Synthesis, Crystal Structure, Cytotoxic, Antileishmanial and Docking Evaluation of 3-(4-Chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one

HAMID Aziz AAMER See FARUKH Jeen ULRICH Florke QURAT-Ul-Ain NADHMAN Akhter

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Synthesis, Crystal Structure, Cytotoxic, Antileishmanial and Docking Evaluation of 3-(4-Chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one

HAMID AzizaAAMER Saeeda①FARUKH JabeenbULRICH FlorkecQURAT-Ul-AindNADHMAN Akhterd

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Chalcone derivatives are of immense importance owing to their superior pharmacological profile. Minor changes in their structures create high degree of diversity that results in a broader spectrum of biological activities. The present research paper presents the synthesis of 3-(4-chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one chalcone (I), single crystal XRD analysis and the evaluation of its possible biological activities like cytotoxic (LD50=5.40 ug/mL) and antileishmanial (LD50=0.81 ug/mL) activities. The results indicate the tested chalcone (I)shows superior inhibition values againstpromastigotes. Further, the possible interaction sites of chalcone (I)with TryR enzyme involved in the redox metabolism of the leishmanial parasite were determined using docking simulation technique.Docking computations demonstrate that the compound establishes prominent binding interactions with the key residues ofTryR and possesses the potential to effectively inhibit the catalytic activities ofenzyme. Thus the results suggest that the chalcone (I) can serve as a potential scaffold for the treatment of leishmaniasis and deserve further development.

chalcone,cytotoxicity,antileishmanial,Claisen-Schmidt condensation,molecular docking;

1 INTRODUCTION

Chalcones are open-chain flavonoids where the bichromophoric molecule is separated by a hetero-vinyl chain[1]. Chalcones exist both inandforms, and can easily be cyclized via Michael addition to flavanones[2]. The presence of double bond conjugated with carbonyl group is chiefly responsible for the biological activities of chalcone. The important pharmacological profile of chalcones includes anticancer, anti-inflammatory, anti-oxidant, anti-microbial, antimalarial[3], leishmanicidal[4]and cytotoxicity[5]. Mostly, single chalcone exhibits several bioactivities, like isoliquiritigenin which shows anti-inflammatory, anxiolytic, and anti-pigmentation activities[3a].Several chalcones have been tested clinically against diseases like hesperidin methylchalcone as vascular protective[3b].

Chalcones are considered as effective cytotoxic agents. Reddyreported the cytotoxicity of resorcinol based chalcones[6]. Similarly, Kolundzijashowed anticancer activity of anthraquinone based chalcones. They argued electron withdrawing substituents as beneficial for cytotoxic activities[7]. Jainargued halogenated chalcones as potent cell growth inhibitors[8]. Hydroxyl chalcones display cytotoxic activity and serve as in vivo cell proli-ferating inhibitors without side effects as compared to their counterparts[9]. Leishmaniasis is a zoonosis that spreads through sand fly. Chalcones offer opportunity for the treatment of Leishmaniasis. The most promising antileishmanial member to date is licochalcone A. Based on this chalcone, several members have been identified as lead compounds[10]. Narender11]reported antileishmanial activity of natural chromenochalcones, notably crotaramosmin, crotaramin and crotin. Boeck P.reported the synthesis and antileishmanial potential of chalcone analogues of xanthoxyline. The synthesized chal- cones displayedpotential against cutaneous leishmaniasis[12].

Thus keeping in view the cytotoxic and antilei-shmanial potential of chalcones, and in continuation of our interest in the synthesis and molecular structure of biologically active compounds[13-16]the present paper reports an efficient synthesis and X-ray crystal analysis of chalcone 3-(4-chloro-3-nitro-phenyl)-1-phenylprop-2-en-1-one chalcone (I). Keeping in view the biological importance of chalcone scaffolds, the synthesized chalcone (I) wasinvestigated for itscytotoxic activity towards human red blood cells and its anti-parasitic ability againstparasites.

2 EXPERIMENTAL

2. 1 Materials and instruments

All of the reagents and solvents required were purchased from sigma-Aldrich. Glassware required for performing chemical reaction was dried in an oven at 80 ℃for minimum of 4 hprior to use.1H and13C nuclear magnetic resonance (NMR) spectra were recorded using DMSO-d6as a solvent on a Bruker spectrometer at 300 and 75 MHz, respec-tively. DMSO-d6was used as solvent and TMS as an internal reference. Chemical shifts are given inscale (ppm). FT-IR spectra were recorded on the Vertex 70 Bruker apparatus. Elemental analysis (CHN) was performed to find out the percentage ofeach element present in the synthesized compound. The progress of the reactions was checked by thin-layer chromatography (TLC) on 2.0cm × 5.0cm aluminium sheets precoated with silica gel 60F254 with a layer thickness of 0.25 mm (Merck). Crystals of the synthesized chalcone (I) were grown by slow evaporation of ethanol at room temperature and the crystal structure was confirmed directly via single crystal XRD.

2. 2 General procedure

Chalcones, a versatile class of natural products, have attracted researchers’ attention for their wide range of biological activities. Electron withdrawing moieties are supposed to further enhance their activities. The best known method for the synthesis of chalcones is the Claisen-Schmidt condensation between ketone and aldehyde in basic media. In this regard, the present paper reports the synthesis of chalcone (I) by Claisen-Schmidt condensation of acetophenone and 4-chloro-3-nitro benzaldehyde using catalytic amount of sodium hydroxide in ethanol[17],as shown in Scheme 1. The synthesized chalcone (I) was purified and recrystallized from ethanol at room temperature as colourless crystals.

2. 3 Syntheses

2. 3. 1 Procedure for the synthesis of chalcone (I)

The title chalcone (I) was synthesized as per the literature reported method with minor modifications. Briefly, a mixture of acetophenone (0.01 mol) and 4-chloro-3-nitrobenzaldehyde (0.01 mol) was dissolved in ethanol (20 mL). Catalytic amount (0.1 gm) of sodium hydroxide was added to the solution dro- pwise with vigorous stirring at room temperature. The reaction mixture was stirred for about 6 h at room temperature. The resultant crude products were filtered, washed successively with distilled water and recrystallized from ethanol to get the corresponding chalcone (I)[13]. Colorless crystals suitable for X-ray diffraction studies were obtained by the slow evaporation technique using ethanol as a solvent.

2. 3. 2 Purification of the synthesized chalcone (I)

Once the chalcone (I) was successfully synthe-sized, the next step involved was the purification of chalcone (I) by recrystallization. For this purpose, chalcone (I) was first purified using column chromatography (30% hexane: ethyl acetate) which yields the purified compounds as solid precipitates. The purified chalcone(I) was further purified byrecrystallization method from ethanol. Briefly, the synthesized chalcone (I) was dissolved in ethanol and its supersaturated solutionwas prepared at 60 °C. The prepared supersaturated solution of chalcone (I) was placed in glass vials covered with a lid to evaporate ethanol slowly from the solution at room temperature. Crystals of chalcone (I) appeared gradually as the evaporation of ethanol started at room temperature. The crystals obtained were separated from the mixture, dried and analyzed using single-crystal XRD to confirm the structure of the chalcone (I). The characterization data of chalcone (I) including1H NMR,13CNMR and CHN analysis are provided below.

2. 3. 3 3-(4-Chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one (I)

Yield 90%; Mol. wt: 287.7;1H NMR (DMSO-d6), 8.20 (s, 1H), 8.08 (d, 1H), 7.83 (d, 1H), 7.81 (d, 2H), 7.48 (d, 1H), 7.408 (m, 2H), 7.64 (d, 1H), 7.5 (d, 1H);13C NMR (DMSO-d6), 190, 148.3, 145.25, 138.5,134.1, 133.4, 134.3, 129.8, 129.6, 129.5, 128.2. 122.7, 121. Anal. Calcd. (%) for C15H10ClNO3:C, 62.62; H, 3.50; Cl, 12.32; N, 4.87; O, 16.68.

2. 4 Biological activities

2. 4. 1 Cytotoxic activity

Cytotoxicity of chalcone (I) was carried out by using a modified method of Nadhman., 2015[18]. Fresh human red blood cells were washed with phosphate buffer solution (PBS) for three times at 3000rpm. The blood cell suspension was incubated with 20μL of chalcone (I) with different concentra-tions at 37oC for 3 hours[19]. After incubation the suspended cells were centrifuged for 10 minutes at 6000rpm. By using UV-visible spectrophotometry (T80, pg instruments), the released hemoglobin was assessed at 576nm by taking the supernatant. Triton X-100 (0.1%) was taken as positive control and the red blood cell suspension in PBS without compound as a negative control. Percent hemolysis was calculated by the following formula:

2. 4. 2 Antileishmanial activity

For anti-leishmanial assay, stock solution of chal-cone (I)was prepared by dissolving it in 1mg/mL of DMSO (20%).KWH23 promastigotes were grown in the M199 (Medium 199). Media were prepared by modified method of Nadhman., 2014[20]. Briefly, cells were cultured in M199, streptomycin, 25mM HEPES buffer(4-(2-hydroxyethyl)-1-piper-azi-neethanesulfonic acid) supplemented with 10% heat inactivated FBS with pH 7.2 and maintained at 24oC. Medium 199 containing culture of promastigotes was suspended in the wells of96-well microtiter plates. After counting cells on Neubauer chamber, promastigotes were suspended to yield 2×106cell/mL in each well. Afterwards, as a reference drug amide compound was treated serially in each well of the plate and 2 wells were left for positive and negative controls. DMSO was taken as negative control and success-sively diluted in the M199 medium. Glucantime was used as positive control and then overnight incubated in the dark at 25oC for 72 h. The number of viable cells was counted on the Neubauer chamber under microscope (Micros, AUSTRIA). All the in-vitro reactions were carried out in triplicate and the results were expressed as a % inhibition in parasite numbers. The drug concentrations required for 50% inhibition, IC50values of the compound, showing anti-leishma-nial activity, were determined by SPSS22 software.

Scheme1. Synthesis of 3-(4-chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one chalcone (I)

2. 5 Crystal structure determination of chalcone (1)

C15H10ClNO3,M= 287.7, colourless crystal, size 0.43mm′0.16mm′0.05mm, monoclinic space group2/c with= 4,= 4.935(6),= 15.159(17),= 17.07(2) ?,= 94.95(3)°,= 1273(3) ?3,D= 1.502 Mg/m3,?= 0.306 mm-1, and(000) = 592. The intensity data were recorded using a Bruker SMART CCD area-detector diffractometer with gra-phite-monochromated Moradiation (= 0.71073 ?) at= 293(2) K. 11725 reflections were collected 1.8≤≤27.9°, including 3034 independent reflections with> 2(),int= 0.11. Structure solution by direct methods[21], full-matrix least-squares refinement based on2and 182 parameters. All but H-atoms were refined anisotro-pically, hydrogen atoms were clearly located from difference Fourier maps and then refined at idealized positions riding on the carbon atoms with isotropic displacement parametersiso(H) = 1.2eq(C) and C–H 0.93 ?. Refinement converged at= 0.047 andw= 0.109. (Δ)max= 0.18, and (Δ)min= 0.37 e/?3.

3 RESULTS AND DISCUSSION

3. 1 X-ray crystal structure description for chalcone (I)

The molecular structure of 3-(4-chloro-3-nitrophenyl)-1-phenylprop-2-en-1-one (I, Fig. 1) is closely related to that of (2E)-3-(2-chloro-5-nitrophenyl)-1-phenylprop-2-en-1-one (LONPIP)[22]but with 4-chloro substitution. The compound 2 shows somewhat twisted conformation with torsion angles C(2)–C(3)–C(11)–C(12) 2.9(4)°, C(2)–C(1)–C(21)–C(26) 9.6(4)° and the C–C=C–C angle C(11)–C(3)–C(2)–C(1) 179.8(3)°; the respective dihedral angle between the two aromatic ring planes is 25.1(2)°. Some selected geometric parameters are given with Fig. 2. The crystal packing (Fig. 2) shows weak intermolecular C(3)–H×××O(1)(–+1,+1, –+1) hydrogen bonds with H×××O of 2.51 ? and C–H×××O of 148.3° that connect molecules into centro-symmetric dimers which are stacked along the-axis. The complete single crystal data including bond distances and angles are given in the supporting information in Tables 1～5.

Fig. 1. Molecular structure of chalcone (I).Anisotropic displacement ellipsoids are drawn at the 50% probability level. Selected geometric parameters are C(21)–C(1) 1.487(4), C(1)–O(1) 1.219(3), C(1)–C(2) 1.453(4), C(2)–C(3) 1.327(3), C(3)–C(11) 1.445(4), C(14)–Cl(1) 1.715(3), N(1)–C(15) 1.471(4), N(1)–O(2) 1.211(3), N(1)–O(3) 1.198(3) ? and C(21)–C(1)–C(2) 119.5(3)°, C(1)–C(2)–C(3) 122.3(3)°, C(2)–C(3)–C(11) 126.8(3)°, O(2)–N(1)–O(3) 123.0(3)°

Fig. 2. Crystal packing of chalcone (I) with main atomic labelling scheme and hydrogen bonding pattern as dotted lines viewed along the-axis. H-atoms not involved and minor orientation of disordered chain are omitted

3. 2 Biological activities of the synthesized chalcone (I)

Thecytotoxic and antileishmanial activi-ties of the synthesized chalcone (I) were evaluated. Cytotoxicity of Iwas performed using human red blood cells. The release of hemoglobin from the red blood cells is the indication of cytotoxicity, which was less with the chalcone compound. Currently, it was found that the chalcone compound is biocom-patible (LD50= 5.40 ug/mL). For anti-leishmanial assay, DMSO was taken as negative control and Glucantimeas positive control. Antileishmanial activity results revealed that chalcone acted as an excellent antileishmanial agent against the promas-tigotes of. chalcone (I) has maximum antileishmanial activity with IC50value 0.81 ug/mL, while the standard drug (Glucantime) showed an IC50of 7.44 ug/mL, which clearly indicates that the current chalcone (I) is a better antileishmanial agent than the standard drug. The higher efficacy of Imight result from the chlorine and nitro groups present in the structure ofchalcone[23].

3. 3 Molecular docking study

Synthesized compound was computationally docked into the active site of trypanothione reductase (TryR) enzyme, required for redox balance of the parasite. Trypanothione reductase (TryR) is involved in the redox metabolism of the leishmanial parasite and is a key drug target enzyme. Inhibition of TryR may disrupt the redox balance of the parasite leading to parasite death[19].Thus, organic molecules may be designed, which can bind the active site of TryR leading to its inhibition.

tools such as virtual screening, Quantita-tive Structure Activity Relationship (QSAR), and molecular docking have been instrumental for the accelerate design and discovery process of thera-peutic agents[24].In current studies,Glide docking module[25], offered by Schrodinger suite, was used to perform molecular docking of small molecule on macromolecular protein by treating the ligand as conformationally flexible.analysis sugges-ted TryR as a possible target for such compounds. The compound showed reasonable docking with acceptable statistics in the active site of TryR. The enzyme (TryR) is a dimer consisting of two active sites and the active site from the chain A was selected for the docking studies. The following residues, such as: Gly13, Ser14, Gly15, Val53, Val55, Gly56, Lys57, Lys 60, Lys61, Leu62, Ser178, Phe182, Tyro 198, Ile199, Glu202, Phe203, GLy326, Asp327, Val332, Leu334, Met333, Thr335, and Ala338, were found around the co-crystallized ligand inside the active site gorge. It can be suggested that these residues can play important functional roles in affecting the catalytic function of the enzyme TryR. Thus, interaction of the ligands with these residues can change the active site structure, leading to the inhibition ofenzyme activates. Fig. 3b represents the binding of ligand inside the pocket of TryR. The detailed analysis of the inhibition mode of the ligand revealed that the oxygen atom of the NO2grouppresentat one of the phenyl groups of the ligand anchored the ligand by establishing the robust hydrogen bonding with Thr 51 and Cys52 (Figs. 4a and 4b). In addition to that tertiary nitrogen of the nitro group formed salt bridge with Asp327. Moreover, chlorine atom present at the same phenyl ring established halogen bonding (shown in pink dashed line, Fig. 4a) with the Ala338. The flanking region of the ligand was stabilized inside the active site by a determinant hydrogen bonding established by carbonyl oxygen with Lys60 in addition to strongstacking (Fig. 4a) of the second phenyl ring of the molecule with the phenyl group of Trp198. Furthermore, compound was found to interact favorably with TryR protein with a very good docking and Glide score of –6.318 kcal/mol.

Fig. 3. (a) Optimized geometry of the chalcone (I), ligand is presented in ball n stick mode in cyan color. (b) Modeling of the ligand with TryR protein. Docked conformation of compound is shown inside the active site of TryR. Receptor is shown in ribbon and cartoon mode and ligand is shown in CPK presentation in cyan and elemental color

Fig. 4. Binding mode of chalcone (I) inside the active site of TryR. a) The docking pose of compound in three-dimensional space. The ligand is shown in ball and stick mode with carbon atoms shown in cyan color. Key segments of the amino acid residues are shown in stick mode. Aromatic hydrogen bonding, hydrogen and halogen bonding of ligand atoms with residue atoms is shown in cyan, yellow and pink colored dashed lines respectively. b) A representation of the binding interactions of compound inside the active site of TryR in 2D space. Details about the image are given in legend

4 CONCLUSION

The present paper describes in detail the synthesis, bio-assay and molecular structure of a novel chalcone (I). The novel chalcone (I) was synthesized by Claisen-Schmidt condensation in a good yield. The colorless crystals of chalcone (I) were grown by slow evaporation of the solvent technique and were analyzed by single crystal XRD. The crystal structure diversities of the compound and various interactions responsible for their crystal stability are described in detail. In addition, the chalcone (I) is also evaluated for in vitro cytotoxic and antileishmanial activity. The compound produced a varied range of inhibition results, which is due to the presence of electron negative halogen and nitro substituents on the phenyl ring. Further, the betterbiocompatibility and antileishmanial activity of the chalcone could be a better choice for further evaluation as therapeutic agents against leishmaniasis upon comparison with the standard drug (Glucantime). Ligand docking studies could provide the first evidence for binding of compound in different areas of TryR. Thus, chalcone (I)can be a possible lead for the development of novel drug against leishmanial parasites. A highly significant inhibitive potential exhibited by the compound proclaimed it to be considered as a potential surro-gate for future investigation as a potential candidate for the treatment of Leishmaniasis.

ACKNOWLEDGMENTS

The author Mr. Hamid Aziz acknowledgesthe Higher Education Commission of Pakistan for indigenous scholarship as a financial support for the research work performed.

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30 November 2017;

13 May 2018 (CCDC1547931)

① Department of Chemistry, Quad-iAzam University Islamabad. 009251-90642128. E-mail: aamersaeed@yahoo.com