Formula I, its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof, or mixtures thereof,

Formula I wherein Ar may be C6-C14 aryl, or C5-C14 heterocyclyl; wherein Ar may be unsubstituted or substituted with one or more of halogen, C1-C6 alkyl, C1-C6 alkoxy, cyano, nitro, -NH2, -N(R1)2, -COOH, -COOR1, or -OH; and wherein R1 may be a C1-C6 alkyl.

[0054] In an embodiment, the Ar may be phenyl or napthyl; wherein the Ar may
be unsubstituted or substituted with one or more of F, CI, Br, methyl, ethyl,
propyl, methoxy, ethoxy, propoxy, cyano, nitro, -NH2, -N(CH3)2, -N(C2Hs)2, -
COOH, -COOCH3, -COOC2H5 or OH.
[0055] In a preferred embodiment, the Ar may be phenyl; wherein the phenyl may
be unsubstituted or substituted with one or more of CI, Br, methyl, methoxy, nitro,
or -N(CH3)2.
[0056] In an embodiment, the Ar may be furanyl, thiophenyl, pyrrolyl, or
pyridinyl; wherein the Ar may be unsubstituted or substituted with one or more of
F, CI, Br, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, cyano, nitro, -NH2, -
N(CH3)2, -N(C2H5)2, -COOH, -COOCH3, -COOC2H5 or OH.
[0057] In a preferred embodiment, the Ar may be unsubstituted furanyl,
thiophenyl, pyrrolyl, or pyridinyl.
[0058] In an embodiment, the compound may be:
8-methyl-2,4-diphenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Bromo-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(2-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-p-tolyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Methoxy-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-
a]carbazole;
8-Methyl-4-(4-nitro-phenyl)-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
Dimethyl-[4-(8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole-4-yl)-
phenyl]-amine;
4-Furan-2-yl-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-thiophene-2-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-(lH-pyrrolo-2-yl)-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-pyridin-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof,
or mixtures thereof.

[0059] 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 invention are prepared in a manner
known to one skilled in the art. Pharmaceutically acceptable salts of the
compound of the present invention include but are not limited to, an acid salt of a
compound of the present invention 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, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.
[0060] In yet another embodiment, the pharmaceutically acceptable salts of the
compound of the present invention 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.

[0061] In an embodiment, the solvate includes hydrates such as monohydrate,
dihydrate, trihydrate, sesquihydrate, or combinations thereof.
[0062] The compounds of the present disclosure have improved planarity and
hydrophobicity compared to Ellipticine. The compounds display anticancer
activity, i.e., cytotoxicity towards cancer cell lines. The activity of the compounds
is comparative to standard anti-cancer drugs - Cisplatin and Ellipticine. In some
embodiments, the compounds display cytotoxicity towards breast cancer cells and
cervical cancer cells.
[0063] The anti-cancer activity of the compounds is due to their strong binding
with ct-DNA via intercalation. In some embodiments, the compounds with higher
hydrophobicity show higher anti-cancer activity due to increase in binding with
ct-DNA.
[0064] The compounds are also anti-microbials. In some embodiments, the
compounds show anti-microbial effect against Enterococcus faecalis and
Klebsiella pneumonia. In some embodiments, the compounds show equipotent
anti-microbial efficacy compared to standard drug - Ciprofloxacin.
[0065] In an embodiment, the present disclosure provides a pharmaceutical
composition comprising a compound of Formula I, its stereoisomer, a tautomer, a
solvate, a pharmaceutically acceptable salt thereof, or mixtures thereof; and one or
more pharmaceutically acceptable excipient(s).

O
Formula I wherein Ar is as defined earlier.

[0066] The present disclosure also relates to a process for the production of the pharmaceutical composition, which includes bringing a compound of Formula (I), into a suitable administration form using a pharmaceutically acceptable excipient and, if appropriate, further suitable pharmaceutically acceptable carriers, additives or auxiliaries. 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.
[0067] In an embodiment, the pharmaceutical compositions can be administered orally, for example in the form of pills, tablets, suspension, solution, powders, coated tablets, capsules, granules or elixirs. Administration, however, can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injectable sterile solutions or suspensions, or topically, for example in the form of ointments or creams or transdermally, in the form of patches, or in other ways, for example in the form of aerosols or nasal sprays.
[0068] For the production of oral dosages form of the compound of Formula (I) such as the pills, tablets, coated tablets and hard gelatin capsules, it is possible to use, for example, lactose, corn starch or compounds thereof, gum arabica, magnesia or glucose, etc. Pharmaceutically acceptable excipients that can be used for soft gelatin capsules and suppositories are, for example, fats, waxes, natural or hardened oils, etc. Suitable pharmaceutically acceptable excipients for the production of solutions, for example injection solutions, or of emulsions or syrups are, for example, water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the said solvents.
[0069] 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) or its pharmaceutically acceptable salt. The amount of the compound of Formula (I) or its pharmaceutically acceptable salt in the pharmaceutical compositions 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, can cover a wide range depending on the type of disease or disorder to be treated. The dose to be administered daily is to be selected to suit the desired effect. A suitable dosage is about 0.01 to 100 mg/kg of the compound of Formula (I) or its pharmaceutically acceptable salt depending on the body weight of the recipient (subject) per day, for example, about 0.1 to 50 mg/kg/day of a compound of Formula (I) or a pharmaceutically acceptable salt of the compound. If required, higher or lower daily doses can also be administered.
[0070] The selected dosage level will depend upon a variety of factors including the activity of a compound of the present invention, or its salt employed, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of the treatment, other concurrently administered drugs, compounds and/or materials, the age, sex, weight, condition, general health and prior medical history of the patient (subject) being treated, and like factors well known in the medical arts.
[0071] In addition to the compound of Formula (I) or its pharmaceutically acceptable salt and the pharmaceutically acceptable carrier substances, the pharmaceutical compositions of the present invention can contain additives such as, for example, fillers, antioxidants, dispersants, emulsifiers, defoamers, flavors, preservatives, solubilizers or colorants. Furthermore, in addition to a compound of Formula (I) or its pharmaceutically acceptable salt, the pharmaceutical compositions can also contain one or more other therapeutically or prophylactically active agents.
[0072] The present disclosure also encompasses within its scope the use of a compound of Formula (I) or its pharmaceutically acceptable salt in combination, with other therapeutically active agents.
[0073] In an embodiment, the combination of compound of present disclosure with another therapeutic agent or treatment includes co-administration of a compound of Formula (I) with the other therapeutic agent or treatment as either a single combination dosage form or as multiple, separate dosage forms, administration of the compound of the present invention 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 invention. Further
therapeutic agents are administered either simultaneously or sequentially.
[0074] In another embodiment of the present invention, 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. The selection of other therapeutic agent(s) is
based upon the particular disease or disorder being treated. Such choice is within
the knowledge of a treating physician. Furthermore, the additional therapeutic
agent may be any agent when administered in combination with the
administration of a compound of the present invention provides benefit to the
subject in need thereof.
[0075] In an embodiment, the present disclosure provides a compound of Formula
I, its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt
thereof, or mixtures thereof for use in treatment of cancer.
[0076] In an embodiment, the present disclosure provides a medicament
comprising the compound of Formula I, its stereoisomer, a tautomer, a solvate, a
pharmaceutically acceptable salt thereof, or mixtures thereof.
[0077] In another embodiment, the present disclosure provides a process of
synthesizing the compounds of Formula I using a one-pot synthesis.
[0078] In an embodiment, the process of synthesizing compounds of Formula I
using a one-pot synthesis, comprises the step of reacting a 8-m ethyl-1,2,3,4-
tetrahydro-9H-carbazole-l-one (1) with N-phenacylpyridinium bromide (2),
aldehyde (3) and ammonium acetate (4) in presence of an organic solvent
followed by microwaving to yield a compound of Formula I as shown in Scheme
I


Formula I
o
Tv?-V

Ar-CHO ^

Scheme I
where Ar is as defined previously.
[0079] In an embodiment, the organic solvent may be acetic acid.
[0080] The process is a one-pot synthesis that gives good yields. In some
embodiments, the yield may be up to above 60%, above 70%, above 80% or
above 90%; preferably the yield may be about 86%.
[0081] The efficiency of microwave heating is that it drastically reduces the
reaction time (reduced from days to hours, hours to minutes or minutes to
seconds). In some embodiments, the process recited above may be microwaved
for about 3 to 15 minutes.
[0082] The process also avoids use of poisonous and expensive solvents and as
such can be environmentally benign and makes manipulation much easier. The
process also avoids exposure to such poisonous chemicals for humans.
[0083] In another embodiment, the present disclosure provides a method of
treatment, amelioration or prophylaxis of cancer comprising administering a
therapeutically effective amount of the compound of Formula (I) or a
pharmaceutical composition as recited above to a subject in need thereof.
[0084] In another embodiment, the present disclosure provides a method of
treatment, amelioration or prophylaxis of an anti-microbial infection comprising
administering a therapeutically effective amount of the compound of Formula (I)
or a pharmaceutical composition as recited above to a subject in need thereof.
[0085] 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
[0086] The present invention is further explained in the form of following
examples. However, it is to be understood that the following examples are merely
illustrative and are not to be taken as limitations upon the scope of the invention.
Example 1: Synthesis and characterization of compounds
[0087] All the compounds were synthesized according to Scheme II below.
Reaction of 8-methyl-l,2,3,4-tetrahydro-9H-carbazole-l-one (1) with N-
phenacylpyridinium bromide (2), aldehyde (3a-31) and ammonium acetate (4) in
presence of acetic acid which yield 8-methyl-2,4-diphenyl-5,6-dihydro-llH-
pyrido[2,3-a]carbazole (5a-51), wherein a-1 define compounds with different Ar
groups.


H,C
Ar-CHO
3a-3l

(4) CH3COONH4+
AcOH, MW, 3-10 min, Yield 56-86%

H,C

Scheme II [0088] General procedure for synthesis of compounds (5a-51): To a 50 mL
flask 6-methyl-2,3,4,9-tetrahydro-lH-carbazole-l-one (1 mmol), N-
phenacylpyridinium bromide (1.2 mmol), aromatic appropriate aldehyde (1.0 mmol), ammonium acetate (3.0 g) and acetic acid (2.0 mL) were added. The mixture was heated in a microwave for about 3-10 minutes (Biotag microwave oven, 90 °C). After cooling, the reaction mixture was diluted with water (50 mL) and the resulting precipitate was collected by filtration. The crude product was recrystallized from ethanol to give the pure solid sample for analysis. The same reaction was carried out with appropriate substituted aldehydes as represented in

Table 1. Table 1 also provides the characterization data, structure and yields of the compounds.
[0089] The IR spectrum of compound 5a displayed absorption bands at 3420 cm"1 (asym, NH), 3340 cm"1 (sym, NH), 2923 cm"1 (aromatic, C-H) and 1644 cm"1 (C=N). In !H NMR spectrum, the carbazole NH proton resonates at S 11.45. The singlet peak appeared at 8 2.38 indicating the presence of methyl protons. The C6-CH2 and C5-CH2 signal appeared as two multiplets at S 2.91 and S 3.04 respectively. The aromatic protons appeared in the expected regions. The total number of protons matched perfectly with its structure. The 13C NMR spectrum revealed the presence of all carbons in 5a.
Table 1: Synthesized pyrido[2,3-a]carbazole compounds

Structure Ti me (mi
n) Yield
(%) Characterization
H3C\^^ /\ ^\ 3 63 Yellow solid; yield: m.p. 237 °C;
FT-IR (KBr, cm"1, v): 3420, 2923, 2360 and 1644 (C=N); ^-NMR (5, ppm, DMSO-de ): 11.45 (s, IH, Nn-H); 8.63-6.99 (m, 14H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 4", 5", 6" -Aromatic); 3.04 (m, 2H, H-5); 2.91 (m, 2H, H-6); 2.38 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-de): 164.88, 159.58, 157.58, 145.01,144.57, 142.36, 140.22, 136.41, 133.18, 128.54, 128.31, 128.13, 125.41, 120.24,116.36, 115.38, 114.67, 112.54, 112.46,30.66,24.67,21.04; MS: m/z (%) 386.23 (M+, 100); Anal. Calcd. For C28H22N2: C, 87.01; H, 5.74; N, 7.25; Found: C, 87.04; H, 5.71;N,7.25%.
t 1 1 1 1
H M
Compound 5a

H3C^/^ ^\ ^?\/CI 5 82 Orange solid; m.p. 240 °C;
FT-IR (KBr, cm"1, v): 3422,
2925, 2360 and 1641
(C=N);
!H-NMR (5, ppm, DMSO-
d6 ): 11.46 (s, IH, Nn-H);
8.35-6.99 (m, 13H, 2', 3',
4', 5', 6', 3,7,9,10, 2", 3",
5", 6" - Aromatic); 3.03 (m,
2H, H-5); 2.91 (m, 2H, H-
6); 2.38 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-
d6): 164.90, 158.53,
157.10,150.06,
145.01,143.71, 142.41,
140.22,136.42, 133.18,
129.51, 128.20, 128.11,
125.55, 120.14, 116.36,
115.38, 114.67, 112.14,
112.77,111.00,30.66,24.87,
21.10.
MS: m/z (%) 420.17 (M+,
100);
Anal. Calcd. For
C28H21CIN2: C, 78.89; H,
5.03; N, 6.66; Found: C,
78.91; H, 5.01; N,6.64%.
XXXXU
Compound 5b

H3^^^ /\ ^^\^Br 7 74 Yellow solid; m.p. 260 °C; FT-IR (KBr, cm"1, v): 3432, 2930, 2361 and 1640 (C=N);
!H-NMR (5, ppm, DMSO-d6 ): 11.47 (s, IH, Nn-H); 8.37-6.92 (m, 13H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 5",6" - Aromatic); 3.10 (m, 2H, H-5); 2.92 (m, 2H, H-6); 2.40 (bs, 3H, CH3) 13C NMR (5, ppm, DMSO-d6): 165.10, 158.62, 157.50,151.07, 145.01,144.57, 142.36,140.32,135.31, 133.18, 128.24, 127.10, 128.12, 125.40, 120.24, 116.30, 115.38, 114.67,
Compound 5c

112.32, 112.21,111.00,
30.68,24.67,21.08.
MS: m/z (%) 464.09 (M+,
100);
Anal. Calcd. For
C28H2iBrN2: C, 72.26; H,
4.55; N, 6.02;Found: C,
72.28; H, 4.55; N,6.08%.
H3C\^^ /\ ^\ 5 78 Orange solid; m.p. 232 °C; FT-IR (KBr, cm"1, v): 3377, 2925, 1640 (C=N) and 735; !H-NMR (5, ppm, DMSO-d6 ): 11.48(s, IH, Nn-H); 8.34-6.91 (m, 13H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 4", 5" - Aromatic); 3.08 (m, 2H, H-5); 2.91 (m, 2H, H-6); 2.38 (bs, 3H, CH3); 13C NMR (5, ppm, DMSO-d6): 164.78, 160.20, 157.58,150.00, 145.01,144.57, 143.31, 140.22,136.40, 133.20, 128.54, 128.13, 128.12, 124.00, 120.24, 116.35, 115.38, 114.67, 112.45, 112.34,111.04,30.81,24.97, 21.09.
MS: m/z (%)420.14 (M+, 100);
Anal. Calcd. For C28H21CIN2: C, 79.89; H, 5.03; N, 6.66;Found: C, 79.90; H, 5.01; N,6.62%.
t 1 1 1 1
H >U A.
Compound 5d

H3C\/^ ^\ ^\^CH3 4 70 Yellow solid; m.p. 197 °C; FT-IR (KBr, cm"1, v): 3378, 2942, 1636 (C=N); !H-NMR (5, ppm, DMSO-d6 ): 11.49(s, IH, Nn-H); 8.39-6.91 (m, 13H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 5", 6" -Aromatic); 3.12 (m, 2H, H-5); 2.91 (m, 2H, H-6); 2.38 (bs, 3H, CH3); 2.13 (bs, 3H, C4"-CH3); 13C NMR (5, ppm, DMSO-
t 1 1 1 1
H SI A,
Compound 5e

de): 163.88, 159.58, 157.14,150.16, 145.01,144.57, 142.36, 140.11,136.21, 133.00, 128.54, 128.30, 128.13, 120.24, 116.56, 115.40, 114.68, 112.54, 112.39, 30.66,24.67,21.04,20.24. MS: m/z (%) 400.19 (M+, 100);
Anal. Calcd. For C29H24N2: C, 86.97; H, 6.04; N, 6.99; Found: C, 86.88; H, 6.07; N,6.71 %.
H3C\^^ /\ ^S^/0CH3 5 75 Brown solid; m.p. 213 °C; FT-IR (KBr, cm"1, v): 3389, 2942, 1639 (C=N), 1254; !H-NMR (5, ppm, DMSO-d6 ): 11.47 (s, IH, Nn-H); 8.34-6.78 (m, 13H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 5", 6" - Aromatic); 3.76 (bs, 3H, OCH3), 3.04 (m, 2H, H-5); 2.92 (m, 2H, H-6); 2.38 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-d6): 162.42, 158.24, 157.18, 145.01,144.57, 142.37,140.20,136.10,128.5 4, 128.31, 128.13, 125.40, 120.20, 116.38, 115.28, 114.60, 112.50, 112.46, 111.09, 49.77, 30.64, 24.67, 21.05.
MS: m/z (%) 416.19 (M+, 100);
Anal. Calcd. For C29H24N2O: C, 83.63; H, 5.81; N, 6.73;Found: C, 83.70; H, 5.75; N,6.70%.
TXXXy
Compound 5f

Compound 5g
Compound 5h

10

86
56

Yellow solid; m.p. 255 °C; FT-IR (KBr, cm"1, v): 3378, 2925, 1636 (C=N), 1546, 1325;
^-NMR (5, ppm, DMSO-d6 ): 11.45 (s, 1H, Nn-H); 8.35-6.90 (m, 13H, 2', 3', 4', 5', 6\ 3,7,9,10, 2", 3",
i ^ i ' i^ i A vi
5", 6" - Aromatic); 3.03 (m,
2H, H-5); 2.91 (m, 2H, H-
6); 2.38 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-
d6): 164.88, 159.58,
157.58, 145.01,144.57,
142.36,140.22,136.41,133.1
8, 128.54, 128.13, 125.41,
120.24, 116.36, 115.38,
114.67, 112.54, 112.46,
30.66,24.67,21.04. MS: m/z (%) 431.16 (M+, 100);
Anal. Calcd. For
C28H21N3O2: C, 77.94; H, 4.91; N, 9.74; Found: C, 77.92; H, 4.91; N,9.72%.
Yellow solid; m.p. 232 °C; FT-IR (KBr, cm"1, v): 3379, 2924, 1645 (C=N); !H-NMR (5, ppm, DMSO-d6 ): 11.47 (s, 1H, Nn-H); 8.26-6.87 (m, 13H, 2', 3', 4', 5', 6\ 3,7,9,10, 2", 3",
1'1^ 1 J
5", 6" - Aromatic); 3.21 (bs, 6H, N(CH3)2); 3.01 (m, 2H, H-5); 2.92 (m, 2H, H-6);2.39 (bs, 3H, CH3); 13C NMR (5, ppm, DMSO-

d6163.17, 159.47, 158.32,
145.04,144.56,142.36,140.2
2,136.41,133.51,133.20,
128.45, 128.13, 125.41,
120.21, 116.34, 115.37,
114.69, 112.50, 112.47,
30.68,24.61,21.03.
MS: m/z (%) 429.22 (M+,
100);
Anal. Calcd. For C30H27N3:
C, 83.88; H, 6.34; N,
9.78;Found: C, 83.89; H,
6.36; N,9.77 %.
H3C^/^ ^\ _ 4 68 Black solid; m.p. 229 °C; FT-IR (KBr, cm"1, v): 3380, 2944, 2360, 1642 (C=N); !H-NMR (5, ppm, DMSO-d6 ): 11.63 (s, IH, Nn-H); 8.33-6.93 (m, 12H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 4" -Aromatic);3.03 (m, 2H, H-5); 2.91 (m, 2H, H-6); 2.38 (bs, 3H, CH3); 13C NMR (5, ppm, DMSO-d6) 164.93, 159.52, 157.56, 145.02,145.51, 142.34, 140.22,136.40,133.18,128.5 4, 128.31, 128.17, 125.40, 120.26, 116.35, 115.39, 114.67, 112.50, 112.44, 30.69,24.69,21.07. MS: m/z (%)376.16 (M+, 100);
Anal. Calcd. For C26H20N2O: C, 82.95; H, 5.35; N, 7.49, Found: C, 82.97; H, 5.34; N,7.47%.
XxxXi3
Compound 5i

H3<^ ^<^ /\ 4 69 Brown solid; m.p. 237 °C; FT-IR (KBr, cm"1, v): 3378, 2942, 1643 (C=N); !H-NMR (5, ppm, DMSO-d6 ): 11.66 (s, IH, Nn-H); 8.36-6.97 (m, 12H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 4" - Aromatic);3.01 (m, 2H, H-5); 2.92 (m, 2H, H-6);
Yxxi J3
Compound 5j

2.39 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-
d6) 164.90, 159.51, 157.54,
145.02,144.57, 142.34,
140.22,136.41,133.18,128.5
4, 128.30, 128.17, 125.41,
120.24, 116.36, 115.38,
114.69, 112.51, 112.44,
30.69,24.68,21.04.
MS: m/z (%)392.13 (M+,
100);
Anal. Calcd. For C26H20N2S:
C, 79.56; H, 5.14; N, 7.14;
5, 8.17; Found: C, 79.56; H,
5.12;N,7.17; S, 8.16%.
H3C^/^ ^\ _ 4 71 Brown solid; m.p. 242 °C; FT-IR (KBr, cm"1, v): 3376, 2942, 1638 (C=N); !H-NMR (5, ppm, DMSO-d6 ): 11.64 (s, IH, Nn-H); 8.36-6.97 (m, 12H, 2', 3', 4', 5', 6', 3,7,9,10, 2", 3", 4" - Aromatic); 5.71 (s, IH, 1-NH); 3.05 (m, 2H, H-5); 2.92 (m, 2H, H-6); 2.37 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-d6) 163.90, 159.51, 155.54, 143.02,144.57, 142.54, 140.12,135.41,133.18,128.7 4, 128.30, 128.17, 125.41, 120.84, 116.36, 115.38, 114.69, 111.51, 112.56, 30.78,25.07,21.13. MS: m/z (%)375 (M+, 100); Anal. Calcd. For C26H21N3: C, 83.17; H, 5.64; N, 11.19; Found: C, 83.19; H, 5.60; N,11.15%.
H J H
Compound 5k

H,C

Compound 51

77

Orange solid; m.p. 257 °C;
FT-IR (KBr, cm"1, v): 3422,
2925, 2360, 1649 (C=N),
and 1641 (C=N);
^-NMR (5, ppm, DMSO-
d6 ): 11.46 (s, 1H, Nn-H);
8.33-6.97 (m, 13H, 2', 3',
4', 5', 6', 3,7,9,10, 2", 3",
5", 6" - Aromatic); 3.05 (m,
2H, H-5); 2.95 (m, 2H, H-
6); 2.35 (bs, 3H, CH3);
13C NMR (5, ppm, DMSO-
d6): 162.90, 158.77,
1, 142.41,
132.41,
128.63,
116.36,
112.17,
156.74,150.23,144.03,143.7
140.22,135.23,
129.72, 128.35, 123.55, 121.21, 115.38, 114.67, 112.12, 110.83,
30.91,23.91,21.11.
MS: m/z (%) 387.12 (M+,
100);
Anal. Calcd. For C27H21N3:
C, 83.69; H, 5.46; N, 10.84;
Found: C, 83.72; H, 5.40; N,
10.89%.

Example 2: Biological Evaluation
[0090] Human breast cancer cell line (MCF7) and Cervix adeno carcinoma cell
line (HeLa) was obtained from National Centre for Cell Science (NCCS), Pune
and grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal
bovine serum (FBS). All cells were maintained at 37°C, 5% CO2, 95% air and
100%) relative humidity. Maintenance cultures were passaged weekly, and the
culture medium was changed twice a week.
[0091] 2.1 Cytotoxicity: The cytotoxicity was evaluated by HeLa (Cervix adeno
carcinoma) and MCF-7 (Breast cancer), by the MTT assay method as previously
well known and practiced (Munusamy, S.; Badavath, V. N.; Maji, S.; Sekar, M.;
Shabbir, M. Novel Halogenated Pyrido [2, 3-a] Carbazole with Enhanced

Aromaticity as Potent Anticancer and Antioxidant: Rationale Design and Microwave Assisted Synthesis. New J. Chem. 2019, No. 44, 17231-17240.). [0092] 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore,the amount of formazan produced is directly proportional to the number of viable cells. After 48h of incubation, 15 uL of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100 uL of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula.
% Cell Inhibition = 100- Abs (sample)/Abs (control) xlOO - (1)
[0093] Nonlinear regression graph was plotted between % Cell inhibition and Log concentration and IC50 was determined using GraphPad Prism software. [0094] The results have been presented in Table 2 below.
Table 2: Cytotoxic activity of the compounds

Compound MCF-7 ICso (uM)a HeLa ICso (uM)a
5a >100 >100
5b 26.4±0.001 1.13±0.024
5c 44.26±0.003 2.18±0.123
5d 30.4±0.021 3.04±0.023
5e >100 >100
5f 37.32±0.012 44.38±0.025
5g 24.63±0.120 0.9±0.123
5h >100 >100
5i 74.31±0.031 78.1±0.013
5j 54.69±0.010 3.65±0.024
5k 59.68±0.020 5.5±0.015
51 178.1±0.31 54.69±0.120
Cisplatin 16.72±0.001 9.81±0.003
Ellipticine 16.31 19.85
aIC5o minimum inhibitory concentration are expressed as mean ± standard deviations of triplicate determination.

[0095] As shown in Table 2, the compounds showed good potent anti-cancer activities against both the tested cells. Among all the compounds, 5g, 5b and 5d showed almost equipotent cytotoxic activity against MCF-7 with IC50 value of 24.63 uM, 26.4 uM and 30.4, uM respectively. The compounds 5f and 5c depicted stronger cytotoxicity activity against MCF-7 with IC50 value of 37.32 and 44.26 uM respectively. The Compounds 5j, 5k, 5i and 51 displayed significant IC50 values of 54.69, 59.68, 74.31 and 178.1 uM against MCF-7, respectively. Compounds 5a, 5e and 5h did not show very significant activity against MCF-7 cancer line.
[0096] The results indicated that the compounds showed selective cytotoxicity against HeLa. Among all the compounds, 5g, 5b and 5d out-performed the positive control cisplatin against HeLa and the compounds 5e, 5j and 5k showed almost equipotent activity with Cisplatin and Ellipticine against HeLa. Furthermore, the compounds 5f and 51 displayed substantial activity (IC50 values 44.38 and 54.69 uM) with cisplatin against HeLa. The Compound 5i displayed significant IC50 value of 78.1 uM against HeLa. The Compounds 5a, 5e and 5h did not show any significant activity against HeLa cancer cell line. In general it was found that all synthesized compounds showed selective cytotoxicity against HeLa cancer cell line. Triplication was maintained and the medium not containing the compounds served as control.
[0097] 2.2 DNA Binding studies: Electronic absorption titration was used to study the DNA binding tendencies of the Compounds 5a-l (as per protocol in Munusamy, S.; Badavath, V. N.; Maji, S.; Sekar, M.; Shabbir, M. Novel Halogenated Pyrido [2, 3-a] Carbazole with Enhanced Aromaticity as Potent Anticancer and Antioxidant: Rationale Design and Microwave Assisted Synthesis. New J. Chem. 2019, No. 44, 17231-17240). The concentration of the compound was kept as 25 uM, and concentration of DNA was varied from 0-50 uM. Absorption spectra of the compounds 5a-51 in the absence and presence of ct-DNA is given in Figure 1. Upon addition of increased amount of ct-DNA, a significant hypochromism and a red shift of about 4-7 nm is observed in the band at 220-340 nm. The hypochromic effects and hypsochromic shifts in the UV

spectra have been considered as one of the evidences of the intercalation of small molecules towards DNA. This can be attributed to a strong interaction between DNA and compounds and it is also likely that these compounds bind to DNA helix via intercalation.
[0098] In order to illustrate quantitatively the consequence, the absorption data was analyzed to evaluate the intrinsic binding constant (Kb) from the plot of DNA/(ea-eb) versus [DNA] (refer Figure 2). Kb is calculated by the ratio of slope to the intercept and the results have been presented in Table 3 below. The magnitude of intrinsic binding constant (Kb) values of compounds 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, 5i, 5j, 5k and 51 were noted to be 5.7xl03, 1.9xl05, 3.8xl04, 1.8xl05, 2.1xl03, 2.2xl04, 2.2xl05, 4.5xl03, 1.3xl04, 1.4xl04, 1.4xl04 and 5.8xl03 M"1 respectively. Further, the observed binding constant values were comparable with the classical intercalator ethidium bromide, Kb=6.58X104 M"1. The compounds 5c, 5d, 5f, 5i, 5j, 5k and 51 show good binding affinity. The nitro and chloro substituted pyrido[2,3-a]carbazole compounds 5g, 5b and 5d strongly bind with ct-DNA by intercalation compared to the other compounds. This could be due to an increase of the polar hydrophobicity of the nitro and chloro derivatives.
Table 3: Intrinsic binding constant (Kb) values of synthesized compounds Compound Binding constant (Kb)
5a 5.7xl03
5b 1.9x10s
5c 3.8xl04
5d 1.8x10s
5e 2.1xl03
5f 2.2xl04
5g 2.2x10s
5h 4.5xl03
5i 1.3xl04
5j 1.4xl04
5k 1.4xl04
51 5.8xl03
[0099] 2.3 Viscosity Measurements: The values of relative specific viscosities of ct-DNA in the absence and presence of the compounds was plotted against

[Compound]/[DNA] (viscosity was measured as in Munusamy, S.; Badavath, V. N.; Maji, S.; Sekar, M.; Shabbir, M. Novel Halogenated Pyrido [2, 3-a] Carbazole with Enhanced Aromaticity as Potent Anticancer and Antioxidant: Rationale Design and Microwave Assisted Synthesis. New J. Chem. 2019, No. 44, 17231-17240). The relative viscosity of ct-DNA bound to compounds 5a-51 increased with increasing compound concentration (refer Figure 3.). Compound (5g) showed a steady state increase in the relative viscosity of ct-DNA with increasing concentration. Also, compounds (5b, 5d, 5c and 5f), showed constant relative viscosity of ct-DNA. Further, compounds (5i, 5j, 5k and 51) slightly increase the relative viscosity. These phenomena further confirm that compounds 5g, 5b, 5d and 5c bind to the ct-DNA through intercalation mode, which is consistent with the forgoing conclusions.
[00100] 2.4 Electrochemical Titration: In the present study, the cyclic voltametric technique was employed to understand the nature of compound 5g in absence and presence of the ct-DNA concentrations (as per protocol in Munusamy, S.; Badavath, V. N.; Maji, S.; Sekar, M.; Shabbir, M. Novel Halogenated Pyrido [2, 3-a] Carbazole with Enhanced Aromaticity as Potent Anticancer and Antioxidant: Rationale Design and Microwave Assisted Synthesis. New J. Chem. 2019, No. 44, 17231-17240). The CV was recorded in the range 1.0 to -2.0 V. The compound exhibited a one electron reduction and oxidation wave, ca. -1.025 and -0.384, respectively. In presence of ct-DNA the cyclic voltammogram of the compound showed a significant cathodic and anodic shift, ca. -1.008 and -0.459 V, with a decrease in the current peaks from 6.3 X 10 "5 A to 5.7 X 10 "5 A (for cathodic current) and -3.5 X 10 "5 A to -2.3 X 10 "5 A (for anodic current), respectively (refer Figure 4.). The decrease in the peak current of the compound, in presence of ct-DNA may be the result of slow diffusion of the compound into the DNA molecules, forming an equilibrium mixture of the free and DNA-bound nitro phenyl group substituted pyrido[2,3-a]carbazole compound 5g to the electrode surface. From these observation CV data suggest that the 8-Methyl-4-(4-nitro-phenyl)-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole (5g) binds the DNA helix via intercalation pathway.

[00101] 2.5 Structural activity relationship: In general, it was, observed that chloro and bromo substitution compounds 5g, 5b, 5d and 5c showed excellent anticancer activity. The morphological changes brought about in the MCF-7 and HeLa cancer cell lines by the compound 5g at varying concentrations (uM) has been provided in Figures 5 and 6, respectively. This may be due to the stronger electron withdrawing tendency of the nitro, chloro and bromo groups, which play an important role in cytotoxicity effect. The effect of substituent at exposition on the p-OCHa group (5f), showed good anticancer activity against both MCF-7 and HeLa cancer cell lines. The investigation on the effect of five member heterocyclic groups was carried out by introducing furan, thiophene and pyrrole (5k, 5i, and 5j) substitution at exposition. The compound 5j exhibited better cytotoxicity compared to 5k and 5i; in addition, 5j portrayed selective cytotoxicity against HeLa cancer cell line. Presence of thiophene moiety boosted the cytotoxicity. Compound 51 with pyridine core also showed significant cytotoxicity against both MCF-7 and HeLa cancer cell lines. In general, it was observed that pyrido[2,3-a]carbazole compounds of the present disclosure with the electron withdrawing group and hetero aryl rings further increase the potency of cytotoxicity compared to electron donating aryl system and unsubstituted aryl groups. Also, all the synthesized compounds showed selective cytotoxicity against HeLa cancer cell line, when compared to standard drug Cisplatin. [00102] 2.6 Percentage growth inhibition: The percentage inhibition in the growth of the cancer line - MCF-7, by the different compounds 5b-5d, 5f-5g, and 5i-51 has been presented in Figure 7. The percentage inhibition in the growth of the cancer line - HeLa, by the different compounds 5b-5d, 5f-5g, and 5i-51 has been presented in Figure 8.
[00103] Thus, the compounds of the present disclosure synthesized using microwave-assisted, one-pot condensation reaction bind with ct-DNA via. intercalative mode, with binding constant ranging from 103-105M_1 as revealed by the absorption spectral techniques. Among the synthesized compounds provided above, chloro substituted pyrido[2,3-a]carbazole compounds 5g, 5b and 5d strongly bind with ct-DNA with the binding constant values of 2.2*105M_1,

l.QxlC^M"1 and l.SxlC^M"1 respectively. Cytotoxicity studies also indicated that the synthesized compounds show good potency in anti-cancer activity against both MCF-7 and HeLa cell lines.
[00104] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein merely for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention and should not be construed so as to limit the scope of the invention or the appended claims in any way.
ADVANTAGES OF THE PRESENT INVENTION
[00105] The present disclosure provides compounds with high hydrophobicity and planarity that renders them effective against cancer cells. [00106] The present disclosure provides compounds with improved cytotoxicity for cancer cells compared to commercial drugs such as Ellipticine. [00107] The present disclosure provides a process of synthesizing anti-cancer compounds using microwave with high yields.
[00108] The present disclosure provides a process of synthesizing anti-cancer compounds in a simple, one-pot synthesis which saves reaction time, avoids use of poisonous solvents and is environmentally benign.

We Claim:

1. An anti-cancer pyrido[2,3-a]carbazole compound of Formula I, its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof, or mixtures thereof,

Formula I wherein Ar is C6-C14 aryl, or C5-C14 heterocyclyl; wherein Ar is unsubstituted or substituted with one or more of halogen, C1-C6 alkyl, C1-C6 alkoxy, cyano, nitro, -NH2, -N(R1)2, -COOH, -COOR1, or -OH; and wherein R1 is a Ci-Ce alkyl.
2. The compound as claimed in claim 1, wherein the Ar is phenyl or napthyl; and wherein the Ar is unsubstituted or substituted with one or more of F, CI, Br, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, cyano, nitro, -NH2, -N(CFb)2, -N(C2H5)2, -COOH, -COOCH3, -COOC2H5 or OH.
3. The compound as claimed in claim 1, wherein the Ar is phenyl; and wherein the phenyl is unsubstituted or substituted with one or more of CI, Br, methyl, methoxy, nitro, or -N(CH3)2.
4. The compound as claimed in claim 1, wherein the Ar is furanyl, thiophenyl, pyrrolyl, or pyridinyl; and wherein the Ar is unsubstituted or substituted with one or more of F, CI, Br, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, cyano, nitro, -NH2, -N(C2H5)2, -COOH, -COOCH3, -COOC2H5 or OH.
5. The compound as claimed in claim 1, wherein the Ar is unsubstituted furanyl, thiophenyl, pyrrolyl, or pyridinyl.
6. A compound selected from:

8-methyl-2,4-diphenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Bromo-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(2-Chloro-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-p-tolyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
4-(4-Methoxy-phenyl)-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-
a]carbazole;
8-Methyl-4-(4-nitro-phenyl)-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
dimethyl-[4-(8-methyl-2-phenyl-5,6-dihydro-HH-pyrido[2,3-a]carbazole-4-yl)-
phenyl]-amine;
4-Furan-2-yl-8-methyl-2-phenyl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-thiophene-2-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-(lH-pyrrolo-2-yl)-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
8-methyl-2-phenyl-4-pyridin-yl-5,6-dihydro-llH-pyrido[2,3-a]carbazole;
its stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof,
or mixtures thereof.
7. A pharmaceutical composition comprising a compound of Formula I, its
stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt thereof, or
mixtures thereof; and one or more pharmaceutically acceptable excipient(s).
8. A process of synthesizing compounds of Formula I using a one-pot synthesis,
wherein the process comprises the step of reacting a 8-methyl-l,2,3,4-tetrahydro-
9H-carbazole-l-one (1) with N-phenacylpyridinium bromide (2), aldehyde (3) and
ammonium acetate (4) in presence of an organic solvent followed by microwaving
to yield a compound of Formula I as shown in Scheme I.

6
1 ' T] Fomiula I
o
W?
HjC>r^ ^-, ^J C*H3COOXHj+(4)
*■
H

,**

Scheme I

9. The process as claimed in claim 8, wherein the organic solvent is acetic acid.