Synthesis, in silico screening and anthelmintic activity of chlorine containing chalcones

INTRODUCTION

The aim of the study was to synthesize and characterize chlorine containing chalcones and evaluate their potential as anthelmintic agents through in vitro testing and in silico molecular docking studies.

Chalcones, which are found in large quantities in many different plants, are thought to be the progenitors of flavonoids and isoflavonoids. The two aromatic rings are linked by a three- carbon, unsaturated carbonyl system to form open-chain chalcones [Figure 1].^[1] Unsaturated ketone moiety is mainly responsible for these actions.^[2]

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Figure 1:

General structure of chalcone.

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There is considerable interest in the incorporation of different substituents into the two aryl rings since it results in a favorable structure-activity relationship.^[3] Other names for chalcones are benzylidene acetophenone and benzalacetophenone. Chalcones consist of an aliphatic three-carbon chain connecting two aromatic rings. The first condensation is reported by Kostanecki and assigned the name “chalcones.”^[4] Kostanecki, who introduced the first condensation, and named it as “chalcones.” Aromatic ketones, substituted aromatic ketones reacts with benzaldehyde, substituted benzaldehyde in the presence of alcoholic alkali to form chalcones and substituted chalcones by Claisen–Schmidt condensation.^[5] Chalcones are biologically significant molecules due to their unsaturated carbonyl system.^[6] Chalcones are frequently used as intermediates in the synthesis of several heterocyclic compounds.^[7] It has been reported that the compounds with the chalcone moiety have antibacterial, anti-inflammatory, analgesic, antiplatelet, antiulcerative, antimalarial, anticancer, antiviral, anti-leishmanial, antioxidant, anti-tubercular, anti-hyperglycemic, and immunomodulatory properties.^[2,7-9]

The two major helminth phyla are nematodes and Platyhelminths. Nematodes are often referred as round worms which include soil-transmitted helminths and filarial worms that causes lymphatic filariasis and onchocerciasis.^[10] Other phyla of platyhelminths, such as schistosomes, flukes, and tapeworms like the porcine tapeworm that causes cysticercosis, are also referred to as flatworms. Tapeworms are referred to as cestodes and flukes as trematodes.^[11,12] Roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura), and hookworms (Ancylostoma duodenale and Necator americanus) are the three helminthiasis-transmitted worms.^[11] An estimated 1 billion people in underdeveloped nations – specifically, sub-Saharan Africa, Latin America, and Asia – are infected with helminths.^[11] In areas with few resources and poor sanitation, these illnesses are most frequently found. Types of worms and causative agents are given below in Table 1.^[11,12]

The study aimed to synthesize chlorine containing chalcones and study their anthelmintic potential toward Eisenia fetida, the model worm used for the study. The specific objectives of the study are to synthesize chalcones by condensing substituted benzaldehydes with p-chloroacetophenone through Claisen–Schmidt condensation, followed by in silico docking studies to analyze their binding interactions with tubulin-colchicine protein using Molegro Virtual Docker. The docking studies intended to identify favorable binding conformations of the synthesized ligands at the colchicine-binding site. The study also focused to assess the in vitro anthelmintic activity of the synthesized chalcones on E. fetida earthworms and comparing their efficacy with that of the standard drug, albendazole. In addition, the research sought to determine the binding affinities of the chalcones to tubulin-colchicine and select the compounds with the highest binding affinity for further testing. Finally, the synthesized compounds were evaluated for the anthelmintic potential by observing the paralysis and death times (PT and DT) in E. fetida at different concentrations of the chalcones with that of albendazole.

MATERIAL AND METHODS

Substituted benzaldehyde, p-chloroacetophenone, sodium hydroxide, ethanol, albendazole, normal saline, and dimethyl formamide were purchased from the local vendor and were used for the synthesis of chalcones and for the in vitro anthelmintic studies.

Procedure

Chalcones were prepared using an earlier published technique, which is described below.^[2,13-15] A solution of sodium hydroxide (0.1 mmoL) in water (20 mL) and ethanol (15 mL) was chilled in an ice bath using a conical flask. Acetophenone (5 mmoL) was added to the cooled solution, followed by benzaldehyde (5 mmoL). The reaction mixture was agitated continuously using a stirrer or vigorous hand shaking, and the temperature should be maintained at 25°C. Thin-layer chromatography was used to evaluate the reaction progress, with hexane and ethyl acetate (90:10) as the mobile phase.^[16] The reaction mixture was stirred until the reaction gets complete. Then, all the contents were slowly poured down into crushed ice with continuous shaking. The separated product was filtered, washed with cold water. Further, the synthesized compounds were recrystallized using chloroform as solvent.^[17] The mechanism of formation of chlorochalcones is as follows [Scheme 1]:

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Scheme 1:

Mechanism involved in the formation of chlorochalcones.

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RESULTS

In silico studies

The previous report on m-chloro substituted chalcones possessing anti-tubercular and anti-oxidant property. The in silico study using Pyrx virtual screening revealed that it has more binding affinity varies from -6.2 to -7.6 compared to -4.8 for isoniazid and -5.4 for pyrazinamide as a reference standard.^[24] Similar to this, the earlier study on the anti-inflammatory properties of chalcone derivatives was conducted in silico using AutoDock Vena and interaction demonstrated with Discovery Studio Ver. 2017 against a reference drug, diclofenac sodium. Superior anti-inflammatory activity is demonstrated by compounds 3b, which have a binding score of −6.5 for hydroxy substitutions. According to molecular docking evaluations, compounds 3g (fluoro) and 3 h (nitro) showed the greatest binding scores, with values of −7.6 and −7.9, respectively.^[25] These findings collectively highlight the versatility of chalcone derivatives, and implies that structural modifications by introduction of halogen substitutions, can enhance the biological activity of chalcones-based compounds, making them desirable candidates for further medicinal research and development. This gained interest on synthesizing p-chloro substituted chalcone derivatives showing anthelmintic activity, which was demonstrated using molecular docking studies by Molegro Virtual Docker, with 1SA0 (tubulin-colchicine protein) as the receptor target.^[26,27]

The Molegro Virtual Docker study helped to predict the interaction between the ligand and the suitable receptor. In the present study, most of the compounds showed comparable docking scores with that of the reference drug albendazole. The notable docking scores indicated the good affinity of the designed molecules with the active site of the tubulin-colchicine protein. The results are shown in Table 2. The two-dimensional structure of the compound and protein interactions are shown in Figures 2a-9b.

Table 2: Binding affinity of the compounds.

Compound code	Protein	Binding affinity	Rerank score	H-bond
Standard (Albendazole)	1SA0 (ABC chain)	−104.391	−82.4306	−1.45972
	1SA0 (DE chain)	−114.631	−92.5765	−2.30178
Compound 1	1SA0 (ABC chain)	−98.1831	−82.0944	−1.96296
	1SA0 (DE chain)	−107.233	−88.9135	0
Compound 2	1SA0 (ABC chain)	−115.442	−91.0031	−3.06872
	1SA0 (DE chain)	−113.789	−88.9763	−2.03616
Compound 3	1SA0 (ABC chain)	−104.7	−84.3090	0
	1SA0 (DE chain)	−109.144	−90.4401	0
Compound 4	1SA0 (ABC chain)	−115.086	−94.1388	−0.98044
	1SA0 (DE chain)	−112.032	−93.123	−2.44852
Compound 5	1SA0 (ABC chain)	−113.996	−93.8205	0
	1SA0 (DE chain)	−103.786	−81.7711	0
Compound 6	1SA0 (ABC chain)	−98.7361	−81.8298	−2.5
	1SA0 (DE chain)	−87.981	−73.9639	−2.5
Compound 7	1SA0 (ABC chain)	−99.6062	−81.2511	0
	1SA0 (DE chain)	−94.9139	−79.7652	0
Compound 8	1SA0 (ABC chain)	−124.531	−62.3269	0
	1SA0 (DE chain)	−109.674	−90.6929	0

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Figure 2:

(a) Compound 1 (ABC-CHAIN) and (b) Compound 1(DE-CHAIN).

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Figure 3:

(a) Compound 2 (ABC-CHAIN) and (b) Compound 2 (DE-CHAIN).

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Figure 4:

(a) Compound 3 (ABC-CHAIN) and (b) Compound 3 (DE-CHAIN).

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Figure 5:

(a) Compound 4 (ABC-CHAIN) and (b) Compound 4 (DE-CHAIN).

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Figure 6:

(a) Compound 5 (ABC-CHAIN) and (b) Compound 5 (DE-CHAIN).

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Figure 7:

(a) Compound 6 (ABC-CHAIN) and (b) Compound 6 (DE-CHAIN).

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Figure 8:

(a) Compound 7 (ABC-CHAIN) and (b) Compound 7 (DE-CHAIN).

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Figure 9:

(a) Compound 8 (ABC-CHAIN) and (b) Compound 8 (DE-CHAIN).

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Synthesis of chalcones

Based on the bond affinity as well as feasibility of the reaction conditions, four compounds were chosen for the synthesis and their physiochemical properties are shown in Table 3.

Table 3: Physicochemical properties of chalcones.

Compound number	Chemical name	Structure	% Yield	Melting point (°C)
Compound 1	(2 E)-1-(4-chlorophenyl)- 3-phenylprop-2-en-1-one		61.88%	99°C
Compound 2	(2 E)-1-(4-chlorophenyl)-3-[4-(dimethyl amino) phenyl] prop-2-en-1-one		98.19%	111°C
Compound 3	(2 E)-1,3-bis (4-chlorophenyl) prop-2-en-1-one		99.83%	148° C
Compound 4	(2 E)-1-(4-chlorophenyl)-3-(4-nitrophenyl) prop-2-en-1-one		94.72%	130°C

In this study, the synthesis of chalcones [Scheme 2] was carried out using substituted benzaldehyde with p-chloro acetophenone using sodium hydroxide and ethanol as the reaction medium. The aromatic aldehydes used for the synthesis includes benzaldehyde, 4-dimethyl amino benzaldehyde, p-chloro benzaldehyde, and p-nitro benzaldehyde. The development of the reaction was evaluated using thin-layer chromatography and the spots were identified using iodine vapors and ultra-violet chamber.

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Scheme 2:

Preparation of chalcones from substituted benzaldehydes.

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In vitro studies

Anthelmintic activity against E. fetida

The anthelmintic activity of synthesized compounds was evaluated on E. fetida, and then, its efficacy is compared with that of albendazole (reference drug). The synthesized compounds 1, 2, 3, and 4 exhibited more anthelmintic activity when compared to the standard drug activity followed by compounds 1 and 4. The anthelmintics activity of synthesized chalcones against E. fetida is shown in Table 4 and Figures 10a - 11d albendazole. Among all the compounds, compounds 2 and 4 showed highest anthelmintic activities.

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Figure 10:

Anthelmintic activity of (a) control experiment (b) Albendazole (reference drug).

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Figure 11:

(a-d) Anthelmintic activities of compounds 1–4.

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Table 4: Anthelmintic activity of chlorine containing chalcones against Eisenia fetida.

Name of the compound	Concentration (mg/mL)	Time of paralysis (min)	Time of death (min)
Control	0	-	-
Albendazole	1.25	99±13.22	152.33±4.04
Compound 1	1.25	75.66±0.57	126.66±27.42
	2.5	23.66±3.51	125.66±1.52
Compound 2	1.25	50±13.89	136.33±1.52
	2.5	24.33±2.51	34.66±0.57
Compound 3	1.25	59.66±1.15	122.66±16.16
	2.5	18±1.0	91±0
Compound 4	1.25	52.33±0.57	85±1.0
	2.5	14.66±0.57	82.66±0.57

± - standard error

DISCUSSION

Based on the in silico results, it was understood that the chalcones have exhibited more or less similar binding energy as that of the standard drug, albendazole. Hence, it was predicted that the proposed compounds could emerge out as good candidate with anthelmintic potential. Interestingly, the results that obtained from the anthelmintic activity of the chosen compounds (1–4) against E. fetida were in consensus with the in silico predictions. The compounds 1–4 caused paralysis (<75.66 ± 0.57 min) and even death (<136.33 ± 1.52 min) of the worms in much lesser time than the standard drug (PT: 99 ± 13.22; DT: 152.33 ± 4.04) at identical concentrations (1.25 mg/mL). The results thus obtained indicated that these chalcones could emerge as promising drug candidates for treating helminthic infections.

CONCLUSION

The present study was aimed at synthesizing chlorine containing chalcones and explores their activity towards helminths. In this perspective, the in silico studies of the proposed compounds were performed using the tubulincolchicine protein and their binding interactions showed promising results, similar to those of the reference drug albendazole. Therefore, the compounds were synthesized using earlier reported procedures. The synthesized compounds were explored for anthelminthic activity using, the E. fetida and showed an enhanced activity than albendazole and thus supports the in silico predictions. From the preliminary results thus obtained, the synthesized compounds could possibly emerge out as promising lead compounds for treating helminthiasis. As a future prospective, the compounds have to be assessed for various toxicity studies and establish its potential in treating various helminthic infections.