INTRODUCTION:

A variety of chemotherapeutic drugs with a broad range of antibacterial activity can be designed using indole and its analogs as excellent pharmacophores^1-4. In both plants and bacteria, the bis-indole family of compounds is widely distributed^5,6.

Numerous natural substances extracted from marine species or compounds identified based on their molecular scaffolds have shown to be promising therapeutic candidates⁷. Over the past ten years, several bis(indole)alkaloids, including those with antibacterial, antiviral⁸ and cytotoxic⁹ properties have been identified in the marine environment¹⁰.

The indole ring system has drawn a lot of attention because of its powerful biological activities, particularly its anticancer properties^11-14. Several synthesized (indolyl)indolin-2-ones have recently been found to exhibit potent anticancer properties without harming normal cell lines^15-16. As shown by K-252a to d¹⁷, rebeccamycin¹⁸ and staurosporine¹⁹, indolocarbazoles are a subclass of bis-indoles that have inhibitory effects on DNA topoisomerases or protein kinases²⁰. It is recognized that bis-indolyl methane (BIM) is one of the scaffolds present in many natural products, which have been obtained from both terrestrial and marine natural sources²¹. BIMs have a wide spectrum of bioactivities, such as anticancer activity against various popular cancer cell lines²². In the BIM family, 3,3,3-bis-indolylmethanes are a key metabolite of the indole-3-carbinol-derived anticancer compound that is present in Brassica vegetable species. It has been demonstrated that it prevents the proliferation of breast cancer cell lines that are both estrogen-dependent and independent^23-24. To target biological processes, we have produced several physiologically active chemicals. This study's objectives included developing novel inhibitors, expanding the bis-indole library, evaluating the compounds' antibacterial efficacy, and performing a bacterial growth inhibition test. In this research, ADME (Absorption, Distribution, Metabolism, and Excretion) studies on this class of new inhibitors are carried out²⁵ and describe the discovery of compounds that act against both Gram-positive and Gram-negative bacteria^26-27.

MATERIALS AND METHODS:

Materials:

Each chemical was purchased from Sigma Aldrich. The solvents were AR grade. The melting temperatures of the generated compounds were determined in clear glass capillaries. IR spectra were recorded using the Bruker alpha FTIR spectrometer. Elemental analysis was performed, and it was found that values were within 0.4% of theoretical values. ¹H NMR spectra were recorded on Brucker (400 MHz, ¹H NMR) spectrometers using deuterated solvents (DMSO-d₆ and CDCl₃) and tetramethyl silane as an internal standard. Mass spectral data were recorded using a Shimadzu LC2010 mass analyzer and CHN analysis using Perkin Elmer PE 2400.

Methods:

General synthesis of 1-(2-aminophenyl)-2-chloroethan-1-one: - To a stirred solution of aluminum trichloride (7.13gm, 0.053mmol) in dry m-xylene (20 mL) was added a solution of aniline (5gm, 0.053mmol) in m-xylene 60mL under ice-cooling. Chloroacetonitrile (4.83gm 0.064mmol) and aluminum tetrachloride (8.94, 0.053mmol) were added and the resulting mixture refluxed for 3h, during which time the brown suspension present did not dissolve. After cooling, ice 2 N HC1 was added and the mixture was warmed at 80^oC for 30min. The cooled mixture was neutralized with 2 N sodium hydroxide at pH 3 to 4 and extracted with dichloromethane repeatedly. The organic layer was washed with water and the solvent was evaporated and dried in an oven at 40^oC.

Yield: 71.5%; M.P: 135 ^oC; IR (Bruker) ν max cm^-1: 3321 (N-H str.), 1413 (O-H), 1340-1200 (C-N str.), 1225-960 (C-H), 770-735 (C-H o-substituted); ¹H NMR (DMSO, 400 MHz): d = 4.90 (s, 1H), 6.53 (s, 2H, -NH₂), 6.80 (dd, 1H), 6.94 (t, 1H), 6.99 (t, 1H), 7.47 (dd, 1H); MS (m/z): 170.01; C₈H₈ClNO requires 169.03. Anal. Calc. for C, 56.65; H, 4.75; N, 8.26; Found C, 56.50; H, 4.20; N,8.11.

General synthesis of Indole: - To a stirred solution of 1-(2-aminophenyl)-2-chloroethan-1-one (3gm, 0.021 mmol) in dioxane (30mL) containing water (10mL) was added sodium borohydride (0.79gm, 0.021mmol) and the solution was refluxed for 2hr. After the removal of the solvent, water was added and the mixture was extracted with dichloromethane. The extract was dissolved in chloroform and passed through a silica gel layer to remove a polar fraction. The eluate with chloroform was concentrated giving indole.

Yield: 75.6%; M.P: 54^oC; IR (Bruker) ν max cm^-1: 3406 (N-H str.), 1512 (C=C), 1336-1352 (C-H bend), 3022-3049 (C-H str.), 1456 (C-C str.); ¹H NMR (DMSO, 400 MHz): d = 6.11 (d, 1H), 6.53 (dd, 2H), 7.02 (d, 1H), 7.30 (d, 1H), 7.65 (dd, 1H), 10.50(s, 1H, NH); MS (m/z): 118.23; C₈H₇N requires 117.06. Anal. Calc. for C, 82.02; H, 6.02; N, 11.96; Found C, 82.68; H, 6.22; N,11.29.

Fig. 1: Reagent and condition of bis-indole derivatives synthesis: (a) AlCl₃, ClCH₂-CN, (CH₂Cl)₂, Reflux 3hr; (b) NaBH₄, Dioxane, Reflux 2hr; (c) Acetic acid, Reflux 6hr.

General synthesis of Bis -indole derivatives: - Charged Indole [2] (2gm, 0.017moles), substituted aldehyde (0.034 moles) and 20mL acetic acid in 250ml-2N-RBF. The reaction mixture was raised to reflux for 6 hr. Allowed to cool at 25-30℃. The reaction mixture was poured into 100ml crushed ice. Filtered the separated product and washed it with petroleum ether. Dried it in the oven at 40℃.

2-(di (1H-Indol-3-yl) methyl) phenol (3a):

Yield: 81.6 %; M.P: 255 ^oC; IR (Bruker) ν max cm^-1: 3321 (N-H Str.), 1413 (O-H), 1340-1200 (C-N Str.), 1225-960 (C-H), 770-735 (C-H o-Substituted); ¹H NMR (DMSO-d₆, 400 MHz): 6.11 (s, 1H), 6.53 (s, 2H), 6.80 (t, 2H, J = 7.2), 6.94 (t, 3H, J = 7.2 ), 6.99 (d, 2H, J = 9.2 ), 7.11 (t, 1H, J = 7.2), 7.24 (d, 1H), 7.26-7.29 (t, 2H, J = 8.4, 4), 7.44 (d, 2H, J = 7.6 ), 9.99 (s, 1H, -OH), 10.50 (s, 2H, NH); ¹³C NMR (DMSO-d₆, 400 MHz): 49.52, 109.10, 113.33, 116.81, 117.80, 120.80, 121.33, 122.32, 123.33, 125.20, 128.11, 128.32, 131.50, 137.55, 157.11 MS (m/z): 339; Requires 338.14, M.F. C₂₃H₁₈N₂O. Anal. Calc. For C, 81.63; H, 5.36; N, 8.28; Found C, 81.68; H, 5.20; N, 8.25.

1-(di (1H-Indol-3-yl) methyl) naphthalen-2-ol (3b):

Yield: 76.9 %; M.P: 92 ^oC; IR (Bruker) ν max cm^-1: 3403 (N-H Str.), 1410-1300 (O-H), 1340-1200 (C-N Str. Aromatic), 1225-960 (C-H),1170-1110 (C-OH) 770-735 (C-H o-Substituted; ¹H NMR (DMSO, 400 MHz): 5.50 (s, 1H), 6.78 (s, 2H), 6.74 (t, 2H, J = 8.4), 6.92 (m, 3H, J = 7.6), 7.01 (d, 2H, J = 7.2), 7.13 (m, 1H, J = 7.2), 7.26 (d, 2H, J = 7.2), 7.49 (t, 1H, J = 7.4), 7.60 (d, 1H, J = 7.2), 7.93 (d, 1H, J = 7.6), 8.06 (d, 1H, J = 9.2), 9.89 (s, 1H, -OH), 10.78 (s, 2H, NH); ¹³C NMR (DMSO, 400 MHz): 47.52, 109.22, 113.15, 116.41, 117.70, 118.80, 120.53, 122.12, 123.92, 124.50, 125.11, 128.92, 129.10, 132.96, 138.55, 154.48; MS (m/z): 389; Requires 388.47. M.F. C₂₇H₂₀N₂O Anal. Calc. For C, 83.48; H, 5.19; N, 7.21; Found C, 83.58; H, 5.22; N, 7.25.

3,3'-((4-chlorophenyl) methylene) Bis(1H-indole) (3c):

Yield: 72.45%; M.P: 80 ^oC; IR (Bruker) ν max cm^-1: 3406 (N-H Str.), 1340-1200 (C-N Str. Aromatic), 1225-1000 (C-H), 860-800 (C-H),750 (C-Cl Str.); ¹H NMR (DMSO, 400 MHz): 5.74 (s, 1H), 6.84 (t, 2H J = 7.2), 6.98 (t, 2H, J = 7.2), 7.09 (d, 2H, J = 7.6), 7.24 (d, 2H, J = 8), 7.35 (s, 4H), 7.48 (d, 2H, J = 8.4), 10.81 (s, 2H, NH); ¹³C NMR(DMSO, 400 MHz): 55.52, 109.11, 111.53, 117.81, 119.80, 122.70, 123.53, 126.42, 128.53, 130.90, 131.71, 135.72, 136.17, 137.55; MS (m/z): 357; Requires 356.11. M.F. - C₂₃H₁₇ClN₂. Anal. Calc. For C, 77.41; H, 4.80; N, 7.85; Found C, 77.60; H, 4.75; N, 7.81.

3,3'-((4-fluorophenyl) methylene) Bis(1H-indole) (3d):

Yield: 69.9%; M.P: 156 ℃; IR (Bruker) ν max cm^-1: 3389 (N-H Str.), 1340-1200 (C-N Str. Aromatic), 1225-1000 (C-H), 823 (C-F), 770-735 (C-H O-Substituted); ¹H NMR (DMSO, 400 MHz): 5.69 (s, 1H), 6.64 (s, 2H), 6.77 (t, 2H, J = 8.4), 7.01 (t, 2H, J = 7.6), 7.14 (d, 2H, J= 7.2), 7.35 (m, 4H, J = 7.6), 7.50 (d, 2H, J = 8.6), 10.47 (s, 2H, NH); ¹³C NMR (DMSO, 400 MHz): 56.77, 112.80, 115.43, 119.10, 122.80, 126.43, 131.62, 133.66, 137.50, 160.91; MS (m/z): 341; Requires 340.14. M.F. - C₂₃H₁₇FN₂ Anal. Calc. For C, 81.16; H, 5.03; N, 8.23; Found C, 81.92; H, 5.06; N, 7.91.

3,3'-((3-bromophenyl) methylene) Bis(1H-indole) (3e):

Yield: 79.05 %; M.P: 66 ℃; IR (Bruker) ν max cm^-1: 3404 (N-H Str.), 1340-1200 (C-N Str. Aromatic), 1170-1000 (C-H), 900-686 (C-H),770-735 (C-H O-Substituted), 600-500(S) (C-Br Str.); ¹H NMR (DMSO, 400 MHz): 5.87 (s, 1H), 6.87 (s, 2H), 7.05 (t, 2H, J = 7.2),7.26 (d, 1H, J = 7.6), 7.30 (m, 2H, J = 8.4), 7.38 (m, 4H, J = 6.8), 7.50 (d, 2H, J = 7.6), 8.04 (s, 1H), 10.90 (s, 2H, NH); ¹³C NMR (DMSO, 400MHz): 54.52, 110.10, 113.83, 117.88, 120.88, 122.50, 124.33, 127.02, 128.33, 128.76, 130.11, 134.35, 136.50, 140.55; MS (m/z): 401; Requires 400.06, M.F. - C₂₃H₁₇BrN₂ Anal. Calc. For C, 68.84; H, 4.27; N, 6.98; Found C, 68.72; H, 4.14; N, 7.48.

RESULTS:

This approach is an important complementary tool in the synthesis of bis-indoles because of its ease of use, easy accessibility of starting materials, diversity of target molecules, environmental sustainability, high atom economy and strong regioselectivity. As a result, we think that a simple and effective technique might be very useful for a wide range of applications as well as for improving the synthetic production of bis-indoles, which has captured the interest of the organic chemistry and medical communities. A growing number of researches have focused on various aspects of indole chemistry, such as creating novel synthesis methods and improving reaction conditions using ligands, bases, additives and solvents. solid-phase synthesis is used, indole libraries are built and indole derivatives are synthesized using these methods. We have developed a straightforward, effective procedure to synthesize unique bis-indole analogs. Synthesis of the novel Bis-indole analogs outlined in Figure 1 begins with aniline as starting material. Reacted with Chloroacetonitrile in presence of aluminium trichloride and m-xylene gave 1-(2-aminophenyl)-2-chloroethane-1-one (1). This was followed by cyclization using sodium borohydride to give indole (2). Indole reacts with substituted benzaldehyde in acidic conditions using acetic acid. Overall yields of the required bis-indole ranged from good to outstanding. This procedure offers the best method for synthesizing bis-indole derivatives.

Antibacterial Activity:

The title compounds were tested for their antibacterial activity against Enterobacteriaceae (gm^-ve), Escherichia. Coli (gm^-ve), Bacillus subtilis (gm^+ve) and staphylococcus aureus (gm^+ve) using disc diffusion method at 100 ppm (10 mg/ml) concentration in DMSO (dimethyl sulfoxide) solvent. Using a disc diffusion technique described by the Kirby-Bauer method²⁸. Ampicillin was used as a standard drug. Each experiment was repeated twice. All compounds showed excellent activity against Enterobacter and Staphylococcus aureus bacteria.

Table. 1: Antimicrobial activity screening result of synthesized compounds.

Antibacterial Activity
Compound no.	Conc. (mg/ml)	Microorganisms and zone of inhibition
		*Gram-negative bacteria*		*Gram-positive bacteria*
		*Enterobacteriaceae*	*E.Coli*	*S.aureus*	*Bacillus subtilis*
3a	10	21	28	27	20
3b	10	20	27	26	18
3c	10	15	17	13	14
3d	10	14	10	21	11
3e	10	16	18	13	21
Standard (ampicillin)	10	10	10	9	10

Table 2: SwissADME results of Bis-indole derivatives and standard Ibuprofen and BHT.

Compound Id	3a	3b	3c	3d	3e	BHT	Ibuprofen
Rotatable bonds	3	3	3	3	3	2	4
H-bond acceptors	1	1	0	1	0	1	2
H-bond donors	3	3	3	2	2	1	1
MR	106.12	123.62	109.11	104.05	111.8	71.97	62.18
TPSA	51.81	51.81	31.58	31.58	31.58	20.23	37.3
MLOGP	3.53	4.18	4.62	4.51	4.72	4.12	3.13
Consensus Log P	4.45	5.35	5.4	5.17	5.48	4.37	3
ESOL Solubility (mg/ml)	6.86 E-04	6.18 E-05	1.32 E-04	3.44 E-04	7.23 E-05	6.00 E-03	9.09 E-02
GI absorption	High	High	High	High	High	High	High
BBB permeant	Yes	No	No	No	No	Yes	Yes
Pgp substrate	Yes	Yes	Yes	Yes	Yes	No	No
Lipinski violations	0	1	1	1	1	0	0
Muegge violations	1	1	1	1	1	2	0
PAINS alerts	0	0	0	0	0	0	0
Leadlikeness violations	1	2	2	1	2	2	1

ADME Studies:

ADME (Absorption, Distribution, Metabolism and Excretion) can be evaluated separately by dedicated methods it is a computational study method for determining drug-likeness in blood strim²⁹. Physiochemical parameters give information on drug-likeness like Lipinski, ghose, Veber, egan, Muegge and the bioavailability score of the compound³⁰. Boil egg representation of the compound gives information on predicted Gastrointestinal Absorption and Brain Penetration of Small Molecules³¹. It is crucial to synthesize compounds with various biological capabilities based on their structural domain to build novel anti-leishmanial drugs. For a specific biological target, we have created a range of biologically active molecules³². Natural Bis indole alkaloids 1, 2, 3, 4 and 5 have good intestinal absorption, according to in silico ADME research^33-34, but the semi-synthetic derivative 9F had medium intestinal absorption.

Fig. 2: BOILED-Egg representation of Bis-indole derivatives and standard BHT and Ibuprofen.

DISCUSSION:

In this study, novel bis-indole derivatives were designed, synthesized and characterized using FTIR and ¹H NMR and their antimicrobial activities were then analyzed through the Disc diffusion method. All the synthesized compound shows better result than reference ampicillin. All the compound shows a positive response in gram -ve bacteria compared to gram +ve bacteria, while compound 3b is more active against all microorganisms. From ADME studies, it is defined that all the compound are nontoxic and doesn’t show any pain alert. While boil-egg represents shows that the yellow York region is predicted to passively permeate through the blood-brain barrier , the white region covers the compound that can be passively reabsorbed from the gastrointestinal tract. Red indicates that the compound is not a substrate for P-glycoprotein while blue suggests the opposite.

CONCLUSION:

Bis-Indole derivatives were synthesized by condensing aromatic aldehyde and indole in acetic acid with good yield. The title compounds were confirmed by TLC, melting point, IR and NMR analyses. The synthesized compound was exposed to antimicrobial activity and showed a positive response that can be developed more for human welfare. The pharmacokinetic characteristics of title compounds are generally inferred from their properties of absorption, distribution, metabolism, excretion and toxicity (ADME/T). All the compounds are found to pass the ADME evaluation and few compounds passed the predicted toxicity evaluation. This work involved an initial approach to identifying potential novel molecules with promising activity with low toxicity and paving the way for further investigation and development.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Dr. Devanshu Patel, President, Parul University for providing necessary facilities.

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Received on 07.04.2023 Revised on 12.03.2024

Accepted on 19.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1469-1473.

DOI: 10.52711/0974-360X.2025.00210

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