Description:FIELD OF THE INVENTION
[0001] The present disclosure generally relates to pharmaceutical organic compounds. Specifically, the present disclosure provides compounds of Formula (I), its stereoisomers, tautomers, pharmaceutically acceptable solvates or pharmaceutically acceptable salts thereof and pharmaceutical compositions comprising them. The present disclosure also provides a process for preparing the compound of Formula (I) its stereoisomers, tautomers, pharmaceutically acceptable solvates or pharmaceutically acceptable salts thereof.

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] 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 in 2030. A large number of carbazole alkaloids exhibit significant anticancer activities. Among them, pyrido, pyrrolo and pyranocarbazoles have considerable biological activities, such as anticancer, and DNA-intercalator. Ellipticine and its derivatives have drawn tremendous attention from synthetic chemists and synthesis of ellipticine and its derivatives have been reported. Ellipticine (ellitpicine acetate), datelliptium, retellipticine (BD-84), pazellipticine, etc., have also been tested for their clinical use. Ellipticine has anti-mutagenic, cytotoxic and anti-tumor activities due to its good interaction with DNA. A number of ellipticine analogs were synthesized successfully resulting in improved anti-cancer and cytotoxic activities against a group of cancer cell lines, and attracted scientific interest to rational design of ellipticine core structure with enhanced planarity and aromaticity. In addition, modified natural products like a number of synthetic ellipticine analogues exhibited significantly improved in vitro as well as in vivo anticancer activity against a panel of cancer cell lines. The derivative 9-hydroxy-2-methyl ellipticine acetate (celliptium), is currently undergoing extensive clinical trials with good solubility, it has found application in the treatment of breast, kidney and thyroid cancer. But there is need to develop compounds with increased aromaticity and planarity.
[0004] Thus there is a need in the art to develop pharmaceutical compounds having increased aromaticity, planarity and are capable of demonstrating strong anti-cancer activity. Additionally, the compounds should also be capable of anti-oxidant activity.

OBJECTS OF THE INVENTION
[0005] An object of the present disclosure is to provide compounds with anti-cancer and anti-oxidant activity.
[0006] An object of the present disclosure is to provide pyrrolo[2,3-a]carbazole-2-carboxylate based compounds that have higher aromaticity and planarity.
[0007] Another object of the present disclosure is to provide a process of preparing the 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] The present disclosure generally relates to organic pharmaceutical compounds. Specifically, the present disclosure provides a pyrrolo[2,3-a]carbazole-2-carboxylate based compound of Formula (I), its stereoisomer, tautomer, solvate, or a pharmaceutically acceptable salt thereof.
[0010] In an aspect, the present disclosure provides an anti-cancer and anti-oxidant compound of Formula (I), a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

Formula I
wherein R1, R2 and R3 may be independently selected from hydrogen, halogen, -(C1-C6)alkyl, (C1-C6)alkoxy, -NH2, -OH, cyano, nitro, -COOH, -OR4, -SO2, and -COOR4; and wherein R4 may be -(C1-C6)alkyl.
[0011] In an embodiment, the R1, R2 and R3 may be independently selected from hydrogen, –CH3, bromine, chlorine, and -O-CH3.
[0012] In an embodiment, the compound of Formula I may be selected from:
Ethyl 3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,7-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,8-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,9-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-chloro-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-bromo-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-methoxy-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate
or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0013] In an aspect, the present disclosure provides a pharmaceutical composition comprising an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof; and a pharmaceutically acceptable excipient.
[0014] In an embodiment, the pharmaceutical composition is in the form of a tablet, pill, sachet, capsule, solution, cream, paste, gel, lotion, suspension, suppositories, emulsion, powder, microemulsion or nanoemulsion.
[0015] In an aspect, the present disclosure provides a process of preparing an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, as shown in general scheme-I below

Scheme I
wherein R1, R2 and R3 are as defined previously.
[0016] In an embodiment, the process of preparing the compound comprises the steps of: (a) adding sodium nitrate to a mixture of glacial acetic acid and a compound of Formula A followed by stirring; and (b) reacting the solution of step (a) with zinc powder and ethyl acetoacetate to give the compound of Formula I.
[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 the UV/visible absorption spectra 1(i)-1(vii) of compounds 1a-g, as per an embodiment of the present disclosure, in the absence (upper line) or presence of calf-thymus DNA (Phoshate buffer pH 7). Arrows (?) refer to hypochromic effect and the inset shows binding isotherms with varying concentrations of ct-DNA. Adsorption titration experiments were performed with fixed concentration of the compounds (25 µM) with varying concentration of DNA (0-50 µM).
[0020] Figure 2 provides the fluorescence spectra 2(i)-2(iii) of compounds 1b, 1e and 1f respectively, as per an embodiment of the present disclosure, with ct-DNA. Arrows indicate the orientations of the fluorescence quenching (?) with varying concentrations of ct-DNA.
[0021] Figure 3 provides the effect of increasing amounts of the compounds (1a-1g), as per an embodiment of the present disclosure, on the relative viscosity of the ct-DNA at 25 ºC.
[0022] Figure 4 demonstrates the cleavage of ct-DNA by compounds, as per an embodiment of the present disclosure, by gel electrophoresis; Lane 1: Control ct-DNA (untreated sample). Lane 2-7: compounds 1a-1g + ct-DNA, respectively.
[0023] Figure 5 provides the plot of the %growth inhibition at various concentrations of the compounds 1b, 1e, 1f, and 1g, as per an embodiment of the present disclosure.
[0024] Figure 6 provides the quantification of anticancer activity (IC50 values) of compounds 1a-1g, as per an embodiment of the present disclosure, along with Ellipticine.
[0025] Figure 7 demonstrates the morphological changes that occur in cancer cells (human breast cancer, MCF-7) induced by the compound 1e, as per an embodiment of the present disclosure, at varying concentrations of (a) 0 µM, (b) 0.1 µM and (c) 50 µM.
[0026] Figure 8 provides a quantification of antioxidant activity (IC50 values) of compounds 1a-1g, as per an embodiment of the present disclosure, along with ascorbic acid.

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 particular 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 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.
[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 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.
[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, "halogen" as used herein refers to chlorine, fluorine, bromine or iodine atom.
[0046] 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.
[0047] 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.
[0048] 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., cancer). Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0049] Aspects of the present disclosure provide compounds based on pyrrolo[2,3-a]carbazoles that are effective as anti-oxidants and have anti-cancer activity.
[0050] In an embodiment, the present disclosure provides an anti-cancer and anti-oxidant compound of Formula (I), a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

Formula I
wherein R1, R2 and R3 may be independently selected from hydrogen, halogen, -(C1-C6)alkyl, (C1-C6)alkoxy, -NH2, -OH, cyano, nitro, -COOH, -OR4, -SO2, and -COOR4; and wherein R4 may be a -(C1-C6)alkyl.
[0051] In an embodiment, R1, R2 and R3 may be independently selected from hydrogen, -(C1-C6)alkyl, halogen, or (C1-C6)alkoxy.
[0052] In a preferred embodiment, (C1-C6)alkyl may be selected from –CH3, -C2H5, or –C3H7. In a preferred embodiment, (C1-C6)alkoxy may be selected from –OCH3, -OC2H5, or –OC3H7.
[0053] In an embodiment, R1, R2 and R3 may be independently selected from hydrogen, –CH3, bromine, chlorine, and -O-CH3.
[0054] In an embodiment, R4 may be selected from –CH3, -C2H5, or –C3H7.
[0055] In an embodiment, the compound of Formula I may be selected from:
Ethyl 3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,7-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,8-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,9-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-chloro-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-bromo-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-methoxy-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate
or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0056] In an 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] The compounds of the present disclosure have enhanced planarity and aromaticity that show anti-cancer activity by demonstrating good cytotoxicity. In a preferred embodiment, the compounds may show IC50 values as low as 3.04µM compared to 16.72µM for standard anti-cancer drug – Ellipticine against MCF-7 cancer cell line (Figure 7).
[0059] The compounds of the present disclosure interact with circulating tumor DNA (ct-DNA) through intercalation with high binding constants.
[0060] In some embodiments, the compounds also demonstrate anti-oxidant or free radical scavenging effect.
[0061] In an embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof; and a pharmaceutically acceptable excipient.
[0062] In an embodiment, the excipient(s) may be selected from diluent, carrier, binder, disintegrant, glidant, lubricant, coating material, filler, solvent, surfactants, solubilizers, emulsifier, preservatives, coloring agent, flavoring agent, 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 disclosure.
[0063] In an embodiment, the pharmaceutical composition is in the form of a tablet, pill, sachet, capsule, solution, suspensions, elixirs, cream, paste, gel, lotion, suspension, suppositories, emulsion, powder, nanoparticles, patches, aerosols, nasal sprays, microparticles, or nanoemulsion.
[0064] In an embodiment, the composition may be administered orally, bucally, intraperitonially, parenterally, intravenously, intramuscularly, subcutaneously, topically, transdermally, rectally, intramuscularly, or their combinations.
[0065] In the formation of the composition for various forms of administration a suitable excipient may be employed in the required quantity. For example, a tablet may be formed by using magnesium stearate, gum, talc, starch, glucose, sucrose, and lactose; gelatin capsules or suppositories may be prepared by using waxes, oils, and fats; solutions, suspensions or elixirs may be prepared by using water, physiological sodium chloride solution, ethanol, glycerol, sugar solution, or a mixture of these solvents.
[0066] In an embodiment, the composition may comprise about 1% to about 90%, for example about 5% to about 70%, about 20% to about 50% by weight of the compound of Formula I. The amount of the compound of Formula I in the pharmaceutical compositions normally is from about 5 mg 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, based on the subject’s weight, past medical records or general health, severity of condition, gender, age, or like factors well known in medical arts.
[0067] In some embodiments, the composition may also be formed along with other pharmaceutically active component(s). In an embodiment, the combination of compound of present disclosure with other active component or treatment includes co-administration of a compound of Formula I with the other component or treatment 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 component or treatment and administration of the other component or treatment first, followed by the compound of present disclosure. Further therapeutic agents are administered either simultaneously or sequentially.
[0068] The pharmaceutical compositions comprising the compound of Formula I according to the disclosure are prepared in a manner known to one skilled in the art.
[0069] In an embodiment, the present disclosure provides a pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof for use as an anti-cancer agent.
[0070] In another embodiment, the present disclosure provides a pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof for use as an anti-oxidant agent.
[0071] 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.
[0072] In an embodiment, the present disclosure provides a process of preparing an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, as shown in general scheme-I below

Scheme I
wherein R1, R2 and R3 are as defined previously.
[0073] In an embodiment, the process of preparing the compound comprises the steps of: (a) adding sodium nitrate to a mixture of glacial acetic acid and a compound of Formula A followed by stirring; and (b) reacting the solution of step (a) with zinc powder and ethyl acetoacetate to give the compound of Formula I.
[0074] In an embodiment, the temperature for step (a) may be about 5°C.
[0075] The process of the present disclosure gives high yields. In some embodiments, the yield may be as high as 81%.
[0076] In another embodiment, the present disclosure provides a method of treatment of anti-oxidant diseases or disorders, or cancer diseases comprising administering a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof.
[0077] In an embodiment, the compound may be effective in treating cancers related to breasts.
[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.
EXAMPLE 1: Synthetic procedure for the synthesis of Compounds of Formula I (1a-1g)
[0080] Seven compounds of the Formula I were synthesized namely, compounds 1a-1g by following the General Scheme II below using compounds Aa-Ag.

Scheme II
wherein,

[0081] Synthesis of Compound 1a: To a mixture of 1-oxo-1,2,3,4-tetrahydrocarbazole (Aa) (1.85 g, 0.01 mol) in 5.00 mL of glacial acetic acid NaNO2 solution (0.68 g, 0.01 mol) was added at 5 ºC. The reaction mixture was stirred for about 30 min under ice cold condition, and then continued stirring for 4h at room temperature. Zinc powder (0.65 g, 0.1mol) and acetic acid solution of ethyl acetoacetate (1.26mL, 0.01mol) was added to the above mixture under vigorous stirring. Then the mixture was refluxed for about 1 h. The cooled reaction mixture was poured on crushed ice and obtained precipitate was filtered and purified by column chromatography by using petroleum ether: ethyl acetate (10:2 v/v) as eluent. The eluted fractions were evaporated and re-crystallized in methanol to give compound 1a in 65% yield.
[0082] Compound 1a exhibited sharp IR band at 3271 cm-1(N-H) and 1639 cm-1(COOEt) (refer Table 1). 1H NMR spectrum of 1a shows two singlets at d 11.45 and 10.50 which account for -NH in carbazole and pyrrolo, respectively. The aromatic protons appear in the range of d7.10-7.40. The appearance of a quartet at d 4.32 (J = 7.2 Hz) is due to C1 substituted ethyl ester CH2 protons. The three protons of C1 substituted ethyl ester CH3 appear as a triplet at d 1.39 (J = 7.2 Hz). From the 1H NMR spectrum, it was inferred that the number of protons appeared in the spectrum was well in agreement with its molecular structure. The 13C NMR spectrum revealed the presence of 18 carbons.
[0083] All other compounds 1b-1g were prepared in similar manner as described above. Structure and characterization data of all the compounds is provided in Table 1.
Table 1: Structure and characterization data of compounds 1a-1g
Compound Characterization data

Ethyl 3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1a) Yellow solid; Yield: 65 %; M.P. 168 oC. Anal. Calcd. For C18H18N2O2 (%): C, 73.47; H, 6.12; N, 9.52. Found (%): C, 73.45; H, 6.16; N, 9.54. IR (KBr, cm-1): 3271 ? (N-H), 2921 ? (C-H), 1639 ? (C=O). 1H-NMR (DMSO-d6, ppm), d 1.39 (t, 3H, CH2CH3,J = 7.2 Hz), 2.10 (s, 3H C2-CH3), 2.74 (t, 2H, C5-CH2, J = 6.0 Hz), 2.91 (t, 2H, C4-CH2, J = 6.0 Hz), 4.32 (q, 2H, CH2CH3, J = 7.2 Hz), 7.10-7.40 (m, 4H, C6, C7, C8, C9-H), 10.50 (s, 1H, 3-NH), 11.45 (s, 1H, 10-NH).13C NMR (DMSO-d6, ppm), 173.2, 140.2, 136.0, 135.2, 134.5, 131.4, 130.6, 129.7, 129.2, 128.4, 125.3, 120.8, 113.0, 61.0, 25.2, 21.1, 20.9, 13.5. MS: m/z (%) 295 (M+, 100)

Ethyl 3,7-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1b) Brown solid; Yield: 78 %; M.P. 197 oC. Anal. Calcd. For C19H20N2O2 (%): C, 74.03; H, 6.49; N, 9.09. Found (%): C, 74.00; H, 6.54; N, 9.08. IR (KBr, cm-1): 3277 ? (N-H), 2921 ? (C-H), 1639 ? (C=O). 1H-NMR (DMSO-d6, ppm), d 1.37 (t, 3H, CH2CH3,J = 7.2 Hz), 2.10 (s, 3H C2-CH3), 2.50 (s, C7-CH3), 2.74 (t, 2H, C5-CH2, J = 6.0 Hz), 2.91 (t, 2H, C4-CH2, J = 6.0 Hz), 4.32 (q, 2H, CH2CH3, J = 7.2 Hz), 7.10-7.40 (m, 3H, C6, C8, C9-H), 10.50 (s, 1H, 3-NH), 11.45 (s, 1H, 10-NH). 13C NMR (DMSO-d6, ppm), 173.5, 140.2, 136.2, 133.3, 134.7, 130.9, 130.4, 129.7, 129.5, 128.4, 124.8, 120.7, 113.0, 60.9, 25.4, 21.3, 20.7, 15.9, 13.7. MS: m/z (%) 308 (M+, 100).

Ethyl 3,8-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1c) Brown solid; Yield: 81 %; M.P. 204 oC. Anal. Calcd. For C19H20N2O2 (%): C, 74.03; H, 6.49; N, 9.09. Found (%): C, 74.00; H, 6.54; N, 9.08. IR (KBr, cm-1): 3278 ? (N-H), 2924 ? (C-H), 1649 ? (C=O). 1H-NMR (DMSO-d6, ppm), d 1.38 (t, 3H, CH2CH3,J = 7.2 Hz), 2.13 (s, 3H C2-CH3), 2.54 (s, 3H, C8-CH3), 2.74 (t, 2H, C5-CH2, J = 6.0 Hz), 2.93 (t, 2H, C4-CH2,J = 6.0 Hz), 4.33 (q, 2H, CH2CH3, J = 7.2 Hz), 7.10-7.40 (m, 3H, C6, C7, C9-H), 10.58 (s, 1H, 3-NH), 11.44 (s, 1H, 10-NH). 13C NMR (DMSO-d6, ppm), 173.1, 140.2, 136.1, 135.1, 134.5, 132.5, 130.8, 129.6, 129.4, 128.5, 124.7, 120.1, 113.2, 61.1, 25.4, 21.3, 20.6, 15.1, 13.5. MS: m/z (%) 308.25 (M+, 100).

Ethyl 3,9-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1d) Brown solid; Yield: 67 %; M.P. 204 oC. Anal. Calcd. For C19H20N2O2 (%): C, 74.03; H, 6.49; N, 9.09. Found (%): C, 74.00; H, 6.54; N, 9.08. IR (KBr, cm-1): 3278 ? (N-H), 2924 ? (C-H), 1649 ? (C=O); 1H-NMR (DMSO-d6, ppm), d 1.39 (t, 3H, CH2CH3,J = 7.2 Hz), 2.10 (s, 3H, C2-CH3), 2.59 (s, 3H, C9-CH3), 2.74 (t, 2H, C5-CH2, J = 6.0 Hz), 2.91 (t, 2H, C4-CH2, J = 6.0 Hz), 4.32 (q, 2H, CH2CH3, J = 7.2 Hz), 7.10-7.40 (m, 3H, C6, C7, C8-H), 10.58 (s, 1H, 3-NH), 11.44 (s, 1H, 10-NH).13C NMR (DMSO-d6, ppm), 172.8, 140.1, 136.1, 135.4, 134.4, 132.8, 130.8, 129.5, 129.4, 128.7, 124.7, 120.3, 113.3, 61.2, 25.4, 21.3, 20.5, 15.8, 13.5. MS: m/z (%) 308.25 (M+, 100).

Ethyl 7-chloro-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1e) Yellow solid; Yield: 72 %; M.P. 200 oC. Anal. Calcd. For C18H17ClN2O2 (%): C, 65.65; H, 5.17; N, 8.51. Found (%): C, 65.70; H, 5.21; N, 8.52. IR (KBr, cm-1): 3271 ? (N-H), 2931 ? (C-H), 1643 ? (C=O); 1H-NMR (DMSO-d6, ppm), d 1.37 (t, 3H, CH2CH3,J = 7.2 Hz), 2.14 (s, 3H C2-CH3), 2.72 (t, 2H, C5-CH2, J = 6.0 Hz), 2.93 (t, 2H, C4-CH2, J = 6.0 Hz), 4.37 (q, 2H, CH2CH3, J= 7.2 Hz), 7.10-7.40 (m, 3H, C6, C8, C9-H), 10.50 (s, 1H, 3-NH), 11.45 (s,1H, 10-NH). 13CNMR (DMSO-d6, ppm), 172.4, 140.2, 138.9, 136.2, 134.4, 132.1, 130.8, 129.5, 129.3, 128.6, 124.5, 120.3, 113.1, 61.0, 25.3, 21.0, 20.4, 13.4. MS: m/z (%) 330 (M+, 100).

Ethyl 7-bromo-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1f) Yellow solid; Yield: 77 %; M.P. 213 oC. Anal. Calcd. For C18H17BrN2O2 (%): C, 75.90; H, 4.56; N, 7.51. Found (%): C, 75.96; H, 4.59; N, 7.54. IR (KBr, cm-1): 3271 ? (N-H), 2924 ? (C-H), 1598 ? (C=O). 1H-NMR (DMSO-d6, ppm), d 1.41 (t, 3H, CH2CH3,J = 7.2 Hz), 2.10 (s, 3H, C2-CH3), 2.78 (t, 2H, C5-CH2, J = 6.0 Hz), 2.87 (t, 2H, C4-CH2, J = 6.0 Hz), 4.32 (q, 2H, CH2CH3, J= 7.2 Hz), 7.10-7.40 (m, 3H, C6, C8, C9-H), 10.59 (s, 1H, 3-NH); 11.48 (s, 1H, 10-NH). 13CNMR (DMSO-d6, ppm), 173.0, 153.1, 140.3, 139.9, 136.4, 135.5, 134.7, 130.8, 129.4, 129.1 128.5, 124.5, 120.3, 113.0, 60.0, 24.9, 21.1, 20.5, 13.2. MS: m/z (%) 373 (M+, 100).

Ethyl 7-methoxy-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate (1g) Brown solid; Yield: 69 %; M.P. 197 oC. Anal. Calcd. For C19H20N2O3 (%): C, 70.33; H, 6.20; N, 8.66. Found (%): C, 70.35; H, 6.21; N, 8.64. IR (KBr, cm-1): 3277 ? (N-H), 2921 ? (C-H), 1639 ? (C=O). 1H-NMR (DMSO-d6, ppm), d 1.39 (t, 3H, CH2CH3,J = 7.2 Hz), 2.15 (s, 3H C2-CH3), 2.72 (t, 2H, C5-CH2, J = 6.0 Hz), 2.90 (t, 2H, C4-CH2, J = 6.0 Hz), 3.93 (s, 3H, C7-OCH3) 4.31 (q, 2H, CH2CH3, J = 7.2 Hz), 7.10-7.40 (m, 3H, C6, C8, C9-H), 10.50 (s, 1H, 3-NH), 11.45 (s, 1H, 10-NH). 13C NMR (DMSO-d6, ppm), 173.1, 140.2, 136.2, 135.6, 134.3, 132.4, 130.8, 129.6, 129.3, 128.5, 124.6, 120.4, 113.2, 60.9, 55.4, 25.2, 21.2, 20.4, 13.3. MS: m/z (%) 325 (M+, 100).

EXAMPLE 2: Biological Evaluation
[0084] 2.1 DNA Binding-Titration experiments: The binding modes of the synthesized compounds were analyzed through absorbance and shift in wavelength (nm) as a function of the added concentration of DNA. Adsorption titration experiments were performed with fixed concentration of the compounds (25 µM) with varying concentration of DNA (0-50 µM). A proper hypochromic and a red-shift of about 3-5 nm was noticed in the band range of 228-315nm, upon increasing ct-DNA concentration (refer Figures 1(i) – 1(vii) corresponding to the compounds 1a-1g), suggesting a strong interaction between the synthesized compounds and DNA helix likely via intercalation. The obtained magnitude of intrinsic binding constant (Kb) values for compounds 1a, 1b, 1c, 1d, 1e, 1f and 1g were 1.2X104 M-1, 2.8X104 M-1, 2.6X104 M-1, 2.4X104 M-1, 2.9X104 M-1, 2.8X104 M-1 and 2.5X104 M-1, respectively. The observed Kb values were compared with the classical intercalator ethidium bromide, having Kb value - 6.58X104 M-1. From the results, it was concluded that the compounds 1e, 1f and 1b possess more binding affinity than the remaining compounds. Further, the planarity and extended p-system of the synthesized compounds leads to the intercalation mode of binding with ct-DNA.
[0085] Emission measurements were carried out by using a JASCO spectrometer. Tris-HCl buffer was used as blank to make preliminary adjustments. The excitation wavelength was fixed and the emission range was adjusted before measurements. Fluorescents spectral measurements were recorded using 25 µM of the compounds incubated in 1 mL of buffer solution in the presence or absence of increasing concentration of ct-DNA (0-50 µM) using a quartz cuvette. Based on the DNA binding absorbance results compounds 1b, 1e and 1f possess more binding affinity than the remaining compounds. In this context, the emission spectral analysis was carried out for the following compounds 1b, 1e and 1f. Hypochromism was observed with a decrease in intensity at 300-340 nm (20-6) (Figures 2(i)-2(iii)). The decrease in fluorescence intensity of the compounds 1b, 1e and 1f is indicative of the intercalative binding mode of DNA.
[0086] 2.2: Viscosity measurements: Hydrodynamic measurements that are sensitive to the length change are regarded as the least ambiguous and the most critical test of binding model in solution in the absence of crystallographic structural data. To classify the binding mode of the compounds 1a-1g to DNA, relative viscosity was determined at fixed DNA concentration with varying concentrations of the compounds. The results obtained are compared to those of classical intercalator ethidium bromide (Et Br), which is able to cause significant increase in viscosity of DNA solution (refer Figure 3). The viscosity data are in good agreement with the absorption titration data. Thus, both absorption titration and viscosity measurement study show better interaction of the compounds with ct-DNA.
[0087] 2.3: DNA cleavage activity: Gel electrophoresis was carried out to analyze ct-DNA cleavage activity of the compounds 1a-1g. It was observed from the studies that compound 1e with chloro substituent cleaved the ct-DNA. This cleavage may be due to intercalation of the carbazole units with the DNA strands as the molecules are reported to bind with AT sequences. Further, the interaction of DNA with compounds (1b, 1d and 1f) showed excellent result in diminishing of the band with a prominent streak indicating the DNA cleavage activity of the compounds. All the other compounds show partial cleavage as shown in Figure 4. Finally, it may be concluded that the intercalation of the compounds destabilizes ct-DNA.
[0088] 2.4: In vitro cytotoxic activity: Cytotoxicity of the synthesized compounds against human breast cancer cell line (MCF-7) was evaluated by MTT assay method. The medium without compounds was taken as control and triplicate was maintained for all concentrations. The results were examined by cell viability curves and have been expressed with IC50 values whose concentration ranges from 0.1-100 µM. The cell growth inhibition (%) obtained with varying concentration and continuous exposure for 48h is provided in Figure 5. The compounds having cytotoxicity were found to be concentration-dependent. The growth inhibition increased with increasing the concentration of compounds 1a-1g, as shown in Table 2 and 3.
Table 2: The cytotoxicity evaluation data of synthesized compounds against MCF-7 cell lines by MTT assay
Concentration (µM) % growth inhibition
1a 1b 1c 1d 1e 1f 1g
0.1 0.794 -1.967 -1.412 -0.706 6.619 -0.441 1.028
1 4.677 1.504 3.706 1.1473 16.416 1.323 3.213
10 11.120 6.481 12.621 4.677 84.819 4.677 9.383
100 28.331 71.412 24.889 29.479 97.979 83.759 68.894

Table 3: Cytotoxic activity of compounds against MCF-7 cell line
Compound IC50 (µM)
1a >100
1b 74.21
1c >100
1d >100
1e 3.04
1f 44.26
1g 54.69
Ellipticine 16.72

[0089] Among all compounds, compound 1e showed higher cytotoxicity effect as shown in Figure 6. Figure 7 shows the morphological changes of cancer cells (human breast cancer, MCF-7) induced by the compound 1e at concentrations ranging from 0 µM to 50 µM. At 0 µM (Figure 7(a)) no dead cells were found, minimum dead cells were found in 0.1 µM(Figure 7(b)) and maximum dead cells were found in 50 µM (Figure 7(c)).
[0090] The cytotoxic behavior of the compounds was noted to be in the order 1e > 1f > 1g > 1b > 1a > 1c > 1d. Compounds (1e and 1f) with chloro and bromo substitution showed much better cytotoxic activity than the derivatives having methyl group due to the electron-withdrawing nature of chlorine and bromine. Cytotoxicity of the synthesized carbazoles was compared with that of the ellipticine whose IC50 value is 16.72 µM. Among all compounds, compound 1e with chloro substitution shows better cytotoxicity than ellipticine.
[0091] 2.5: Antioxidant activity: Compounds were evaluated for their free radical (DPPH-1,1-diphenyl-2-picrylhydrazyl) scavenging capacity and compared with standard ascorbic acid as a positive control. All the compounds show good to moderate capacity for scavenging DPPH with the concentration beyond 100µg/mL. The compounds (1g, 1e and 1f) with groups -OCH3, -Cl and -Br respectively, showed excellent activity as shown in Figure 8. Methyl group substituted compound 1b showed moderate activity compared to ascorbic acid. Compounds 1a, 1c and 1d showed the least activity when compared with standard Ascorbic acid as provided in Table 4 below.
Table 4: Antioxidant activity of compounds
Compound IC50(µg/mL)
1a 110.7
1b 99.4
1c 143.8
1d 108.2
1e 78.2
1f 85.9
1g 71.5
Ascorbic acid 66.2

[0092] 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.

ADVANTAGES OF THE INVENTION
[0093] The present disclosure provides compounds with anti-cancer and free radical scavenging activity.
[0094] The present disclosure provides compounds that destabilize ct-DNA and show better cytotoxicity than Ellipticine.

We Claims:

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

Formula I
wherein R1, R2 and R3 are independently selected from hydrogen, halogen, -(C1-C6)alkyl, (C1-C6)alkoxy, -NH2, -OH, cyano, nitro, -COOH, -OR4, -SO2, and -COOR4; and wherein R4 is -(C1-C6)alkyl.
2. The compound as claimed in claim 1, wherein R1, R2 and R3 are independently selected from hydrogen, -(C1-C6)alkyl, halogen, or (C1-C6)alkoxy.
3. The compound as claimed in claim 1, wherein R1, R2 and R3 are independently selected from hydrogen, -CH3, bromine, chlorine, and -O-CH3.
4. The compound as claimed in claim 1, wherein the compound of Formula I is selected from:
Ethyl 3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,7-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,8-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 3,9-dimethyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-chloro-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-bromo-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate;
Ethyl 7-methoxy-3-methyl-1,4,5,10-tetrahydropyrrolo[2,3-a]carbazole-2-carboxylate
or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
5. A pharmaceutical composition comprising an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof; and a pharmaceutically acceptable excipient
.

Formula I
6. A process of preparing an anti-cancer and anti-oxidant pharmaceutical compound of Formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,
wherein the process comprises the steps of: (a) adding sodium nitrate to a mixture of glacial acetic acid and a compound of Formula A followed by stirring; and (b) reacting the solution of step (a) with zinc powder and ethyl acetoacetate to give the compound of Formula I