Potential N-(2- (3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) Acetamide as Anticancer Agent

Abstract

Title: Potential N-(2- (3-methoxyphenyl) amino) methyl)-1H benzo[d]imidazol-5-yl) Acetamide as Anticancer Agent The present invention relates to overcome the loopholes of existing molecules used as anticancer treatment includes a novel seven-membered N-(2- (3-methoxyphenyl) amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide compound through a one-pot reaction with the sequential addition of reagents, wherein R substituted with 3 methoxy, to get final compound of N-(2- (3-methoxyphenyl) amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide, demonstrates significant anticancer activity against using human epitheloid carcinoma cervix (HeLa) cells.

Specification

Description:Title of the invention:
"Potential N-(2- (3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide as Anticancer Agent"

Field of the invention
The present invention to pharmaceutical compound comprising of N-(2- (3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide synthesized via a one pot reaction with the sequential addition of reagents, ensuring efficiency and simplicity in its production and demonstrate anticancer activity against human epitheloid carcinoma cervix (HeLa) cells.
Background of invention:
Cancer remains one of the most devastating global health challenges, with approximately 19.3 million new cases and nearly 10 million cancer-related deaths reported in 2023 alone. The persistent challenges in treating various forms of cancer, coupled with the genotoxicity and cytotoxicity of existing chemotherapeutic treatments, highlight the urgent need for novel and more effective anticancer agents. Current therapies often fall short due to severe side effects and limited efficacy, making it crucial to discover new generations of chemotherapeutic drugs with enhanced potency and reduced toxicity.
A promising direction in anticancer drug development lies in the discovery of 2, 5-Disubstituted Benzimidazole analogues, which present a novel therapeutic approach with notable anticancer potential. These compounds have shown specific efficacy against human epitheloid carcinoma cervix (HeLa) cells, positioning them as potential breakthroughs in cancer treatment. The development of various 2,5-Disubstituted Benzimidazole derivatives, each featuring a precise arrangement of functional groups, has been designed to enhance their anticancer properties. This structural innovation, combined with their efficient synthesis methods, strengthens their potential as candidates for further development, offering the promise of safer and more effective cancer therapies in the future.
The compound is synthesized using one pot reaction method that involve the sequential addition of reagents resulting in a molecule N-(2-(3-methoxyphenyl) amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide. These structural features are critical for the compounds biological activity. The compound has been shown to exhibit significant anticancer activity, particularly against human epitheloid carcinoma cervix (HeLa) cells.
Objective of invention:
The principal objective of the invention is to develop a compound of synthetic origin preferably consist of N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide through experimentation to test its efficacy in human epitheloid carcinoma cervix (HeLa) cells as anticancer agent.
Secondary objective of the invention that the seven membered N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide compound through a one-pot reaction with the sequential addition of reagents
Further objective of the invention is to identify from physical characteristics, and characterize the prepared molecule using IR, 1H-NMR and Mass spectroscopy.
Another objective of the invention is that N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide compound evaluated for cytotoxicity using human epitheloid carcinoma cervix (HeLa) cells
Summary of the Invention
The present invention relates to seven-membered N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide compound through a one-pot reaction with the sequential addition of reagents, demonstrates significant anticancer activity against human epitheloid carcinoma cervix (HeLa) cells.
A novel N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide comprising:
Formula (I)




wherein, N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide here in the preparation process includes the following synthesis scheme and steps:

A) Synthesis Scheme




B) Synthetic Steps:
I ) Step I: Synthesis of 2-(chloromethyl)-1H-1,3-benzimidazole
heating O-phenylene diamine (0.25 mol) and chloro carboxylic acid (0.34 mol) on a water bath at 100o C for 6-8 h, monitoring through TLC; cooling the reaction mixture and basifying to pH from 7 to 8, using 10% sodium hydroxide solution; filtering obtained crude 2-(chloromethyl)-1H-1,3-benzimidazole using vaccum pump, subsequentely washed with ice cold water; dissolving the crude product in 400 ml of boiling water and 2 g of decolorizing carbon with warm heating for 15 min;filtering the solution during hot, cooling it to about 10o C and purifying the product by washing with 25 ml of cold water and drying at 100o C. to get 2-(chloromethyl)-1H-1,3-benzimidazole;

II ) Step II: Synthesis of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole
taking 7.5 ml of conc. nitric acid (HNO3) in three necked round bottom flask fitted to a mechanical stirrer; immersing the flask in ice cold water by adding slowly conc. H2SO4 (7.5 ml) from down the condenser with slow stirring; adding 2-(chloromethyl)-1H-1,3-benzimidazole (0.028 mol) ( step I) in a portion over a period of 1 h at a rate that the temperature did not exceed 35ºC;under continuous stirring for 12 h, pouring the reaction mixture very slowly over crushed ice with vigorous stirring to get crude product; in a sequential manner filtering, washing with cold water and recrystallizing the crude product from ethanol to obtain 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole;

III ) Step III: Synthesis of 2-(chloromethyl)-1H-benzo[d]imidazol-5-amine
taking solution of 0.5 g of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole ( step II) in 15 ml of rectified sprit in round bottom flask, to it adding 5 ml of 20 % sodium hydroxide and 2.5 g of zinc dust powder; refluxing the reaction mixture for 4.5 h, until colour of the solution changed from deep red to colourless and filtering during hot; separately extracting the zinc residue remains after reaction with 10 ml of hot rectified sprit for two times; combining both extracts and in a sequential manner evaporating the solvent and recrystallizing using methanol to get brown solid of 2-(chloromethyl)-1H-1,3-benzimidazol-5-amine, effectively used as an anticancer agent, more preferably in human epitheloid carcinoma cervix (HeLa) cells;

IV) Step IV: Synthesis of 5-acetamido-1H-benzo[d]imidazol-2-yl) methyl hypochlorite
mixing compound (0.01 mole) with acetic anhydride (0.02 mole) in a 250 mL RBF and refluxed for 3 hrs; pouring the solution into ice-cold water with vigorous stirring to get solid precipitate; heating the precipitate to boiling to decompose excess of anhydride and cooled to obtain solid product; drying and purifying by recrystallization from ethanol to get pure white crystals; and

V) Step V: Synthesis N-(2-(((3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide
Stirring in a 250 ml Round Bottom Flask (RBF), 2-Chloromethyl-1H-benzimidazole (1.665, 0.01 mol) and Potassium carbonate K 2 CO 3 (0.02mol, 2.76gm) at room temperature in dimethyl formamide (DMF, 20 ml) for half an hour with adding pinch of KI and 3-aminophenyl nitrate ( 0.01 mol); refluxing the reaction for 12 hrs until TLC showed completion of reaction; pouring the reaction mixture into water (20 ml) and extracting the mixture from ethyl acetate (20 ml); washing the organic extracts from water; drying over anhydrous sodium sulphate; concentrating to obtain crude product; and recrystallizing the crude product from diethyl ether to give final pure compound.

Brief Description of Drawings: The following thorough explanation of the various aspects of the invention, taken in conjunction with the corresponding drawing that represents various aspects and other features of the disclosure invention.
Figure 1: Flow chart preparation of N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide
Figure 2: IR Spectrum of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Figure 3: HNMR Spectrum of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Figure 4: 13CNMR Spectrum of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Figure 5: Mass Spectrum of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Detailed Description of the Invention
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration through explanation and figures for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the ingredients described without departing from the scope of the invention.
The use of "including", "comprising" or "having" variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
Important steps involved in synthesis process

O-Phenylenediamine (0.25 mol) and the appropriate carboxylic acid (0.34 mol) were heated on a water bath at 100°C for 6-8 hours. The progress of the reaction was monitored by thin-layer chromatography (TLC). In a separate procedure, concentrated nitric acid (7.5 mL) was placed in a three-necked round-bottom flask fitted with a mechanical stirrer. The flask was immersed in an ice-cold water bath, and concentrated sulfuric acid (7.5 mL) was added dropwise through the condenser with gentle stirring. Upon completion of the addition, 2-substituted benzimidazoles were obtained.

Next, a solution of 0.5 g of 5-nitro-2-substituted benzimidazoles in 15 mL of rectified spirit was prepared in a round-bottom flask. To this, 5 mL of 20% sodium hydroxide solution and 2.5 g of zinc dust were added. The reaction mixture was then refluxed until the solution changed from deep red to colorless, which took approximately 4.5 hours. After this, the hot mixture was filtered, and the zinc residue was returned to the flask and extracted twice with 10 mL portions of hot rectified spirit to isolate the desired compound.

Detailed Procedure for synthesis:
A) Synthesis Scheme

B) Synthetic Steps:
I ) Step I: Synthesis of 2-(chloromethyl)-1H-1,3-benzimidazole
heating O-phenylene diamine (0.25 mol) and chloro carboxylic acid (0.34 mol) on a water bath at 100o C for 6-8 h, monitoring through TLC; cooling the reaction mixture and basifying to pH from 7 to 8, using 10% sodium hydroxide solution; filtering obtained crude 2-(chloromethyl)-1H-1,3-benzimidazole using vaccum pump, subsequentely washed with ice cold water; dissolving the crude product in 400 ml of boiling water and 2 g of decolorizing carbon with warm heating for 15 min;filtering the solution during hot, cooling it to about 10o C and purifying the product by washing with 25 ml of cold water and drying at 100o C. to get 2-(chloromethyl)-1H-1,3-benzimidazole;

II ) Step II: Synthesis of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole
taking 7.5 ml of conc. nitric acid (HNO3) in three necked round bottom flask fitted to a mechanical stirrer; immersing the flask in ice cold water by adding slowly conc. H2SO4 (7.5 ml) from down the condenser with slow stirring; adding 2-(chloromethyl)-1H-1,3-benzimidazole (0.028 mol) ( step I) in a portion over a period of 1 h at a rate that the temperature did not exceed 35ºC;under continuous stirring for 12 h, pouring the reaction mixture very slowly over crushed ice with vigorous stirring to get crude product; in a sequential manner filtering, washing with cold water and recrystallizing the crude product from ethanol to obtain 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole;

III ) Step III: Synthesis of 2-(chloromethyl)-1H-benzo[d]imidazol-5-amine
taking solution of 0.5 g of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole ( step II) in 15 ml of rectified sprit in round bottom flask, to it adding 5 ml of 20 % sodium hydroxide and 2.5 g of zinc dust powder; refluxing the reaction mixture for 4.5 h, until colour of the solution changed from deep red to colourless and filtering during hot; separately extracting the zinc residue remains after reaction with 10 ml of hot rectified sprit for two times; combining both extracts and in a sequential manner evaporating the solvent and recrystallizing using methanol to get brown solid of 2-(chloromethyl)-1H-1,3-benzimidazol-5-amine, effectively used as an anticancer agent, more preferably in human epitheloid carcinoma cervix (HeLa) cells;

IV) Step IV: Synthesis of 5-acetamido-1H-benzo[d]imidazol-2-yl) methyl hypochlorite
mixing compound (0.01 mole) with acetic anhydride (0.02 mole) in a 250 mL RBF and refluxed for 3 hrs; pouring the solution into ice-cold water with vigorous stirring to get solid precipitate; heating the precipitate to boiling to decompose excess of anhydride and cooled to obtain solid product; drying and purifying by recrystallization from ethanol to get pure white crystals; and

V) Step V: Synthesis N-(2-(((3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide
Stirring in a 250 ml Round Bottom Flask (RBF), 2-Chloromethyl-1H-benzimidazole (1.665, 0.01 mol) and Potassium carbonate K 2 CO 3 (0.02mol, 2.76gm) at room temperature in dimethyl formamide (DMF, 20 ml) for half an hour with adding pinch of KI and 3-aminophenyl nitrate ( 0.01 mol); refluxing the reaction for 12 hrs until TLC showed completion of reaction; pouring the reaction mixture into water (20 ml) and extracting the mixture from ethyl acetate (20 ml); washing the organic extracts from water; drying over anhydrous sodium sulphate; concentrating to obtain crude product; and recrystallizing the crude product from diethyl ether to give final pure compound. (Figure 1)
Invented molecule
By using above general procedure, the characterized molecule is summarized as follows:
N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5yl)acetamide
IR (cm-1) 3259 N-H str. (2° amide);3043 =C-H str. (Aromatic); 1658 C=O str. (2° amide) 1543C=N str. (imidazole); 1435 N-H bend (2° amide); 1195 C-N str. (2° amide); 746 disubtituted benzene; 1H NMR (500 MHz, DMSO) δ [ppm] 1.54-1.68 (d, 3H), 1.72-1.78 (m, 4H), 1.87-1.98 (d, 1H), 2.64-3.41 (s, 2H), 3.51- 3.85 (s, 1H); 3.94-4.00 (d, H). Molecular Ion peak m+1) 310 found for C17H18N4O2
( Table 1,2 3, and 4; Figure 2,3 4, and 5)


Table 1- IR interpretation of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide

Sr.No. Peak(cm-1) Corresponding Functional group
1. 3259.81 N-H str. (2° amide)
2. 3043.77 =C-H str. (Aromatic)
3. 1658.84 C=O str. (2° amide)
4. 1543.10 C=N str. (imidazole)
5. 1435.09 N-H bend (2° amide)
6. 1195.91 C-N str. (2° amide)
7. 746.48 disubtituted benzene ring


Table 2- 1HNMR Interpretation of N-(2-(((3 methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Sr. No. Protons Splitting pattern Chemical shift position
1 =CH (Imidazole) Doublet (d) 1.54-1.68
2 =CH (Imidazole) Doublet (d) 1.72-1.78
3 =CH Aromatic Doublet (d) 1.87-1.98
4 =CH-N Singlet (s) 2.64-3.41
5 CH2 Doublet (d) 3.51-3.85
6 N-H proton Doublet (d) 3.94-4.00

Table 3- 13CNMR Interpretation of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide

Sr. No. Protons Splitting pattern Chemical shift position
1 =CH
(Imidazole) Doublet (d) 23.2-284
2 =CH
(Imidazole) Doublet (d) 30.6-39
3 =CH Aromatic Singlet (s) 53.33-57.1
4 =CH-N Singlet (s) 65.3-79.6
5 CH2 Singlet (s) 161.8-171.4





Table 4- Mass Spectrum Interpretation of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide

Sr. No. m/e ratio Fragment Structure
1 (Molecular Ion peak m+1) 325
2 324

3 323


4 322

5 212

6 123



Results and discussion
Synthesis of N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide are presented in reaction Scheme I (fig 1). The Compounds were obtained via a one-pot reaction with the sequential addition of reagents, ensuring efficiency and simplicity in its production. Microorganism culture: HRV2 (Human rhinovirus (family: Picornaviridae) was Typical Culture Collection (Code: Manassas, VA, USA) pathological lab and it was propagated in human epitheloid carcinoma cervix (HeLa) cells at 32℃ Procedure: Assays of antiviral activity and cytotoxicity were evaluated by the SRB method using cytopathic effect reduction, already reported. Briefly, 1 day prior to infection. The 50% cytotoxic concentration for Hela cells in μg/mL; yConcentration of compound in μg/mL producing 50% inhibition of virus-induced cytopathic effects; zTherapeutic index CC50/IC50; IC50 value within the concentration of the compound to be tested not determined due to maximum inhibition rate under 50%. Results are presented as the mean IC50 values obtained from three independent experiments carried out in triplicate SD. Whereas: HRV-1 human rhinovirus; IC50 Z 50% inhibition concentration; ± SD standard deviation; therapeutic index.
Table 5. Cytotoxicity study of synthesized compound N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide
Compound CC50 IC50 TI
N-(2-(((3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl)acetamide 161.43 19.45± 1.29 2.07
Ribavirin 93.16 34.26± 1.29 3.19

It has been proved that the synthesized compound is less as compare to ribavirin standard
Scope of the Invention
The present invention relates to overcome the loopholes of existing formulations used as anticancer treatment includes a novel seven-membered N-(2- (3-methoxyphenyl) amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide compound prepared through a one-pot reaction with the sequential addition of reagents, to get wherein R substituted with 3 methoxy aniline, to get final compound of N-(2- (3-methoxyphenyl) amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide, demonstrates significant anticancer activity against using human epitheloid carcinoma cervix (HeLa) cells, signify a potential therapy in cancer in future.
It is to be understood that the present invention is not limited to the embodiments described above, it should be clear that various modifications and alterations can be made along with various features of one embodiment included in other embodiments, within the scope of the present invention.
, Claims:We claim,
1- A potential N-(2-(3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-
yl) acetamide comprising:
Formula (I)



wherein, N-(2-(((3-methoxyphenyl) amino) methyl)-1H-benzo[d] imidazol-5-yl) acetamide, effectively used as an anticancer agent.
2-The N-(2-(3-methoxyphenyl)amino)methyl)-1H-benzo[d]imidazol-5-yl) acetamide as claimed in claim 1, wherein formula ( I ) compound effectively used in human epitheloid carcinoma cervix (HeLa) cells.
3- The N-(2-(((3-methoxyphenyl) amino) methyl)-1H-benzo[d] imidazol-5-yl) acetamide as claimed in claim 1, prepared through a process includes the following synthesis scheme and steps: wherein,






A) Synthesis Scheme



B) Synthetic Steps:
I ) Step I: Synthesis of 2-(chloromethyl)-1H-1,3-benzimidazole
heating O-phenylene diamine (0.25 mol) and chloro carboxylic acid (0.34 mol) on a water bath at 100o C for 6-8 h, monitoring through TLC; cooling the reaction mixture and basifying to pH from 7 to 8, using 10% sodium hydroxide solution; filtering obtained crude 2-(chloromethyl)-1H-1,3-benzimidazole using vaccum pump, subsequentely washed with ice cold water; dissolving the crude product in 400 ml of boiling water and 2 g of decolorizing carbon with warm heating for 15 min;filtering the solution during hot, cooling it to about 10o C and purifying the product by washing with 25 ml of cold water and drying at 100o C. to get 2-(chloromethyl)-1H-1,3-benzimidazole;

II ) Step II: Synthesis of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole
taking 7.5 ml of conc. nitric acid (HNO3) in three necked round bottom flask fitted to a mechanical stirrer; immersing the flask in ice cold water by adding slowly conc. H2SO4 (7.5 ml) from down the condenser with slow stirring; adding 2-(chloromethyl)-1H-1,3-benzimidazole (0.028 mol) ( step I) in a portion over a period of 1 h at a rate that the temperature did not exceed 35ºC;under continuous stirring for 12 h, pouring the reaction mixture very slowly over crushed ice with vigorous stirring to get crude product; in a sequential manner filtering, washing with cold water and recrystallizing the crude product from ethanol to obtain 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole;

III ) Step III: Synthesis of 2-(chloromethyl)-1H-benzo[d]imidazol-5-amine
taking solution of 0.5 g of 2-(chloromethyl)-5-nitro-1H-1,3-benzimidazole ( step II) in 15 ml of rectified sprit in round bottom flask, to it adding 5 ml of 20 % sodium hydroxide and 2.5 g of zinc dust powder; refluxing the reaction mixture for 4.5 h, until colour of the solution changed from deep red to colourless and filtering during hot; separately extracting the zinc residue remains after reaction with 10 ml of hot rectified sprit for two times; combining both extracts and in a sequential manner evaporating the solvent and recrystallizing using methanol to get brown solid of 2-(chloromethyl)-1H-1,3-benzimidazol-5-amine, effectively used as an anticancer agent, more preferably in human epitheloid carcinoma cervix (HeLa) cells;

IV) Step IV: Synthesis of 5-acetamido-1H-benzo[d]imidazol-2-yl) methyl hypochlorite
mixing compound (0.01 mole) with acetic anhydride (0.02 mole) in a 250 mL RBF and refluxed for 3 hrs; pouring the solution into ice-cold water with vigorous stirring to get solid precipitate; heating the precipitate to boiling to decompose excess of anhydride and cooled to obtain solid product; drying and purifying by recrystallization from ethanol to get pure white crystals; and

V) Step V: Synthesis N-(2-(((3-methoxyphenyl) amino) methyl)-1H-benzo[d]imidazol-5-yl) acetamide
Stirring in a 250 ml Round Bottom Flask (RBF), 2-Chloromethyl-1H-benzimidazole (1.665, 0.01 mol) and Potassium carbonate K 2 CO 3 (0.02mol, 2.76gm) at room temperature in dimethyl formamide (DMF, 20 ml) for half an hour with adding pinch of KI and 3-aminophenyl nitrate ( 0.01 mol); refluxing the reaction for 12 hrs until TLC showed completion of reaction; pouring the reaction mixture into water (20 ml) and extracting the mixture from ethyl acetate (20 ml); washing the organic extracts from water; drying over anhydrous sodium sulphate; concentrating to obtain crude product; and recrystallizing the crude product from diethyl ether to give final pure compound.

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