WO2023209677A2

WO2023209677A2 - A novel aryl azo pyrazole compound and its synthesis

Info

Publication number: WO2023209677A2
Application number: PCT/IB2023/054469
Authority: WO
Inventors: Arpan B PATEL
Original assignee: Zenal Pharmachem Llc
Priority date: 2022-04-29
Filing date: 2023-04-28
Publication date: 2023-11-02
Also published as: WO2023209677A3

Abstract

The present invention relates to novel aryl azo pyrazole compound, having the following structural formula (I-3) which can be used as antimicrobial, anti-inflammatory and antipyretic agent. The present invention also provides novel process for synthesis of novel aryl azo pyrazole compound of structural formula (I-3).

Description

NOVEL ARYL AZO PYRAZOLE COMPOUND AND ITS SYNTHESIS FIELD OF THE INVENTION The invention relates to a novel aryl azo pyrazole compound of formula (I-3), or a pharmaceutically acceptable salt thereof. Particular aspects of the invention relates to a method of synthesizing the compounds and the use of those compounds in treating, ameliorating, or preventing microbial infection and inflammation.

BACKGROUND OF THE INVENTION Antimicrobial agents are natural or synthetic substances that kills or inhibits the growth of microorganisms such as bacteria, fungi and algae. The production and use of the antibiotic penicillin in the early 1940s became the basis for the era of modern antimicrobial therapy and since then many antibiotics and other types of antimicrobials have been found. Major discovery following the introduction of these agents into medicine was the finding that their basic structure could be modified chemically to improve their characteristics. Widespread use of antimicrobial agents in recent decades has led to proliferation of pathogens having multiple drug resistance, often encoded by transmissible plasmids, and therefore capable of spreading rapidly between species. Many previously useful antimicrobial agents are no longer effective against infectious organisms isolated from human and animal subjects. The spectre of epidemic forms of bacterial diseases such as tuberculosis and fungal diseases, which are refractory to known antibiotic agents, may be realized in the near future. Development of novel antimicrobial compounds is a continuing urgent public health need. The discovery and development of new antimicrobial agents have been for decades a major focus of many pharmaceutical companies. Nonetheless, in more recent years there has been an exodus from this area of research and drug development resulting in very few new antimicrobials entering the market. This lack of new antimicrobial is particularly disturbing, especially at a time when bacterial resistance to current therapies is increasing both in the hospital and community settings. Thus, there is a need to develop new antimicrobial compounds. The present invention arose due to the inventor's interest in pyrazole heterocyclic moiety and developed novel aryl azo pyrazole compound as Antimicrobial and Antifungal Agent. OBJECTIVE OF THE INVENTION The main objective of the present invention is to provide novel aryl azo pyrazole compound, having the following structural formula (I-3).

Another objective of the present invention is to provide a process of synthesizing the novel aryl azo pyrazole compound of formula (I-3). Another objective of the present invention is to provide novel aryl azo pyrazole compound of formula (I-3) which can be used as an antimicrobial agent, anti- inflammatory agent and anti-pyretic agent. SUMMARY OF THE INVENTION The main aspect of the present invention is to provide a compound represented by the formula (I-3)

Another aspect of the present invention is to provide the process for preparing the compound having formula (I-3). Another aspect of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I-3), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. In another embodiment, the present invention relates to methods of synthesizing the compound of formula (I-3) and the use of it in treating, ameliorating, or preventing a microbial infection and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: FTIR Sprectra of compound (I-3) Figure 2.1: Mass Sprectra of compound (I-3) (+ve ion) Figure 2.2: Mass Sprectra of compound (I-3) (-ve ion) Figure 3: ¹H NMR Sprectra of compound (I-3) DESCRIPTION OF THE INVENTION The present invention is about aryl azo pyrazole compound as antimicrobial agent, as described herein. The phrase “pharmaceutically acceptable salt(s)”, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulphate, phosphate, acid phosphate, iso-nicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, and ascorbate. Succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, Saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate i.e., 1,1'- methylene-bis-(2- hydroxy-3-naphthoate) salts. The compounds of the present invention that include a basic moiety, such as an amino group, may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. The invention also relates to base addition salts of the compounds of the invention. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds of the invention that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water- soluble amine addition salts such as N-methylglucamine-(meglu mine), and the lower alkanol ammonium and other base salts of pharmaceutically acceptable organic amines. Suitable base salts are formed from bases which form non-toxic salts. Non-limiting examples of suitable base salts include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, Sodium, tromethamine and Zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. The phrase “Antimicrobial Agents” that kills microorganisms or stops their growth. Microorganisms include bacteria, fungi, virus and parasite. The phrase “Anti-inflammatory Agents” are the drugs or substance that reduces inflammation (redness, swelling and pain) in the body. The phrase “Antipyretic Agents” are the drugs or substances that reduces fever in the body. As per the main embodiment, the present invention provides a novel compound (E)- 4-(2-(4-bromophenyl)hydrazono)-1-(4-methoxybenzoyl)-3-methyl-1H-pyrazol- 5(4H)-one represented by the formula (I-3) or pharmaceutically acceptable salts thereof.

As per another main embodiment, a process for preparation of (E)-4-(2-(4- bromophenyl)hydrazono)-1-(4-methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one having formula (I-3) comprises the steps of: a) Preparation of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3- oxobutanoate (Intermediate-1) comprising following steps: i. Dissolving 70g of 4-bromoaniline in a 170 ml of 17% hydrochloric acid and 48 ml of methanol and cool the mixture at 0-5^ºC under stirring. ii. Dissolving 31g of sodium nitrite in 150ml of water and dropwise add the mixture in mixture of step (i). iii. Stirring the mixture of step (ii) at 0-5^ºC for 30 minutes. iv. Dissolving 130g of sodium acetate in 200 ml of water and stirring the mixture to get a clear solution and cooling the solution to 0-5^ºC. v. Adding 53.2g of ethyl acetoacetate to the solution of step (iv) and cooling the mixture to -5-0^ºC. vi. Dropwise adding the mixture of step (v) to that of the mixture of step (iii) to get the white precipitate of product while keeping the temperature below 0-5^ºC for 30 minutes. vii. Stirring the reaction mixture of step (vi) at below 5^ºC for 30 minutes. viii. Filtering the reaction mixture of step (vii) and washing the precipitates with 2500ml of cold water. ix. Drying the compound of step (viii) under vacuum at 45-50^ºC to get 125.5 g of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3- oxobutanoate (Intermediate -1). b) Preparation of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2) comprising following steps: i. Dissolving 31.3g of Intermediate-1 and 16.6g of 4- methoxybenzohydrazide into 100ml of acetic acid at room temperature. ii. Stirring the reaction mixture of step (i) at 25-30^ºC for 2-3 hours (After 1 hour the reaction mixture becomes clear solution and then precipitation occurs after 2 hours). iii. Cooling the reaction mixture of step (ii) to 20-25^ºC and stirring at same temperature for 1 hour. iv. Filtering the precipitated compound of step (iii) and washing it with cold water (2 X 50 mL). v. Drying the compound of step (iv) at 45-50^ºC to get 36.6 g of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2). c) Preparation of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3) comprising following steps: i. Dissolving 36.0g of Intermediate-2 in 360.0ml of toluene and stirring the mixture at room temperature. ii. Refluxing the solution of step (i) under stirring for 4-5 hours. iii. Cooling the mixture of step (ii) to 25-30^ºC and continuously stirring at same temperature for 1 hour. iv. Filtering the crystallized compound of step (iii) and washing it with 80ml of cold toluene. v. Drying the compound of step (iv) under reduced pressure at 45- 50^ºC to get 30.0g of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one Formula (I- 3). The complete reaction scheme of the synthesis of derivatives of the present invention is as follows:

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I-3), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, and suspension, for parenteral injection as a sterile solution, suspension, or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc. Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99% by weight, more preferably from 0.1 to 70% by weight of the active ingredient, and, from 1 to 99.95% by weight, more preferably from 30 to 99.9 weight % of a pharmaceutically acceptable carrier, all percentages being based on the total composition. The pharmaceutical composition may additionally contain various other ingredients known in the art, for example, a lubricant, stabilising agent, buffering agent, emulsifying agent, viscosity-regulating agent, surfactant, preservative, flavouring or colorant. In another embodiment, the present invention relates to methods of synthesizing the compound of formula (I-3) and the use of it in treating, ameliorating, or preventing a microbial infection and inflammation. In another embodiment, the present invention relates to methods of synthesizing the compound of formula (I-3), wherein the compound exhibit Prostaglandin-E2 Synthase-1 inhibitors and non-opioids analgesic activity. Compounds described herein may be in various forms, including but not limited to, amorphous forms, milled forms and nano-particulate forms. In addition, compounds described herein include crystalline forms, also known as polymorphs. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization Solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate. EXAMPLES The following working examples represent preferred embodiments of the present invention. All temperatures are expressed in degrees Centigrade unless otherwise indicated. Example 1: Preparation of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3): a) The process for preparation of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3- oxobutanoate (intermediate-1) comprises the following steps:

i. Dissolving 70g of 4-bromoaniline in a 170 ml of hydrochloric acid and 48 ml of methanol and cool the mixture at 0-5^ºC under stirring. ii. Dissolving 31g of sodium nitrite in 150ml of water and dropwise add the mixture in mixture of step (i). iii. Stirring the mixture of step (ii) at 0-5^ºC for 30 minutes. iv. Dissolving 130g of sodium acetate in 200 ml of water and stirring the mixture to get a clear solution and cooling the solution to 0-5^ºC. v. Adding 53.2g of ethyl acetoacetate to the solution of step (iv) and cooling the mixture to -5-0^ºC. vi. Dropwise adding the mixture of step (v) to that of the mixture of step (iii) while keeping the temperature below 0-5^ºC for 30 minutes. vii. Stirring the reaction mixture of step (vi) at below 5^ºC for 30 minutes. viii. Filtering the reaction mixture of step (vii) and washing the precipitates with 2500ml of cold water. ix. Drying the compound of step (viii) under vacuum at 45-50^ºC to get 125.5 g of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3-oxobutanoate (Intermediate -1). b) Preparation of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2) comprising following steps:

i. Dissolving 31.3g of Intermediate-1 and 16.6g of 4- methoxybenzohydrazide into 100ml of acetic acid at room temperature. ii. Stirring the reaction mixture of step (i) at 25-300C for 2-3 hours. iii. Cooling the reaction mixture of step (ii) to 20-250C and stirring at same temperature for 1 hour. iv. Filtering the precipitated compound of step (iii) and washing it with 100ml of cold water. v. Drying the compound of step (iv) at 45-500C to get 36.6 g of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2). c) Preparation of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3) comprising following steps:

i. Dissolving 36.0g of Intermediate-2 in 360.0ml of toluene and stirring the mixture at room temperature. ii. Refluxing the solution of step (i) under stirring for 4-5 hours. iii. Cooling the mixture of step (ii) to 25-300C and continuously stirring at same temperature for 1 hour. iv. Filtering the crystallized compound of step (iii) and washing it with 80ml of cold toluene. v. Drying the compound of step (iv) under reduced pressure at 45-500C to get 30.0g of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3). Example 2: Analysis of the compound The chemical formula of the compound i. (E)-4-(2-(4- bromophenyl)hydrazono)-1-(4-methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3) is C18H15BrN4O3 having the molecular weight 415.24. Figure 1 illustrates FTIR spectra, Figure 2.1 and 2.2 illustrates Mass spectra, and Figure 3 illustrates ¹H NMR spectra of compound 23. Example 3: Biological Evaluation Antifungal activity: The biological activity such as antifungal activity of compound I-3 was tested on a potato dextrose agar (PDA) medium on each of these vegetable pathogenic strains. The fungicidal activity of compound I-3 was studied at 1000 ppm concentration in vitro. Plant pathogenic organisms used were Aspergillus Niger, Nigrospora Species, Rhizopus Nigricum, Botrydepladia Theobromae and Fusarium Oxyporium. Such a Potato agar medium contained dextrose 20g, potato 200g, agar 20g and water 10 ml. The Compounds to be tested were hovering (1000 ppm) in a PDA medium and autoclaved at 100 °C for 14 minutes at 12 atm. pressure. Old cultures were employed five or more days. These Potato agar media were mixed into sterile Petri plates and the microorganisms were immunized after cooling the Petri plates. The % zonal inhibition calculated for fungi was after five days using the formula given below: % of inhibition = 100 (X-Y) / X Where, Y = test plate Area of colony X = control plate Area of colony Table 1: Antifungal activity of 4-[4-Methoxybenzoyl]-3-methyl-1-(4- bromophenyl)-2-pyrazoline -5-one (Compound I-3)

Antibacterial activity: It was mixed with 0.3-0.8 ml of 48 hour mixed well and old culture especially by normal stirring before adding on the Petri dish sterilized (50 ml in each dish). Nutrient agar broth was added in an aqueous beaker and boiled to 55 °C with occasional shaking to form well mixing. The melted mass was allowed to mix (1.5 hour) and after the mixing the “cups” were made by pressing into the agar with spooning out the pressed part of agar and sterile cork borer. Into this cups 0.10 ml of prepared test solution (prepared by dissolving 10.0 gm of sample in 10 ml Dimethyl formamide) was added by micropipette which was sterile. The plates were noted by appropriate codes. Table 2: Antibacterial activity of 4-[4-Methoxybenzoyl]-3-methyl-1-(4- bromophenyl)-2-pyrazoline -5-one (Compound I-3)

Example 3: Oral Toxicity Study Acute oral toxicity for the compound with Formula (I-3) was done to study of adverse effects of a test item that results either from a single oral exposure or from multiple exposures within 24 hours. The study was conducted on 9 female Wistar Rats that are 8-12 weeks old in age. The animals were subjected to 12 hours light and 12 hours dark cycle maintaining the temperature at 20 – 24.3°C and relative humidity of 49-64%. Treatment Procedure: Animals were fasted overnight before the treatment. The dose volume was calculated based on individual animal body weight. The test item was administered orally. After the test item administered, animals were fasted for further 3-4 hours. Single animal was dosed in sequence usually at 48 hours intervals based on AOT 425 Software and as per the selected slope. As the animal survives, the second animal was receiving a higher dose until the stopping criteria met.

Results: The test compound of Formula I-3 administered by oral route and did not show any clinical sign and mortality/morbidity at any dose level under the tested conditions. Based on the results, the LD50 of the test compound of Formula I-3 for acute oral toxicity was found greater than 5000 mg/kg b.wt and stopping criteria met 3 consecutive animals survive at the upper bound/limit dose.

Claims

CLAIMS We claim, 1. A novel aryl azo pyrazole compound, comprising the formula (I-3),

or a pharmaceutically acceptable salt thereof. 2. A process for preparation of compound claimed in claim 1, comprising the following steps: a) Preparation of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3- oxobutanoate (Intermediate-1) comprising following steps: i. Dissolving 70g of 4-bromoaniline in a 170 ml of 17% hydrochloric acid and 48 ml of methanol and cool the mixture at 0-5^ºC under stirring. ii. Dissolving 31g of sodium nitrite in 150ml of water and dropwise add the mixture in mixture of step (i). iii. Stirring the mixture of step (ii) at 0-5^ºC for 30 minutes. iv. Dissolving 130g of sodium acetate in 200 ml of water and stirring the mixture to get a clear solution and cooling the solution to 0-5^ºC. v. Adding 53.2g of ethyl acetoacetate to the solution of step (iv) and cooling the mixture to -5-0^ºC. vi. Dropwise adding the mixture of step (v) to that of the mixture of step (iii) to get the white precipitate of the compound while keeping the temperature below 0-5^ºC for 30 minutes. vii. Stirring the reaction mixture of step (vi) at below 5^ºC for 30 minutes. viii. Filtering the reaction mixture of step (vii) and washing the precipitates with 2500ml of cold water. ix. Drying the compound of step (viii) under vacuum at 45-50^ºC to get 125.5 g of (E)-ethyl 2-(2-(4-bromophenyl)hydrazono)-3- oxobutanoate (Intermediate -1). b) Preparation of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2) comprising following steps: i. Dissolving 31.3g of Intermediate-1 and 16.6g of 4- methoxybenzohydrazide into 100ml of acetic acid at room temperature. ii. Stirring the reaction mixture of step (i) at 25-30^ºC for 2-3 hours (After 1 hour the reaction mixture becomes clear solution and then precipitation occurs after 2 hours). iii. Cooling the reaction mixture of step (ii) to 20-25^ºC and stirring at same temperature for 1 hour. iv. Filtering the precipitated compound of step (iii) and washing it with cold water (2 X 50 mL). v. Drying the compound of step (iv) at 45-50^ºC to get 36.6 g of ethyl 2-(2-(4-bromophenyl)hydrazono)-3-(2-(4- methoxybenzoyl)hydrazono)butanoate (Intermediate-2). c) Preparation of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Formula I-3) comprising following steps: i. Dissolving 36.0g of Intermediate-2 in 360.0ml of toluene and stirring the mixture at room temperature. ii. Refluxing the solution of step (i) under stirring for 4-5 hours. iii. Cooling the mixture of step (ii) to 25-30^ºC and continuously stirring at same temperature for 1 hour. iv. Filtering the crystallized compound of step (iii) and washing it with 80ml of cold toluene. v. Drying the compound of step (iv) under reduced pressure at 45- 50^ºC to get 30.0g of (E)-4-(2-(4-bromophenyl)hydrazono)-1-(4- methoxybenzoyl)-3-methyl-1H-pyrazol-5(4H)-one (Compound 23). 3. The novel aryl azo pyrazole compound as claimed in claim 1, wherein the said compound is used to prepare pharmaceutical composition comprising a pharmaceutically active compound of Formula (I-3), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. 4. The novel aryl azo pyrazole compound as claimed in claim 1, wherein the pharmaceutical composition is an anti-microbial agent for treating microbial infection. 5. The aryl azo pyrazole derivatives as claimed in claim 1, wherein the said derivatives having LD505000mg/kg.