Synthesis and Biological Properties of 4-Chloro-N1-(5-Chloro-1H-Indol-1-yl)-N2-(2-Chlorophenyl) benzene-1,2-Diamine Derivatives

Indole, sometimes called benzopyrrole, is an aromatic compound that has ten π-electrons (two from a lone pair on N and eight from=bonds). On indole, electrophilic substitution happens easily because of high π-electrons delocalization, much like it does on the benzene ring. An essential heterocyclic system, indole supplies the structural framework for alkaloids derived from plants, Strychnine and Lysergic acid Diethylamide (LSD).¹ They have distinct smells and are colourless and crystalline in nature. When the indole nucleus is added to pharmaceuticals with a physiologically active pharmacophore, it becomes a significant heterocyclic molecule with wide-ranging biological action. As a result, scientists were interested in creating alternative indole scaffolds for the purpose of screening diverse pharmacological actions.² Indole serves as the parent nucleus in a number of natural chemicals, including tryptophan. Higher plants break down tryptophan to create the plant hormone indole-3-acetic acid. Indole derivatives are of great interest. Indoles having a chemical formula of C₈H₇N and a molecular weight of 117.15 g/mol.³ Compared to standard medicine, the molecule with electron-donating and electron-withdrawing groups attached to the indole rings had a stronger inhibitory capacity against bacterial and fungal strains. Indole has been found to possess broad-spectrum antibacterial, anti-inflammatory, analgesic, anti-tubercular, antihypertensive, anticonvulsant and antiviral properties following a thorough examination of several research and review papers.⁴ Indole is manufactured by a range of bacteria and is found in large quantities in the natural world. As a result, various research organisations have worked to find novel antimicrobial medications and the WHO and young scientists worldwide have made significant efforts to treat these infections,⁵ such as anti-inflammatory, antibacterial, antifungal, herbicidal and antiviral activity, however, is represented pharmacologically by indole, along with its derivatives.⁶ In the Figure 1 below, indole was shown. These days, indoles have a broad variety of pharmacological properties. We have attempted to provide a summary of the significant pharmacological activities of derivatives of indole here. The goal of this study is to provide an overview of the current status of indole syntheses, with a special emphasis on the most recent advancements in novel synthetic methodologies.⁷ Preparing natural goods or bioactive chemicals that include the indole unit is emphasised. We give an overview of the synthesis of indole from a historical viewpoint, highlighting the methods used to choose the nitrogen precursors. The review is structured to provide indole synthesis techniques by using various aromatic substrates that include nitrogen-containing functional groups as a source of nitrogen for the indole moiety.⁸ The scant attention paid to some functional groups and typical reactions in earlier evaluations on this subject has led to their being especially emphasised and underlined. The most recent published results are highlighted, although other synthetic methodologies that have been more often utilised and addressed in thorough, outstanding studies on the subject are summarised. We are especially interested in the indolization processes and the various ring closure techniques; we pay little attention to changing the indole structures from the beginning.⁹ Novel indole syntheses are always being found and the scientific community places a high value on these discoveries because of the tactical significance of molecules and significant attention and uses because of their biological and pharmacological properties.¹⁰

Figure 1:
Structure of Indole derivatives.

Benzoic acid and ethanoic acid, propionic acid, hydroxylamine, 2,5 Dichloro aniline, Egg albumin solution, Phosphate buffered saline, distilled water/DMSO are used for the synthesis of indole derivatives Every chemical was of the analytical variety. All of the chemicals were bought from Modern Chemicals in Nashik, while others are offered by the college.

Every derivative of indoles was created using a traditional process. Melting point was confirmed using the capillary open tube technique. The chemicals’ purity was examined using Thin-Layer Chromatography (TLC). Using KBr pellets and a Perkin Elmer Spectrum FTIR equipment, IR spectra have been acquired. A Bruker AVANCE III 500 MHz (AV 500) spectrometer was used to record 1 H-NMR spectrum. Physical Data of Indole derivatives were given in Table 1; Scheme of 4-chloro-N1-(5-chloro-1H-indol-1-yl)-N2-(2-chlorophenyl) benzene-1,2-diamine were given in Figure 2 and Scheme of 4-chloro-N1-(5-chloro-1H-indol-1-yl)-N2-(2-chlorophenyl) benzene-1,2-diamine derivatives were given in Figure 3.

2 g of 5-chloro-1H-indol-1-amine and 7 mL of 2,5-dichloroaniline were combined in a flask with a round bottom. For 2 hr, the mixture was heated to 100° in a heating mantel. Once the liquid had cooled, 10% sodium hydroxide solution was gradually added until it was barely litmus-alkaline. Reaction flask was rinsed with ice-cold water. After recrystallizing the crude product with cold water, it was cleaned using around 25 mL of cold water.

Add 2 g of the product and two chloroaniline to a round-bottom flask. Heat the reaction mixture for 3 hr at 100°C in a heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated.

Add benzene to a flask with a round bottom, add 2 g of product and heat the reaction mixture for 3 hr at 100°C in a heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated.

Add acetic acid to a flask with a round bottom, add 2 g of product and heat the reaction mixture for 3 hr at 100°C in a heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated. Synthesis of 2-({5-chloro- 2-[(5-chloro-1H-indol-1-yl)amino]phenyl}amino)phenyl propanoate: NA3 (Scheme 1B).

In a round-bottomed flask, 2 g of product and add ethanoic acid and treat the reaction mixture for 4 hr at 100°C in heating mantel. After cooling, the reaction mixture was filtered and recrystallized with ethanol. The Product was washed with ice cold water and filtered and dried in hot air oven for 10 min and later naturally. Synthesis of 2-({5-chloro-2-[(5-chloro-1H-indol-1-yl) amino]phenyl}amino)phenyl formate: NA4 (Scheme 1B).

In a round-bottomed flask, 2 g of product and add formic acid and treat the reaction mixture for 3 hr at 100°C in heating mantel. After cooling, the reaction mixture was filtered and recrystallized with ethanol. The Product was washed with ice cold water and filtered and dried in hot air oven for 10 min and later naturally. Synthesis of N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol -1-yl)-4-aminobenzene-1,2-diamine:NA5 (Scheme 1B).

In a round-bottomed flask, 2 g of product and add 4 nitro aniline and treat the reaction mixture for 3 hr at 100°C in heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated. Synthesis of N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chlo ro-1H-indol-1-yl)-3-aminobenzene-1,2-diamine: NA6 (Scheme 1B).

Put 2 g of the product and 3 g of nitro aniline into a round-bottom flask. Heat the reaction mixture for 3 hr at 100°C in a heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated.

Synthesis of N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol -1-yl)-2-aminobenzene-1,2-diamine: NA7 (Scheme 1B)

Add 2 g of the product and 2 g of nitro aniline to a round-bottom flask. Heat the reaction mixture for 3 hr at 100°C in a heating mantel. The reaction mixture was cooled, filtered and then recrystallized with ethanol. The product was cleaned in ice-cold water, filtered and dried for ten minutes in a hot air oven before it naturally dehydrated.

FTIR (KBr) ν cm^-1: 1600.92 C=C Stretch (Aromatic), 2952.84 C-H Stretch (Aromatic), 2828.10 C-N bend (Aromatic), 3537.45 N-H Stretch (Aromatic), 2423.21 C-H Stretch (Aliphatic), 771.87 C-Cl Stretch (Aromatic), 1521.84 N-H bend (Aromatic); 1H NMR (400 MHz, DMSO): δ 11.7 N-H (s, 1H, J=11.1), δ 12.4 N-H (s, 1H, J=11.4), δ 7.3-7.9 Ar C-H (m, 12H, J=14.3).

FTIR (KBr) ν cm^-1: 1498.45 C=C Stretch (Aromatic), 1160.01 C-C Stretch (Aromatic), 1301.72 C-N Stretch (Aromatic), 3313 N-H Stretch (Aromatic), 799.5 C-Cl Stretch, 2844 C-H (Aromatic); 1H NMR (400 MHz, DMSO): δ 12.5 N-H (s, 1H, J=11.7), 13.3 N-H (s, 1H, J=11.4), 7.0-7.8 Ar C-H (m, 17H, J=14.3).

FTIR (KBr) ν cm^-1: 1586 C=C Stretch (Aromatic), 1132.01 C-C Stretch (Aromatic), 1332.83 C-N Stretch (Aromatic), 3359 N-H Stretch (Aromatic), 1728.10 C=O (Ester), 782 C-Cl bend (Aliphatic); 1H NMR (400 MHz, DMSO): δ 12.5 N-H (s, 1H, J=11.7), 13.3 N-H (s, 1H, J=11.4), 7.1-7.9 Ar C-H (m, 15H, J=14.3), 2.5 CH₃ (s, 3H, J=11.0).

FTIR (KBr) ν cm^-1: 1718.26 C=C Stretch (Aromatic), 1191.63 C-C Stretch (Aromatic), 1266.86 C-N Stretch (Aromatic), 3259.13 N-H Stretch (Aromatic), 747.0 C-Cl bend (Aliphatic), 1728.15 C=O (Ester); 1H NMR (400 MHz, DMSO): δ 2.2 CH₂ (s, 2H, J=11.7), 13.3 N-H (s, 1H, J=11.4), 12.7 N-H(s, 1H, J=11.4), (7.0-7.8 Ar C-H (m, 13H, J=14.3), 2.8 CH₃ (s, 3H, J=11.0).

FTIR (KBr) ν cm^-1: 1632.29 C=C Stretch (Aromatic), 1232.5 R-O-R (Aliphatic), 2835.81 C-N bend (Aromatic), 998.02 N-H Bend (Aromatic), 3247.7 O-H bend (Aliphatic), 1728.1 C=O (Diketones), 1550 N-O (Aromatic), 778.4 C-H bend (Aliphatic); 1H NMR (400 MHz, DMSO): δ 3.3 CH₃ (s, 3H, J=11.4), 6.4 CH₂ Group (s, 2H, J=13.1), 7.0-7.9 Ar C-H (m, 14H, J=14.3); Mol.Wt. 374.35.

FTIR (KBr) ν cm^-1: 1679.69 C=C Stretch (Aromatic), 1182.78 C-O Stretch (Aromatic), 2828.10 C-N stretch (Aromatic), 1646.80 N-H bend (Aromatic), 3289.2 O-H bend (Aliphatic), 1722.46 C=O (Diketones), 3289.2 O-H Stretch (Aliphatic), 620.66 C-Cl (Aliphatic); 1H NMR (400 MHz, DMSO): δ 2.5 CH₃(s, 3H, J=11.7), 3.3 CH₃ (s, 3H, J=11.4), 7.7-7.98 Ar C-H (m, 16H, J=14.3); Mol.Wt.353.24.

FTIR (KBr) ν cm^-1: 1594.6 C=C Stretch (Aromatic), 1262.2 C-C Stretch (Aromatic), 2828.10 C-N bend (Aromatic), 1496.6 N-H bend (Aromatic), 3279.2 O-H bend (Aliphatic), 1134.5 C=O (Diketones); 1H NMR (400 MHz, DMSO): δ 2.5 CH₃ (s, 3H, J=11.7), 3.3 CH₃ (s, 3H, J=11.4), 7.5-7.98 Ar C-H (m, 15H, J=14.3); Mol.Wt.374.35.

FTIR (KBr) ν cm^-1: 1495.45 C=C Stretch (Aromatic), 1145.12 C-C Stretch (Aromatic), 2431 C-N Stretch (Aromatic), 3313 N-H Stretch (Aromatic), 1699.5 C=O Stretch (Carboxylic Acid), 3471 and 3537 O-H Stretch (Aliphatic), 1670.10 C=O (Diketones), 2844 C-H(Aromatic); 1H NMR (400 MHz, DMSO): δ 3.63 CH₃ (s, 3H, J=11.7), 3.71 CH₃ (s, 3H, J=11.4), 5.44 CH₂ Group (s, 2H, J=13.1), 6.36-6.38 CH₂ Group (s, 2H, J=13.1), 7.18-7.25 Ar C-H (m, 15H, J=14.3); 5.98-5.56 OH (2H, s, J=17.4); Mol.Wt. 404.46

A 1% egg albumin solution may be made using either fresh hen’s eggs or easily found egg albumin powder from shops. To correctly make egg-albumin solution from a fresh hen’s egg, fracture it gently, then add 1 mL of the transparent part to 100 mL of distilled water with a thorough swirling motion. Egg albumin is the name of the transparent part of the egg. When creating the solution, the water has to be cold. When water reaches a boil, it will coagulate.

Figure 2:
Scheme of 4-chloro-N¹-(5-chloro-1H-indol-1-yl)-N2-(2-chlorophenyl)benzene-1,2-diamine.

Graphical Representation of Albumin Denaturation Inhibitory Activity of Synthesized Compounds were given in Figure 4.

Protein denaturation is one of the most well-known causes of inflammation. As part of the investigation, seven test samples (NA1-NA7) had their ability to reduce inflammation assessed. A test for denaturation of proteins has been carried out. Multiple concentrations (50, 100, 200 and 300 μg/mL) of test samples were produced. 0.5 mL of 1.5 mg/mL Bovine Serum Albumin (BSA) was added to each reaction mixture and it was then incubated for 20 min at 37°C. The reaction mixtures were then heated for three minutes to 57°C. Before adding 250 μL of phosphate buffer (0.5 M, pH 6.3), each mixture had to be thoroughly blended. When all of the molecules in each reaction mixture had been evenly distributed, 100 μL of each mixture was transferred into separate test tubes and the same amount of Folin-Ciocalteu’s reagent was added. The tubes were allowed to cool after a 10 min incubation time at 55°C. The absorbance at 650 nm was then measured using a UV/vis spectrophotometer, with triple distilled water acting as the blank. The collected data was juxtaposed to the reference medicine, indomethacin sodium (100 μg/mL). The inhibition percentage of protein denaturation was calculated using the procedure given table number 2 below.

Research undertaken globally has demonstrated a remarkable influence on scientists who are working on indole derivatives to create indole derivatives that are permitted for use in commerce. Many pharmacological compounds with an indole moiety are being studied for their potential to manage various diseases.¹¹ To establish and support the formulator are the clear features that form the basis and reasoning of the study topic.¹² We reviewed a number of studies published over the past few decades that addressed topics such as anticancer, antifungal, antiplatelet, antidiabetic, antimalarial, antimicrobial, antidiabetic, antiviral, antifungal, anticonvulsant, antifertility, anti-inflammatory, antidepressant, antioxidant, antiestrogenic and antitubercular.^13–15 Researchers looking for a tactical overview of new indole derivatives with a variety of exciting biological activity and less toxicity and side effects might use this review article as a foundation. Because of its many metabolic activities, indole is an exceptional heterocyclic molecule that is very flexible and has a wide range of pharmacological effects. The amazing indole moiety shares similarities with many different protein architectures.^16,17 Indole’s intriguing molecular structure makes it a good candidate for medication development. Indole derivatives have the ability to bind reversibly to a variety of enzymes and imitate the structure of peptides, which presents a wealth of options for the development of new medications with unique mechanisms of action. Since the core structures of many compounds contain many physiologically relevant molecules that are active in various illnesses, the existence of nitrogen-based heterocycles in various drugs has been extensively studied.¹⁸ Following scheme 1B, the synthesis of Novel Substituted Indole derivatives from NA1 to NA7 was started. A comparative chart (refer to Table 2 above) demonstrates the anti-inflammatory properties of novel substituted indole-derived compounds at various doses, such as 50, 100, 200 and 300 μg/ mL, as well as how well they can stop the spread of inflammation. indomethacin was prescribed as usual medication. Table provided a snapshot of the anti-inflammatory findings for the named compounds (NA1-NA8). Compounds NA5 and NA6 had greater absorbance at lower dosage levels of 50 μg/mL and 100 μg/mL. Compound 2-({5-chloro-2-[(5-chloro-1H-indol-1-yl) amino]phenyl} amino) phenyl formate NA4 (Scheme 1B) and N2-(biphenyl-2-yl)-4-chloro-N1-(5-chloro-1H-indol-1-yl)-2-aminobenzene-1,2-diamine NA7 (Scheme 1B) displayed lower absorbance at higher dose level of 300 μg/mL. Compound 4 N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol-1-yl)benzene-1,2-diamine NA1 (Scheme 1B) and 2-({5-chloro-2- [(5-chloro-1H-indol-1-yl)amino]phenyl}amino)phenyl acetate NA2 (Scheme 1B) containing aromatic group, showed higher absorbance at lower dose level of 200 μg/mL. Table displayed the outcomes of the produced compounds’ anti-inflammatory tests. The anti-inflammatory properties of recently synthesised compounds have been investigated in relation to the Albumin Denaturation Inhibitory Activity. Tests have been performed on all the substances at 50, 100, 200 and 300 μg/mL. Table 4 summarises the study’s findings. It is interesting to point out that all the compounds of the present series showed some anti-inflammatory activity. 2-({5-chloro-2-[(5-chloro-1H-indol-1-yl)amino]phenyl} amino) phenyl formate NA4 (Scheme 1B) and N²-(biphenyl-2- yl)-4-chloro-N¹-(5-chloro-1H-indol-1-yl)-2-aminobenzene- 1,2-diamine NA7 (Scheme 1B) had more anti-inflammatory properties than the ones that matched them. Nitro aniline compounds showed more activity than other derivatives, while their indole derivatives showed less percentage inhibition by albumin denaturation inhibitory action. It has been observed that the N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol-1 -yl)-3-aminobenzene-1,2-diamine NA6 (Scheme 1B) and N²– (biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol-1-yl)-4-amin obenzene-1,2-diamine NA5 (Scheme 1B) and N2-(biphenyl-2- yl)-4-chloro-N1-(5-chloro-1H-indol-1-yl)-2-aminobenzene- 1,2-diamine NA7 (Scheme 1B) having amino at ortho, meta and para position on phenyl ring was the most active and it was studied at three graded doses (50, 100, 200 and 300 μg/mL). The compound N²-(biphenyl-2-yl)-4-chloro-N¹-(5-chloro-1H-indol -1-yl)-4-aminobenzene-1,2-diamine NA5 (Scheme 1B) showed the strongest and most dose-dependent anti-inflammatory action of all the newly created compounds. Because indole derivatives have so many uses in medicinal chemistry, both traditional and green synthetic techniques have been devised for their production. Compared to conventional procedures, the green methodology offers several benefits such as quick reaction times, low costs for reagents, high yields and environmental friendliness. To get beyond the limitations of traditional techniques, researchers in both academia and the pharmaceutical industry are now working on creating a variety of green methodologies for the chemical synthesis of molecules based on indole. This study incorporates the advancements over the past ten years in environmentally friendly techniques for the synthesis of indole derivatives, including the use of microwaves, ionic liquids, water, ultrasound, nanocatalysts, green catalysts, multicomponent reactions and solvent-free reactions.¹⁹ Additionally, this research has demonstrated the use of green chemistry in the production of medicines containing indole and their biological investigations. One of the busiest fields of heterocyclic chemistry research has been indole chemistry. Particularly, 3-substituted indole derivatives have drawn a lot of interest as starting points for the synthesis of several naturally occurring substances as well as additional substances with biological activity. In this paper, a three-component reaction involving aromatic amine and carboxylic acid with a catalytic quantity of acid under convectional conditions effectively created a synthetic process for multi-substituted indole derivatives. The benefits of this process are high atom utilisation, easy operation and easily accessible raw materials.

Figure 3:
Scheme of 4-chloro-N¹-(5-chloro-1H-indol-1-yl)-N²-(2-chlorophenyl)benzene-1,2-diamine derivatives.

Figure 4:
Graphical Representation of Albumin Denaturation Inhibitory Activity of Synthesized Compounds.

Convectional technique was used to synthesised several Indole derivatives. The seven Indole derivatives in all were synthesised. Good yields of every chemical were produced. The albumin denaturation inhibitory activity was used to provide the biological anti-inflammatory effect. According to this study, indole derivatives exhibited more potent anti-inflammatory activity against various forms of inflammation. Several of the compounds generated have been demonstrated to have potent anti-inflammatory characteristics. When synthesised compounds were compared to other Indole, higher activity was observed. There are several compounds with a variety of biological applications that possess the indole moiety. Many synthetic medicinal compounds embrace an indole nucleus as part of their pharmacophore structure, which helps pharmaceuticals bind to the residual residues of target molecules’ binding sites. Derivatives having indole core have a variety of biological consequences, including antidiabetic, anticancer, antibacterial, anti-HIV, antiviral, anti-inflammatory, antioxidant, anticholinesterase, antitubercular and antimalarial defining features. In their hunt for novel chemical entities, these behaviours have attracted indole to the attention of researchers. These ingredients might be used to create more effective and less hazardous drugs for an assortment of ailments.

The authors are thankful to Dr. S.B. Bhawar, Pravara Rural College of Pharmacy, Pravaranagar.