FIELD OF THE INVENTION
The present invention relates to novel 3,5-dimethyl pyrazole compounds capable of being used as anticancer compounds. The invention also relates to a process of synthesis of novel 3,5-dimethyl pyrazole compounds. The invention further relates to pharmaceutical composition for treating cancer comprising novel 3,5-dimethyl pyrazole compounds or a pharmaceutically acceptable salt thereof as an active ingredient.

BACKGROUND OF THE INVENTION
Cancer is a term used for large group of diseases caused by rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs; the latter process is referred to as metastasis. Cancerous cells spread into, or invade, nearby tissues and can travel to distant places in the body to form new tumors. Types of cancer are usually named for the organs or tissues where the cancers form. For example, lung cancer starts in the lung, and brain cancer starts in the brain. Cancers also may be described by the type of cell that formed them, such as an epithelial cell or a squamous cell. Some categories of cancers are sarcoma, carcinoma, germ cell tumor, lymphoma, leukemia and blastoma.
According to World Health Organization (WHO) (3 February 2022), Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020, or nearly one in six deaths. The most common cancers are breast, lung, colon and rectum and prostate cancers.

The prognosis for treatment of many forms of cancer remains poor. Conventional cancer chemotherapy is seriously limited by tumor cells exhibiting multidrug resistance, caused by the over expression of integral membrane transporters (eflux pumps), such as Pgp and MDR associated proteins (MRPs) which decreases the drug accumulation and cell death and toxic effects.
Improvement in treatment and prevention has led to a decrease in cancer death. Therefore, number of new drug synthesis and their pre clinical trials are rising. There are various class of compounds that have been evaluated for potential anticancer activity. Some of these are quinazoline, pyrimidine, pyrrole, guanine, chalcone etc.

Pyrazoles are also a promising scaffold for many anticancer agents. A number of clinical anticancer therapeutics, such as crizotinib, ruxolitinib, niraparib, encorafenib, and darolutamide, currently consist of a pyrazole moiety. However, there is a continuous quest to find new pyrazole compounds having better therapeutic profile as anticancer agents.

Abdulrhman Alsayari et. al.; “Anticolon Cancer Properties of Pyrazole Derivatives
Acting through Xanthine Oxidase Inhibition”; Journal of Oncology Volume 2021, discloses pyrazole derivatives as anticancer compounds for colon cancer. It optimized the anticancer potential of arylhydrazono-pyrazole derivatives with different substitutions at the phenyl ring. The compounds were evaluated for antiproliferative activity against three human tumor cell lines, namely, hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116), and breast cancer (MCF-7) by the sulforhodamine B assay and autodocking studies. It identifies 1-isonicotinoyl-3-methyl-4-[2-(4-nitrophenyl) hydrazono]-2-pyrazolin-5-one, as a potential anticancer lead molecule.

US10039749B1 discloses substituted pyrazole derivatives having anticancer properties against hepatic cancer (HePG-2) cell line. The document in particular identifies (4Z)-4-((1-(3-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4yl)methylene)-4,5-dihydro-5-imino-1-phenyl-1H-pyrazol-3-amine (Structure A) as a potential compound for therapeutic treatment.

Structure A
WO2006021881 discloses pyrazole-substituted aminoheteroaryl compounds (Structure B) as protein kinase inhibitors.

Structure B
The lead compounds are potent inhibitors of the c-Met protein kinase, and are useful in the treatment of cancer.

Nashwa M. Saleh et. al.; “Novel Anticancer Fused Pyrazole Derivatives as EGFR and VEGFR-2 Dual TK Inhibitors”; Front. Chem., 24 January 2020 discloses fused pyrazole derivatives useful as anticancer compounds. The compounds act by inhibiting EGFR and VEGFR. Some of the compounds (Such as structure C , D and E) showed nearly 10 fold more activity than erlotinib with IC50 ranging from 0.31 to 0.71µM.
????????
Structure C Structure D Structure E

Farid Abrigach et. al.; “Library of synthetic compounds based on pyrazole unit: design and screening against breast and colorectal cancer”; Bentham Science Vol. 11, Issue 8, 2014; identified new active molecules from a synthetic library of 14 nitrogen compounds. All these compounds exert antiproliferative activity against breast and colorectal cancer cell lines with varying IC50 values (the half-maximal inhibitory concentration, which is a measure of the effectiveness of a compound in inhibiting biological or biochemical function). Researchers found a one log order difference in activity among the different tested compounds. The most active compound 7 showed an IC50 values equal to 8.5µg/ml in both MDA-MB 231(breast cancer) and LOVO (colorectal cancer) cell lines.

In spite of many anticancer moieties, there is still search for novel compounds that can have better therapeutic profile.

A wide array of biological potentials of 3,5-dimethyl pyrazole has motivated strong worldwide efforts to develop their structural derivatives, where dissimilar establishment and biological activity could permit them to be used as novel chemotherapeutic agents. There has also been a considerable interest in the Boc, secondary amine, chalcone incorporated heterocyclic systems, both with regard to their chemistry and pharmacological activities. The present invention is based on strategic experimentation based on biological potentials of bioactive pharmacophores of 3,5-dimethyl pyrazole as chemotherapeutic agents to develop novel compounds effective in the treatment of different categories of cancers.

OBJECTS OF THE INVENTION
The main object of the present invention is to provide novel 3,5-dimethyl pyrazole compounds capable of being used as anticancer compounds.

Another object of the present invention is to provide novel 3,5-dimethyl pyrazole compounds represented by Formula I, its isomer, tautomer, solvate and hydrate thereof, pharmaceutically acceptable salts, pharmaceutically acceptable derivatives including crystalline solid or amorphous forms capable of being used as anticancer compounds.

Yet another object of the present invention is to provide novel compound (5) 3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one for use in treatment of cancer.

Yet another object of the present invention is to provide a process to obtain novel 3,5-dimethyl pyrazole compounds represented by Formula I capable of being used as anticancer compounds.

Yet another object of the present invention is to provide novel pharmaceutical composition comprising 3,5-dimethyl pyrazole compounds for use in treatment of cancer.

Yet another object of the present invention is to provide novel pharmaceutical composition comprising novel 3,5-dimethyl pyrazole compounds or a pharmaceutically acceptable salt thereof as an active ingredient.

SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, process, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Accordingly, in a first aspect, the present invention provides 3,5-dimethyl pyrazole compounds for use as anticancer compounds. More specifically, the present invention relates to compound of Formula (I):

Formula (I)
wherein R represents a group selected from bromine, chlorine, nitrogen dioxide, methoxy or hydroxyl.

The invention also relates to the compound of Formula (I), its isomers if exist, and pharmaceutically acceptable salts thereof.

In a second aspect, the present invention provides compound of Formula (I) wherein R represents methoxy group (-OMe), its isomers if exist, and pharmaceutically acceptable salts thereof.

The invention covers all pharmaceutically acceptable derivatives of the compound of Formula (I).

In a third aspect, the invention provides crystalline solid or amorphous forms of the pharmaceutically acceptable salts of compounds of Formula (I).

In a fourth aspect, the invention provides pharmaceutical compositions for preventing or treating cancer or related conditions in a mammal. The composition contains a therapeutically effective amount of one or more compounds of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.
In a fifth aspect, the pharmaceutical composition is in the form of a tablet, capsule, drop, suppository, oral solution, injectable solution or ointment.

In a sixth aspect, the invention provides a method for preventing or treating cancer or related conditions in a mammal by administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

In a seventh aspect, the invention provides methods for preparing compounds of Formula (I), their crystalline solid or amorphous forms and pharmaceutical compositions for preventing or treating cancer or related conditions in a mammal.

In an eighth aspect, the pharmaceutical composition of the present invention is capable of being used in treating the wide categories of cancers such as pancreatic cancer, breast cancer, brain tumor, glioma, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, esophageal cancer, stomach cancer, kidney cancer, endometrial cancer, cervical cancer, ovary cancer, retinoblastoma, prostate cancer, testicular tumor, liver cancer, skin cancer, colon cancer and rectal cancer.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EXAMPLES AND EMBODIMENTS:
The present invention provides novel 3,5-dimethyl pyrazole compounds of Formula (I), their derivatives and crystalline solid and amorphous forms thereof, and process of synthesis thereof. The compounds of Formula (I) and pharmaceutical salts thereof are used for preventing or treating wide categories of cancer or related conditions in a mammal.

Definitions
In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.

The phrase "a" or "an" entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more",and "at least one" can be used interchangeably herein.

The term "polymorphs" as used herein means crystal structures in which a compound can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, *infrared spectra, melting points/endotherm maximums, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate.

The term "solid form" as used herein means crystal structures in which compounds can crystallize in different packing arrangements. Solid forms include polymorphs, hydrates, and solvates as those terms are used in this invention. Different solid forms, including different polymorphs, of the same compound exhibit different x-ray powder diffraction patterns and different spectra including infra-red, Raman, and solid-state NMR. Their optical, electrical, stability, and solubility properties may also differ.

The term “mammal” includes, without limitation, humans, domestic animals (e.g., dogs or cats), farm animals (cows, horses, or pigs), monkeys, rabbits, mice, and laboratory animals.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-l 4 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

As used herein, the term "preventing" refers to the prophylactic treatment of a patient in need thereof. The prophylactic treatment can be accomplished by providing an appropriate dose of a therapeutic agent to a subject at risk of suffering from an ailment, thereby substantially averting onset of the ailment.

As used herein, the term "treating" refers to providing an appropriate dose of a therapeutic agent to a subject suffering from an ailment.

As used herein, the term “therapeutically effective amount" refers to an amount of a therapeutic agent that is sufficient to affect the treatment of a subject suffering from an ailment.

As used herein. the term "condition" refers to a disease state for which the compounds, compositions and methods of the present invention are being used against.

Compound of Formula (I) below represent one embodiment of the present invention:

Formula (I)
wherein R is selected from a group consistng of bromine, chlorine, nitrogen dioxide, methoxy or hydroxyl.
.
The invention provides compound represented by Formula I, polymorphs, isomers, tautomers, solvates and pharmaceutically acceptable salts and hydrates thereof for use in preventing and treating cancer in mammals.
The invention also covers all pharmaceutically acceptable derivatives of the compounds of Formula (I) and pharmaceutically acceptable salts thereof.

Within the description above are a number of embodiments, consisting of group compounds 1 to 24.

In one group of preferred embodiments, R is selected from bromine, chlorine, nitrogen dioxide, methoxy or hydroxyl, represented herein as compounds 5a to 5e:

5a 5b 5c

5d 5e
In one of the most preferred embodiments of the compound of Formula (I), the R is methoxy.

5d
In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 9a, 9b, 9c, 9d, 9e as illustrated below:

9a 9b

9c 9d

9e
In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 11a, 11b, 11c, 11d, 11e as illustrated below:

11a 11b 11c

11d 11e

In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 12a, and 12b as illustrated below:

12a 12b

In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 13, 14a, 14b, 14c as illustrated below:

13 14a

14b 14c

In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 18, 19, 20 as illustrated below:

18 19 20
In another group of embodiments, the group of compounds of 3,5-dimethyl pyrazole are selected from group compounds 21, 22, 23 and 24 as illustrated below:

21 22

23 24
The schemes 1 to 6 have been drawn to show the strategies which have been employed for the synthesis of the 3,5-dimethylpyrazole compounds.

As illustrated in Scheme 1, 3,5-dimethylpyrazole (1) has been allowed to react with bromo acetophenone in the presence of alkali metal carbonate in organic solvent at a temperature in the range of 80oC - 95oC to obtain compound (3). Compound (3) is reacted with compound from a group selected from compound(4(a-e)) to yield compound (5(a-e)) in the presence of alkali metal hydride in an organic solvent at a temperature in the range of 75oC - 90oC. The compound (4a-e)) is selected from a group of p-bromo benzaldehyde, p-chloro benzaldehyde, p-nitro benzaldehyde, p-methoxy benzaldehyde, p-hydroxy benzaldehyde.
Scheme 1: Synthesis of compounds 5(a-e)
wherein (a) represents reaction conditions of obtaining compound (3) from compound (1) and (2) and (b) represents reactions conditions of obtaining compound (5(a-e)) from compound (3) and compound (4(a-e).

In a preferred embodiment, (a) represents presence of K2CO3 and acetonitrile and a temperature of 85oC for 5 h, and (b) represents presence of NaH and DMF at 81oC for 10 h.

Scheme 2 illustrates method of preparing of compounds (9(a-e)) and compounds (11(a-e)). 3,5-dimethylpyrazole has been allowed to react with different derivatives of bromo acetophenone (6(a-e)) to get compounds 7(a-e). The compounds (7)a-e)) reacts with compound (8) to synthesize compounds (9(a-e)). The compounds (7)a-e)) reacts with compound (10) to synthesize compounds (11(a-e)).
Scheme 2: Synthesis of compounds 9(a-e) and 11(a-e)
wherein reagents and conditions are represented by (c) and (d), wherein (c) is acetonitrile, K2CO3, reflux, 5h, 85oC; and (d) is MeOH, NaH, 0 o C, stirring 12 h; e = MeOH, NaH, 0 o C, stirring 12 h.

Scheme 3 illustrates method of preparing the compounds 12(a-b).

Scheme 3: Synthesis of compounds 12(a-b)
wherein reagents and conditions are represented as (f) being NH2OH.HCl, reflux, 5 h, 85 oC.

Scheme 4 illustrates the method of preparing compounds 13 and 14a, 14b and 14c. The compound (1) has been acetylated at 4-position in the presences of polar aprotic solvent to get compound (13). Compound 13 was introduced into Mannich reaction, as it is the only position for reaction in pyrazole with polar (hydrophilic) aprotic solvents as well as in polar protic solvents with secondary amines in the presence of formaldehyde (14(a-c)).
Scheme 4: Synthesis of compound 13 and 14(a-c)
wherein reagents and conditions being (g) = 96% H2SO4, reflux, 10 h, HCl; (h) = 50% ethanol, 37% Formalin, rt/1h, 100 o C, 3h.
Scheme 5 illustrates the method of preparing compound 18, 19 and 20.
Scheme 5: Synthesis of various substituted chalcone intermediate (18-20)
wherein reagents and conditions are (i) = acetonitrile, K2CO3, reflux, 5h, 85o C; (j) = acetonitrile, K2CO3, reflux, 5 h, 85 o C; (k) = acetonitrile, K2CO3, reflux, 5h, 85 o C.
Scheme 6 illustrates method of preparing compounds 21, 22, 23 and 24.
Scheme 6: Synthesis of substituted chalcone derivative (21-24)
wherein reagents and conditions are: (l) = ethanol, 40% KOH, 10 o C, 4-5 h; (m) = ethanol, 40% KOH, 10 o C, 4-5 h; (n) = ethanol, 40% KOH, 10 o C; (o) = ethanol, 40% KOH, 10 o C

Synthesized compounds have been tested for in vitro anticancer activities employing Human ovary cancer cell line (SK-OV-3), Human lung cancer cell line (A-549) and Human breast cancer cell lines (MCF-7) using Sulforhodamine-B colorimetric assay.

According to the invention, compounds of Formula (1) may be further treated to form pharmaceutically acceptable salts.

The invention also provides pharmaceutically acceptable isomers, hydrates, and solvates of compounds of formula (I). Compounds of formula (I) may also' exist in various isomeric and tautomeric forms including pharmaceutically acceptable salts, hydrates and
solvates of such isomers and tautomers.

Anticancer Activity
The anti- proliferative effect of synthesized compounds has been studied on selected cell line using Sulforhodamine-B assay. Adriamycin has been taken as standard.

The Sulforhodamine-B gets attached to a protein part of the cells in the test sample that have been set to tissue culture plates by tricholoacetic acid (TCA). In order to perform in vitro anticancer activity of novel synthesized compounds, the cell lines namely Human Ovary cancer cell line (SK-OV-3), Human Lung cancer cell line (A-549), Human Breast cancer cell lines (MCF-7) have been employed.

The test compound was firstly dissolved in dimethyl sulfoxide (DMSO) medium at 100mg/ml and diluted to 1mg/ml with the help of water and stored frozen as frozen concentrate prior to use.
At the time of addition of test compound in test sample, an aliquote of the frozen concentrate (1mg/ml) was thawed and diluted to 100 µg/ml, 200 µg/ml, 400 µg/ml and 800 µg/ml with DMSO. Aliquots of 10 µl of these different dilutions were added to the appropriate microliter wells already containing 90 µl of medium, resulting in the required final test compound concentrations i.e.10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml.

The test sample is prepared by growing cell lines in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. For present screening experiment, cells were inoculated into 96 well microliter plates in 100 µL at plating densities, depending on the doubling time of individual cell lines. After cell inoculation, the microliter plates were incubated at 37°C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of the test compound.

After addition of the test compound, plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold trichloroacetic acid (TCA). Cells were fixed in situ by the gentle addition of 50 µl of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded. The plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1% acetic acid was added to each of the wells and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma (2-Amino-2-(hydroxymethyl)-1,3-propanediol) base and the absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm reference wavelength.

Under mild acidic condition SRB binds to cell protein and under the mild basic condition it detached or recovered from cell proteins and solubilized for measurement of absorbance. After staining, a bounded dye was recovered with trizma base (10mM), and the residual dye was removed by washing five times with 1% acetic acid.
The absorbance was recorded by micro-plate reader at a wavelength of 540 nm with
690 nm reference wavelength.

Calculations for measurement of anticancer activity are carried out using Six absorbance measurements method. This is precise measurement of the absorbance at many wavelengths allow the identification of substance via absorption spectroscopy.

Terms, abbreviations and symbols for calculation:
The terms, abbreviations and symbols used in the screening of anti-cancer activity and in the calculation are:

? Std: Standard
? T: The optical density of the test well after a 48-h period of exposure to test drug.
? Tz: Absorbance of SRB/ optical density of test well at at Time zero/ Ti: Initial Time (both are same)
? Tz (ADR): Absorbance of SRB/ optical density of test well at Time zero for cells treated with std. drug Adriamycin.
? T (test): Absorbance of SRB/ optical density of test well at Time zero for cells treated with test compounds.
? C: Absorbance of SRB/ optical density of test well of Control growth (untreated cells: cell lines without std/test treatment)
? Tf: is the final absorbance of the solubilized Sulforhodamine B (SRB) (representing the number of cells) / final optical density of test well of the four different concentrations (Four concentration levels) of the compound used in the std/test to see the growth in cells
? Concentration level: A series of concentration are known as levels. These are presented on the X axis of a graph. The percentage growth was measured at each of the compound concentration levels. Percentage cell growth was calculated on a plate-by-plate (well) basis. The test wells were correlated to control wells.
? Concentration response parameter selected for the study:
o LC50: LC signifies a cytotoxic effect. LC50 is the concentration of drug causing 50% cell death indicating a net loss of cell following treatment. This indicates the concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to the beginning.
o GI50: GI signifies growth inhibitory power. GI50 is the concentration of drug resulting in 50% inhibition of cell growth/ net protein increase (as measured by SRB staining) in control cells during the drug incubation.
o TGI: TGI signifies a cytostatic effect. TGE is the concentration of drug causing Total Inhibition of Cell growth, indicating net loss of protein at the end of the drug treatment.
? ADR: Adriamycin, Positive control compound
? NE: Non-evaluable data
? SK-OV-3: Human Ovary cancer cell line
? A-549: Human Lung cancer cell line
? MCF-7: Human Breast cancer cell lines
? NE: Non- evaluable data. Experiment needs to be repeated using different set of drug concentrations.
? NA: Non-Analyzable due to microbial contaminant

Formulae for Measurement:
1. Percentage cell growth =
Average absorbance of the test well x 100
____________________________
Average absorbance of the control wells

2. Percent growth inhibition =100 – percentage cell growth
3. Percentage cell growth when four concentration levels is very high than initial time ie Tf = Ti

GI50 = = 50
4. Percentage cell growth when four concentration levels is very less than initial time ie Tf80 >80 74.1
5c NE NE 61.7
5d 47.7 <10 <10
5e NE NE >80
12a NE NE 13.1
12b NE NE NE
13 NE NE >80
14a NE >80 >80
14b >80 >80 55.1
14c NE NE >80
18 NE NE >80
19 NE NE >80
20 NE NE >80
21 NE >80 >80
22 NE NE >80
23 NE >80 >80
24 NE >80 >80
9a NE NE 74.1
9b NE NE >80
9c >80 65.5 39.5
9d NE >80 >80
9e NE >80 >80
11a NE >80 >80
11b NE >80 >80
11c NE >80 >80
11d NE >80 >80
11e NE >80 71.3
ADR NE <10 <10

Table 2 shows that the compound 5d shows optimum anticancer activity in terms of LC, GI and TGI at the concentration in the range of 20 µg/ml to 80 µg/ml. The optimum concentration of compound 5d showing anticancer activity is 40 µg/ml.

Table 3: In vitro anticancer activity of test compounds against human ovarian cancer cell lines (SK-OV-3)

Compounds Std/Test compound Concentrations (µg/ml)
10 20 40 80
Average percentage Control Growth
5a 85.3 94.1 83.3 47.3
5b 141.9 129.2 108.4 89.1
5c 95.2 99.8 97.4 88.2
5d -40.1 -42.7 -59.1 -54.6
5e 119.3 141.2 118.4 111.1
12a 73.5 72.9 34.7 -9.1
12b 86.7 96.8 94.3 89.8
13 113.7 127.1 120.1 109.3
14a 94.3 85.6 78.6 50.9
14b 104.7 119.3 -30.7 -53.0
14c 113.0 122.5 116.0 104.8
18 122.9 122.7 122.7. 114.8
19 106.2 119.6 108.1 118.1
20 101.1 111.8 88.3 114.5
21 114.6 117.6 114.6 119.4.
22 111.9 138.2 118.4 132.1
23 128.5 105..6 112.9 125.6
24 111.4 111.9 115.9 116.4
9a 111.9 138.2 118.4 132.1
9b 117.5 103.0 107.3 114.8
9c 58.1 55.2 23.8 7.2
9d 106.0 113.4 117.5 144.0
9e 111.0 111.9 116.0 136.7
11a 122.1 109.0 117.8 121.5
11b 128.2 103.0 124.4 143.3
11c 122.9 122.7 122.7 100.3
11d 115.7 118.4 114.6 117.1
11e 93.1 84.6 75.3 43.3
ADR -39.2 -43.3 -57.7 -57.1

Table 3 shows that the synthesized 3,5-dimethyl pyrazole compounds show anticancer activity at the concentration rate of 20 µg/ml to 80 µg/ml. The compound 5d shows optimum anticancer activity at the concentration of 20 µg/ml to 80 µg/ml. The optimum concentration of compound 5d showing anticancer activity is 40 µg/ml.

Table 4: In vitro anticancer activity of test compounds against human ovarian cancer cell lines (SK-OV-3)

Compounds LC50 TGI GI50*
5a NE NE >80
5b NE NE >80
5c NE NE NE
5d 41.6 <10 <10
5e NE NE >80
12a NE 71.9 31.9
12b NE NE NE
13 NE NE >80
14a NE >80 >80
14b 71.0 51.3 31.6
14c NE NE >80
18 NE NE >80
19 NE NE >80
20 NE NE >80
21 NE >80 >80
22 NE NE >80
23 NE >80 >80
24 NE >80 >80
9a NE NE >80
9b NE NE >80
9c >80 >80 19.3
9d NE >80 >80
9e NE >80 >80
11a NE >80 >80
11b NE >80 >80
11c NE >80 >80
11d NE >80 >80
11e NE >80 71.9
ADR 40.2 <10 <10

GI50 value of = 10^-6 molar (i.e. 1 µmolar) or = 10µg/ml is considered to demonstrate activity in case of pure compounds.

Table 4 shows that the compound 5d shows optimum anticancer activity in terms of LC, GI and TGI at the concentration in the range of 20 µg/ml to 80 µg/ml. The optimum concentration of compound 5d showing anticancer activity is 40 µg/ml.

Table 5: In vitro anticancer activity of test compounds against human lung cancer cell lines (A-549)

Std/test compound Std/Test compound Concentrations (µg/ml)
10 20 40 80
Average percentage Control Growth
5a 116.5 117.2 104.0 66.1
5b 99.5 81.3 72.8 61.6
5c 87.3 96.4 101.7 98.3
5d 12.7 13.2 -30.7 -15.3
5e 103.5 91.3 78.8 59.6
12a 100.4 88.6 56.5 26.5
12b 107.4 116.1 115.2 105.9
13 92.8 94.5 83.3 77.4
14a 105.2 103.4 98.6 99.8
14b 84.1 84.1 80.1 75.2
14c 95.8 87.3 37.8 -46.3
18 100.4 100.5 104.5 106.8
19 47.2 45.1 24.7 20.1
20 64.0 42.5 27.6 22.8
21 100.5 96.5 98.2 100.5
22 97.5 95.3 85.8 91.6
23 105.4 104.5 100.5 98.2
24 102.4 98.2 96.3 83.6
9a 97.5 95.3 85.8 91.6
9b 95.8 91.3 80.0 83.5
9c 100.5 106.3 98.8 77.9
9d 101.6 105.9 96.5 96.7
9e 100.2 100.1 83.3 79.4
11a 100.5 107.5 100.1 99.7
11b 101.1 108.4 103.4 105.5
11c 103.3 107.4 99.9 99.2
11d 104.6 105.1 98.6 103.0
11e 105.1 89.2 63.2 53.2
ADR -23.9 -32.9 -16.7 -18.3

Table 5 shows that synthesized 3,5-dimethyl pyrazole compounds shows anticancer activity at the concentration of 20 µg/ml to 80 µg/ml. The compound 5d shows optimum anticancer activity at the concentration of 20 µg/ml to 80 µg/ml. The optimum concentration of compound 5d showing anticancer activity is 40 µg/ml.

Table 6: In vitro anticancer activity of test compounds against Human Lung cancer cell lines (A-549)

Compounds LC50 TGI GI50*
5a NE NE >80
5b NE NE >80
5c NE NE NE
5d NE 26.4 <10
5e NE NE >80
12a NE NE 54.5
12b NE NE NE
13 NE NE >80
14a NE >80 >80
14b NE NE >80
14c 82.1 58.3 34.5
18 NE NE >80
19 NE NE <10
20 NE NE 16.4
21 NE >80 >80
22 NE NE >80
23 NE >80 76.9
24 NE >80 >80
9a NE NE >80
9b NE NE >80
9c NE NE >80
9d NE >80 >80
9e NE >80 >80
11a NE >80 >80
11b NE >80 >80
11c NE >80 >80
11d NE >80 >80
11e NE >80 76.7
ADR NE <10 <10

Table 6 shows that the compound 5d shows optimum anticancer activity in terms of LC, GI and TGI at the concentration in the range of 20 µg/ml to 80 µg/ml. The optimum concentration of compound 5d showing anticancer activity is 40 µg/ml.

In a best embodiment, among synthesized compounds screened for anticancer activity, (Z)-3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one has been found most active against all the three human cancer cell lines used.
The effective concentration/dosage varies according to the age and weight of the patient, the administration route, the nature and severity of the disorder, and the administration of any associated treatments and ranges from 20 µg/ml to 80 µg/ml per day in one or more administration.

The invention also provides a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula (I) or derivatives or polymorphs or isomers or tautomers thereof or pharmaceutically acceptable salts thereof
Formula I
The pharmaceutical composition is in the form of a tablet, capsule, drop, suppository, oral solution, injectable solution or ointment. The composition comprises a therapeutically effective amount of a compound of Formula I, and a pharmaceutically acceptable salt thereof.

The composition comprises compound 3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one as an active ingredient. More specifically, the pharmaceutical composition comprises (Z)-3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one as an active ingredient.

The pharmaceutical composition of the present invention in used in preventing and treating pancreatic cancer, breast cancer, brain tumor, glioma, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, esophageal cancer, stomach cancer, kidney cancer, endometrial cancer, cervical cancer, ovary cancer, retinoblastoma, prostate cancer, testicular tumor, liver cancer, skin cancer, colon cancer and rectal cancer.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

We Claim:

1. A compound represented by Formula I, polymorphs, isomers, tautomers, solvates and pharmaceutically acceptable salts and hydrates thereof,

Formula I
wherein R is selected from Chloro (-Cl), Bromo (-Br), hydroxyl (-OH), methoxy (-OCH3) and Nitro (-NO2) group.
2. The compound as claimed in claim 1 wherein R is methoxy group (-OCH3).
3. The compound as claimed in claim 1 wherein the compound is 3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one represented as compound (5d) for use in treatment of cancer.

Compound 5(d)
4. The compound as claimed in claim 1 wherein said compound is (Z)-3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one.
5. The compound as claimed in claim 1 wherein said compound includes all pharmaceutically acceptable derivatives of the compounds of Formula (I).
6. The compound as claimed in claim 1 wherein the compound includes crystalline solid or amorphous forms of the pharmaceutically acceptable salts of compounds of Formula (I).
7. The compound as claimed in claim 1, wherein said compound is capable of being used in the manufacture of a medicament for the treatment of cancer.
8. A process for the preparation of compounds of formula I, said process comprising:
(i) reacting 3,5-dimethylpyrazole (compound 1) with bromoacetophenone (compound 2) to obtain 1-benzoyl-3,5-dimethyl 1H-pyrazole (compound 3) in the presence of alkali metal carbonate and organic solvent at a temperature ranging from 80oC to 95oC,
(ii) reacting 1-benzoyl-3,5-dimethyl-1H-pyrazole (compound 3) with compound (4(a-e)) to obtain compound of Formula I (5(a-e)) in the presence of alkali metal hydride and organic solvent at a temperature ranging from 80oC to 95oC,

9. The process as claimed in claim 8, wherein in step (i) said alkali metal carbonate is K2CO3 and said organic solvent is acetonitrile, and said temperature is 85oC for 5 h.
10. The process as claimed in claim 8, wherein in step (ii) said alkali metal hydride is NaH and said organic solvent is DMF, and said temperature is 81oC for10 h.
11. The process for the preparation of compounds of formula I as claimed in claim 8, wherein the compound (4) is selected from a group of p-bromo benzaldehyde, p-chloro benzaldehyde, p-nitro benzaldehyde, p-methoxy benzaldehyde, p-hydroxy benzaldehyde.
12. The process for the preparation of compounds of formula I as claimed in claim 8, wherein -R in compound (5) is -OCH3.
13. A pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula (I) or derivatives or isomers or tautomers thereof or pharmaceutically acceptable salts thereof
Formula I
14. The pharmaceutical composition of claim 13, wherein the composition is in the form of a tablet, capsule, drop, suppository, oral solution, injectable solution or ointment.
15. The pharmaceutical composition of claim 13 comprising a therapeutically effective amount of a compound of Formula I, and a pharmaceutically acceptable salt thereof.
16. The pharmaceutical composition of claim 13, wherein said compound is 3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one.
17. The pharmaceutical composition of claim 13, wherein said compound is (Z)-3-(4-methoxyphenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-phenylprop-2-en-1-one.
18. The pharmaceutical composition as claimed in claim 13 wherein the cancer is pancreatic cancer, breast cancer, brain tumor, glioma, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, esophageal cancer, stomach cancer, kidney cancer, endometrial cancer, cervical cancer, ovary cancer, retinoblastoma, prostate cancer, testicular tumor, liver cancer, skin cancer, colon cancer and rectal cancer.