Looga AM; Ambassa P; Kamga J; Hortense GK; Ngadjui BT; Ngameni B

Research Article

Austin J Anal Pharm Chem. 2019; 6(2): 1116.

Synthesis and Evaluation of Antimicrobial Properties of Some Novel Indole Pyridine Based Chalcones

Looga AM¹, Ambassa P^2*, Kamga J², Hortense GK³, Ngadjui BT² and Ngameni B¹*

¹Department of Pharmacognosy and Pharmaceutical Chemistry, University of Yaoundé I, Yaoundé, Cameroon

²Department of Organic Chemistry, University of Yaoundé I, Yaoundé, Cameroon

³Department of Microbiology, parasitology, hematology and infectious diseases, University of Yaoundé I, Yaoundé, Cameroon

*Corresponding author: Ambassa P, Department of Organic Chemistry, University of Yaoundé I, Yaoundé, Cameroon, Email: [email protected]

Ngameni B, Department of Pharmacognosy and Pharmaceutical Chemistry, University of Yaoundé I, Yaoundé, Cameroon, Email: [email protected]

Received: April 22, 2019; Accepted: July 15, 2019; Published: July 22, 2019

Abstract

A series of novel substituted Indolylchalcone derivatives 2a-d were synthesized via Claisen-Schmidt condensation between some selected indolic aldehyde and pyridynic acetophenone. The structures of the products were elucidated by spectroscopic analysis (¹H and ¹³C NMR). All compounds were screened for their antibacterial and antifungal activity against six different bacterial strains such as Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 700603, Staphylococcus aureus ATCC 25923, Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa and against one fungal strain Candida albicans. The Results reveal that all compounds exhibited moderate to good antibacterial and antifungal activities.

Keywords: Synthesis; Chalcone; Indole; Pyridine; Antibacterial & Antifungal Activity

Introduction

Chalcones or 1,3-diphenyl-2-propen-1-one derivatives are a class of open chain flavonoids in which two aromatic rings are linked by a three carbon a,β-unsaturated carbonyl skeleton. Chalcone are an important class of natural products which exhibit interesting pharmacological activities [1]. Some of the chalcones have been reported to possess many biological properties such as antimicrobial [2-4], anticancer [5], antimalarial [6], anti-inflammatory [7], antituberculosis [8], antioxidant [9,10], analgesic [11], anticonvulsant [12] and antidiabetic [13].

A good safety profile, possibility of oral administration [14] and easy synthesis are the major factors contributing to the increasing interest in exploring the pharmacological activities of chalcones. During the last decade, the antimicrobial resistant have represented the major problem facing the world, so that several new antibiotics and antifungal agents are accepted each year to help for the treatment of infectious diseases.

Indole derivatives have recently been reported to have potent antibacterial and antifungal property against human pathogens resistant to classic antibiotic [15]. In the other hand pyridine derivatives have been reported for variety of biological activities and number of compounds are in clinical uses [16]. Moreover some of those derivatives possess good antibacterial property against staphylococcus epidermis, streptococcus mutans and streptococcus sanguinis and are only slightly cytotoxic [17].

In our continuing effort to discover new antimicrobial agents [4] with excellent therapeutic index, we carry out the present research in order to combine the antimicrobial effect of chalcone moiety with those of indole and pyridine derivative. Hence, this research illustrated the synthesis of novel Indole pyridine chalcone derivatives and their biological screening against some gram positive and gram negative bacterial species including fungus specie by standard methods.

Experimental

Chemistry

All starting materials and solvents were purchased from Sigma- Aldrich and Alfa Aesar and used without further purification. Purity of the compounds was checked on silica gel G TLC plates of 2mm thickness using n-hexane and ethyl acetate as solvent system. The visualization of spot was carried out under UV light. NMR study was performed on Brucker AMX-300 using DMSO-d6 as solvent at 300MHz. Tetramethylsilane (TMS) was used as internal reference and chemical shifts are expressed in d ppm.

The key method of our synthesis involves the Claisen-Schmidt condensation of equimolar quantities of a substituted acetophenone with substituted aldehydes in the presence of anhydrous alcoholic alkali.

The various derivatives were synthesized according to procedure described by [18].

Synthesis of (E)-3-(1H-indol-3-yl)-1-(pyridin-3-yl) prop-2- en-1-one (2a): 0.173mL of 3-acetylpyridine (1.58mmol) was added dropwise with stirring to a solution of indole-3-carbaldehyde (153.87mg, 1.06mmol) in anhydrous ethanol (15mL). The mixture was treated with piperidine (0.155mL, 1.58mmol) and refluxed for 28 h. Upon completion, the mixture was cooled by pouring crushed ice in it to complete the crystallization. The solid was filtered by Buchner filtration and washed with ice-cold MeOH (30mL) and dried at room temperature for 24-48 h to yield 21.67% of a bright yellow powder

¹HNMR (300 MHz; DMSO-d6) dppm (m, J(Hz)): 11.98 (1H; .s; H-3’), 9.29 (1H; d; J=3Hz; H-2), 8.80 (1H; dd; J=3Hz et 6Hz; H-4), 8.45 (1H; dd; J=3Hz and 6Hz; H-6), 8.17 (1H; s; H-2’), 8.14 (1H; m; H-5), 8.11 (1H; d; J=15Hz; H-β), 7.65 (1H; d; J=15Hz; H-a), 7.60 (1H; dd; J=3Hz and 6Hz; H-8’), 7.49 (1H; m; H-5’), 7.26 (1H; m; H-6’), 7.24 (1H; m; H-7’). ¹³CNMR (75 MHz; DMSO-d6) dppm (m, J(Hz)): 187.9 (-C=O), 152.7 (C-4), 149.3 ( C-2), 139.9 ( C-β), 137.6 ( C-4’), 135.5 ( C-6), 134.0 ( C-1), 133.7 (C-2’), 125.1 (C-9’), 123.9 (C- a), 122.8 (C-5), 121.3 (C-6’), 120.6 ( C-7’), 115.0 ( C-8’), 112.9 ( 2C; C-1’ and C-5’).

Synthesis of (E)-3-(1-methyl-1H-indol-3-yl)-1-(pyridin-3-yl) prop-2-en-1-one (2b): 0.173mL of 3-acetylpyridine (1.58mmol) was added dropwise with stirring to a solution of N-methylindole-3- carbaldehyde (168.54mg, 1.06mmol) in anhydrous ethanol (15mL). The mixture was treated with piperidine (0.155mL, 1.58mmol) and refluxed for 28 h. Upon completion, the mixture was cooled by pouring crushed ice in it to complete the crystallization. The solid was filtered by Buchner filtration and purified by column chromatography using silica gel as adsorbent and a mixture of hexane and ethyl acetate as eluent. After recrystallization in 10% Hexane-ethyl acetate we obtained (2b) as a yellow powder with 67.33% yield.

¹HNMR (300 MHz; DMSO-d6) dppm (m, J(Hz): 9.29 (1H; d; J=3Hz; H-2), 8.81 (1H; dd; J=3Hz and 6Hz; H-4), 8.46 (1H; dd; J=3Hz and 6Hz; H-6), 8.17 (1H; s; H-2’), 8.16 (1H; dd; J=3Hz and 6Hz; H-8’), 8.07 (1H; d; J=15Hz; H-β), 7.65 (1H; d; J=15Hz; H-a), 7.59 (1H; m; H-5), 7.58 (1H; m; H-5’), 7.34 (1H; m; H-6’), 7.32 (1H; m; H7’), 3.88 (3H; s; Me-N). ¹³CNMR (75MHz; DMSO) dppm: 187.7 (C=O), 152.6 ( C-4), 142.2 (C-3), 139.1 (C-β), 138.0 (C-4’), 137.2 ( C-2’), 135.6 (C- 6), 133.6 ( C-1), 125.5 (C-9’), 123.8 (C-5), 122.8 ( C-6’), 121.5 (C-7’), 120.7 ( C-8’), 115.0 (C-a), 111.8 (C-1’), 110.8 (C-5’), 33.1(Me).

Synthesis of (E)-3-(1-methyl-1H-indol-3-yl)-1-(pyridin-4-yl) prop-2-en-1-one (2c): 0.173mL of 4-acétylpyridine (1.58mmol) was added dropwise with stirring to a solution of N-methylindole-3- carbaldehyde (168.54mg, 1.06mmol) in anhydrous ethanol (15mL). The mixture was treated with piperidine (0.155mL, 1.58mmol) and refluxed for 28 h. Upon completion, the mixture was cooled by pouring crushed ice in it to complete the crystallization. The solid was filtered by Buchner filtration and purified by column chromatography using silica gel as adsorbent and a mixture of hexane and ethyl acetate as eluent. After recrystallization in 10% Hexane-ethyl acetate we obtained (2c) as a yellow powder with 31.32% yield.

¹HNMR (300 MHz; DMSO-d6) dppm (m, J(Hz): 8.85 (2H; d; J=6Hz; H-3 and H-5), 8.17 (1H; s; H-2’), 8.15 (1H; dd; J=3Hz and 6Hz; H-8’), 8.07 (1H; d; J=15Hz; H-β), 7.99 (2H; d; J=6Hz; H-2 and H-6), 7.58 (1H; m; H-5’), 7.57 (1H; d; J=15Hz; H-a), 7.34 (1H; m; H-6’), 7.32 (1H; m; H-7’), 3.88 (3H; s; N-Me). ¹³CNMR (75 MHz; DMSO) dppm: 188.3 ( C=O), 150.6 (2C; C-3 and C-5), 144.6 (C-1), 140.1 ( C-β), 138.1 ( C-4’), 137.7 ( C-2’), 125.5 ( C-9’), 122.9 (C-6’), 121.8 (C-7’), 121.7 (2C; C-2 and C-6), 120.7 ( C-8’), 114.6 (C-a), 111.8 ( C-1’), 110.9 (C-5’), 33.1 ( Me-N).

Synthesis of (E)-3-(1H-indol-3-yl)-1-(pyridin-4-yl) prop- 2-en-1-one (2d): 0.173mL of 4-acetylpyridine (1.58mmol) was added dropwise with stirring to a solution of indole-3-carbaldehyde (153.87mg, 1.06mmol) in anhydrous ethanol (15mL). The mixture was treated with piperidine (0.155mL, 1.58mmol) and refluxed for 28 h. Upon completion, the mixture was cooled by pouring crushed ice in it to complete the crystallization. The solid was filtered by Buchner filtration and purified by column chromatography using silica gel as adsorbent and a mixture of hexane and ethyl acetate as eluent. After recrystallization in 10% Hexane-ethyl acetate we obtained (2d) as a yellow powder with 25.18% yield.

¹HNMR (300 MHz; DMSO-d6) dppm(m, J(Hz): 12.03 (1H; s; H-3’), 8.82 (2H; d; J=6Hz; H-3 and H-5), 8.18 (1H; s; H-2’), 8.11 (1H; d; J=15Hz; H-β), 8.10 (1H; m; H-8’), 7.77 (2H; d; J=6Hz; H-2 and H-6), 7.58 (1H; d; J=15Hz, H-a), 7.51 (1H; m; H-7’), 7.27 (1H; m; H-5’), 7.23 (1H; m; H-6’).

Biology

Paper disc diffusion technique

Anti-bacterial and anti-fungal activities of synthesized compounds were assessed by paper disc diffusion method [19,20]. Here, all the synthesized compounds’ antimicrobial activities were calculated from the zone of inhibition in Mueller-Hinton (anti-bacterial) and Sabouraud dextrose culture media (antifungal). First, the aforesaid sterilised (120°C for 30 min in autoclave) culture medium (40-50°C) was prepared and poured into a Petri dish (3-4mm), solidified and seeded with different micro-organisms (Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Staphylococcus aureus(ATCC 25923), Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa and Candida albicans) in on each plate. Simultaneously, different dilutions of synthesized compounds and standard solutions were prepared in dimethylsulfoxide (DMSO). To identify the anti-microbial activities, the paper impregnated with synthesized compounds were placed on the solidified Mueller- Hinton/Sabouraud dextrose medium and incubated at 37°C for 24 hours.

In addition to that, Norfloxacin (100mg/disc) and Nystatin (100mg/disc) were also placed on the Petri dish and had been considered as a standard for antibacterial and antifungal activities, respectively. The solvent control was charged separately on a Petri dish. For each compound, anti-microbial activity has been measured from its zone of inhibition against standard drugs.

Minimum inhibitory concentration

Minimum Inhibitory Concentration (MIC) is the least concentration of an antimicrobial agent having the potency to inhibit the detectable growth of a microorganism after an overnight period of incubation in appropriate culture media [20,21]. It is used to detect the potency and drug resistance nature of anti-microbial agents. A highly potent anti-microbial agent had the least value of MIC and needed less quantity to inhibit the visible growth of the microorganism. An anti-microbial agent that is called as less active needs high concentration to kill the micro-organism and to have greater MIC value. Here, the anti-microbial activities of different concentrations of the synthesized compounds in dimethylsulfoxide (DMSO) were calculated through MIC value.

Each concentration of the synthesized compounds (1000, 500, 250, 125, 62.5 and 31.25μg/mL) was tested with bacteria/fungal seeded culture medium (Mueller-Hinton and Sabouraud dextrose) for 24 hours/48 hours at 37°C. The different microbial inoculums were used to prepare compared Mc Farland 0.5 standard bacteria suspensions.

The minimal inhibitory concentration was read by observing the opacity of the wells. The inhibition of microbial growth by the test compounds left some wells clear (not turbid), except in the negative control. The least concentrated well in which no turbidity was observed was considered the minimum inhibitory concentration of the test compound.

Result and Discussion

Chemistry

The entire synthesized compounds were prepared from the mentioned synthetic scheme that is depicted in Scheme 1. The novel bi-heterocyclic chalcone derivatives were prepared by a Claisen- Schmidt condensation reaction between substituted acetophenone derivatives and 2 substituted 1H-indole-3-carbaldehyde.

Scheme : General reaction and mechanism of chalcone synthesis.

    
    
    Scheme : General reaction and mechanism of chalcone synthesis.

The chemical structure of all our synthesized compounds was elucidated by 1HNMR, ¹³CNMR, and elemental analysis. The number of protons and carbons present in the synthesized compounds was identified by ¹HNMR and ¹³CNMR spectroscopy, respectively from the chemical shift (d in ppm). The proton spectra showed a doublet at d 7.60 and 8.11ppm corresponding to the a and β hydrogen respectively of the unsaturated carbon with a coupling constant of 15Hz which confirmed their trans stereochemical configuration; a doublet at d 7.77-8.82ppm corresponding to aromatic protons (Ar-H); a doublet of doublet at d 8.80-8.81ppm corresponding to aromatic protons (Ar-H); a multiplet at d 7.59-8.14ppm corresponding to aromatic protons (Ar-H) and a singlet at d 11.98-12.03ppm corresponding to NH-proton; a singlet of three protons at d 3.88 ppm corresponding to N-CH3-protons. The composition of the atom present in the synthesized compounds was confirmed through element analysis.

The ¹³CNMR reveals the pic of the carbonyl group around 187.0ppm as the most deshielded.

Anti-microbial activity

In the recent times, many organisms are developing resistance to available antibiotics. Therefore, there is a need to prepare a novel agent that is resistant free in nature, acts specifically on microorganisms and causes the least harm to the host function. The synthesized chalcone derivatives possessed all the advantages as stated above due to the presence of pyridine and indole nucleus as part of the molecular structure. The synthesized compounds (2ac) had been screened for anti-microbial activity against Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Staphylococcus aureus (ATCC 25923), Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa and Candida albicans using zone of inhibition (mm) and minimum inhibitory concentration (μg/mL) level. The amount of Compound 2d synthesized was not enough to be screen for antimicrobial activity and had just been used for structural analysis. We found that most of the synthesized compounds showed significant anti-microbial activity against different organisms and the results are demonstrated in Tables 1,2. Among the different synthesized compounds, compounds 2b showed significant antimicrobial activity against various organisms as compared to standard drugs like Norfloxacin and Nystatin. Particularly compound 2b had a zone of inhibition against S. typhi (20mm), P. mirabilis (19mm) and the lowest MIC value for S. typhi (31.25μg/mL) were recorded with compound 2c.

Synthesis and Evaluation of Antimicrobial Properties of Some Novel Indole Pyridine Based Chalcones

Abstract

Introduction

Experimental

Chemistry

Biology

Paper disc diffusion technique

Minimum inhibitory concentration

Result and Discussion

Chemistry

Anti-microbial activity

Conclusion

References

Compounds	*S. aureus*	*E. coli*	*P. mirabilis*	*P. aeruginosa*	*S. typhi*	*K. pneumoniae*	*C. albicans*
2a	10	9	18	8	9	8	20
2b	12	9.5	19	13	20	9	19
2c	9	9.5	17	12.7	17	7	21
Norfloxacin	19	15	22	17	16	12	-
Nystatin	-	-	-	-	-	-	23

Compounds	*S. aureus*	*E. coli*	*P. mirabilis*	*P. aeruginosa*	*S. typhi*	*K. pneumoniae*	*C. albicans*
2a	250	500	125	125	1000	125	62.5
2b	125	62.5	125	250	62.5	125	125
2c	250	250	250	250	31.25	125	62.5
Norfloxacin	250	62.5	125	31.25	1000	31.25	-
Nystatin	-	-	-	-	-	-	15.625