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European Journal of Medicinal Chemistry 43 (2008) 1530e1535http://www.elsevier.com/locate/ejmech

Short communication

Synthesis of novel substituted 1,3-diaryl propenone derivativesand their antimalarial activity in vitro

Nidhi Mishra a, Preeti Arora a, Brajesh Kumar a, Lokesh C. Mishra b,Amit Bhattacharya b, Satish K. Awasthi a,*, Virendra K. Bhasin b,**

a Chemical Biology Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, Indiab Department of Zoology, University of Delhi, Delhi 110007, India

Received 5 May 2007; received in revised form 7 September 2007; accepted 13 September 2007

Available online 29 September 2007

Abstract

The synthesis of novel 1,3-diaryl propenone derivatives and their antimalarial activity in vitro against asexual blood stages of human malariaparasite, Plasmodium falciparum, are described. Chalcone derivatives were prepared via ClaiseneSchmidt condensation of substituted aldehydeswith substituted methyl ketones. Antiplasmodial IC50 (half maximal inhibitory concentration) activity of these compounds ranged between 1.5and 12.3 mg/ml. The chloro-series, 1,2,4-triazole substituted chalcone was found to be the most effective in inhibiting the growth of P. falcipa-rum in vitro while pyrrole and benzotriazole substituted chalcones showed relatively less inhibitory activity. This is the first report on antiplas-modial activity of chalcones with azoles on acetophenone ring.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Chalcones; Synthesis; Antimalarial activity; Plasmodium falciparum; In vitro

1. Introduction

Malaria continues to be one of the major public healthproblems in many tropical countries causing extensive mor-bidity and loss of life [1]. Annual malaria mortality due toPlasmodium falciparum costs 1e2.7 million lives in Africaalone, comprising of mainly young children [2]. Artemisinin(Fig. 1), contained in the decoction prepared from the aerialparts of a plant Artemisia annua, used for over thousand yearsfor fever resolution has been re-discovered as the most potentantimalarial drug [3]. Resistance to this drug has not been clin-ically encountered so far [4,5]. Chloroquine (Fig. 1), the onlysynthetic antimalarial drug which cured malaria for decades,rather than centuries, has fallen to resistance [6]. With the de-mise of chloroquine there is renewed interest to look for

* Corresponding author. Tel.: þ91 11 27662104.

** Corresponding author. Tel.: þ91 11 27667989; fax: þ91 11 27667524.

E-mail addresses: awasthisatish@yahoo.com (S.K. Awasthi),

virendrabhasin@hotmail.com (V.K. Bhasin).

0223-5234/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.ejmech.2007.09.014

alternate synthetic and inexpensive medicines for malaria.Synthesis of artemisinin is commercially unviable at present.One of the strategies adopted by malariologist to delay theemergence of resistance to artemisinin and its derivatives isto use them in combination with other novel antimalarial(s).Artemisinin based combination therapies (ACTs) instead ofartemisinin monotherapy are being advocated and supportedby WHO. Currently used ACTs are in demand and by nomeans the ideal combinations [5,7]. There is scarcity of arte-misinin globally. Thus there is a need to search for novel, in-expensive antiplasmodials as suitable synergistic partners forartemisinin to reduce dependence on artemisinin for malarialtreatment.

Chalcone, a biosynthetic product of shikimate pathway, isa class of privileged structure that has a wide range of biologicalproperties. Chalcones are precursors of various flavones and keyintermediates for combinatorial assembly of different heterocy-clic scaffolds. Chalcone (1,3-diaryl propenone or 1,3-diphenyl-2-propen-1-one) constitutes an important group of naturalproducts, and some of them possess a wide range of biological

Table 1

Antimalarial activity of chalcone derivatives

Compounds R R1 R2 R3 Mean IC50� SEa (mg/ml)

1N

H Cl H 2.93� 0.08

2

NN

NH Cl H 2.5� 0.27

3 NN H Cl H 7.76� 0.42

4 N H Cl H 6.01� 0.13

5

O

NH Cl H 9.1� 0.10

6

N

NH Cl H 8.26� 0.06

7

NN

NH Cl H 1.52� 0.04

8 N H Cl H 5.15� 0.07

HN

Cl

NEt2

OO

OO

H

O

HHO

O

OH

O

Chloroquine Artemisinin Licochalcone A

Fig. 1. Structure of chloroquine, artemisinin and licochalcone A.

1531N. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 1530e1535

activities, such as anti-bacterial [8,9], anti-fungal [10], anti-viral [11,12], anti-inflammatory [13], anti-tumor [14,15], anti-oxidant [16], insect anti-feedent [17], and act as tyrosinaseinhibitors [18]. Interest in chalcones as antimalarials was initi-ated by the discovery of antiplasmodial activity of licochalconeA (Fig. 1), an oxygenated chalcone isolated from the roots of theChinese licorice during routine screening [19]. Computationalstructural analysis also identified chalcones as potential plasmo-dial cysteine protease inhibitors consistent with the experimen-tal data [20,21]. The model proposed [21] is presented in Fig. 2.Since then attempts have been made to find new syntheticantimalarial analogues [22,23].

In our earlier work we have evaluated novel artemisininbased combinations that target multiple sites in the parasiteas antiplasmodial agents [24]. In diversifying our work wehave designed and synthesized new chalcone derivatives andevaluated their antiplasmodial ability in vitro, with an aim touse the potent chalcones in combination with artemisininsubsequently.

9 NN H OMe H 12.33� 0.88

10

NN

NH OMe H 6.8� 0.11

11 N OMe OMe OMe 7.16� 0.10

12 NN OMe OMe OMe 6.0� 0.05

13

O

NOMe OMe OMe 4.6� 0.15

2. Chemistry

The ClaiseneSchmidt condensation method [20] was em-ployed for the synthesis of 14 chalcone derivatives (com-pounds 1e14, Table 1). Two step synthesis protocols, asdepicted in Fig. 3, were used to prepare the compounds. Thefirst step involved treatment of 4-fluoroacetophenone with var-ious cyclic amines in dimethylformamide (DMF) and potas-sium carbonate at 110 �C for 18 h to yield substitutedacetophenone as either liquid (compounds 1, 2, 3, 6) or solid(compounds 4, 5, 7). In the next step, the substituted acetophe-none was condensed with a suitable aldehyde using solid so-dium hydroxide as catalyst in methanol at room temperatureto yield chalcone [18]. These conditions were found to be

N

H

C

O

R NR'

H

C

O

RR'+

Nucleophilicattack

CH2

CH2

SH..

:

Enzyme

HN :

NCH2

CH2

SH..

:

Enzyme

HN :

N

His

Cys

His

Cys

Fig. 2. Peptide bond cleavage by the enzyme, cysteine protease.

14

NN

NOMe OMe OMe 8.03� 0.2

a SE, standard error (n¼ 3).

satisfactory for the synthesis of chalcones in good yields. Inmost cases, products were formed immediately after the addi-tion of sodium hydroxide pellets to the stirred solution of alde-hyde and substituted methyl ketone. If the starting materialwas insoluble in methanol, tetrahydrofuran (THF) or 1,4-diox-ane was used as a co-solvent. To ensure the formation of solid,

F

O

R

O O

R R2R3

R1

H

O

R3

R2

R1

(a) (b)

(a) c yclic amines, K2CO3, DMF, 18 hrs., 110°C (b) NaOH, methanol, 16-20 hrs, rt.

iii) R1= R2= R3= -OCH3

i) R1=H, R2=Cl, R3=Hii) R1=H, R2= -OCH3, R3=H

Fig. 3. General procedure for the synthesis of chalcones.

1532 N. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 1530e1535

a minimal amount of methanol was used. The product, a,b-un-saturated ketone, is almost always obtained in the trans-alkeneform (E-form). The yields were not optimized and ranged be-tween 56 and 78%.

The homogeneity of the final compounds was ensured bycolumn chromatography (silica, mesh size 60e120, CDH) us-ing chloroform as eluent. The compounds were characterizedby Macromass G and 1H NMR (300 MHz, Brucker), the datawere found consistent with the structures and the microanalyt-ical results were within �0.4 from theoretical values. The Rf ofall compounds was recorded in 1% methanol in chloroform.The biophysical parameters of the compounds are presentedin Table 1.

3. Results and discussion

Chalcone is an exceptional chemical template having mul-tifarious biological activities. Antimalarial property of somechalcone derivatives is derived from their ability to inhibitthe parasitic enzyme, cysteine protease [20]. The enzyme ca-tabolizes globin into small peptides within the acidic food vac-uole of the intra-erythrocytic malaria parasite. Withoutcysteine protease action osmotic swelling occurs, food vacuo-lar functions are impaired, and parasite death ensues. Homol-ogy-based computer model structure also predicts malariablood stage cysteine protease as the most likely target enzymeof chalcones [25]. The chalcones are conjugates of a,b-unsat-urated ketones that assume linear or near planar structure. Thisstructure is stable in acidic food vacuolar environment wheremalarial cysteine protease acts, and structural conformationmay fit well into the long cleft of the active site of the enzyme.Considering these facts we designed and synthesized variouschalcones. Three substituents chosen at aldehyde ring were4-chloro, 4-methoxy and 3,4,5-trimethoxy functions. In theacetophenone moiety fluoro group was replaced systematicallywith pyrrole, imidazole, benzotriazole, pyrazole, pyrrolidine,morpholine and piperidine to get new compounds. The anti-plasmodial IC50 values of these 14 compounds are depictedin Table 1. Within the chloro-series compounds 1, 2 and 7showed marked antiplasmodial activity. Compound 7, a tria-zole substituted chalcone was found to be the most effectiveagainst the parasites, and pyrrole and benzotriazole showedcomparable activities. The morpholine substituent in chloro-series was found to be the least active (compound 5). In themonomethoxy series, the benzotriazole group (compound 10)

showed moderate activity while pyrazole showed least activity(compound 9). In the trimethoxy series, compound 13 havingmorpholine substituents unlike in chloro-series showed signifi-cant activity and it is comparable with compounds 1 and 2 inchloro-series. Morpholine substituent was found to be theleast effective in chloro-series. Compound 14 containing1,2,4-triazole in trimethoxy series showed moderate activity,whereas it was the most active in chloro-series. The activitycan be attributed to the presence of azoles, although reasonsfor increased activity are unclear.

Compound 7, containing triazole and chloro substituents,was found to be the most potent antiplasmodial derivativeevaluated, suggesting that small lipophilic groups containingsingle or multiple nitrogen can enhance antimalarial activityin vitro. Compound 2 showed lower IC50 value compared tocompound 1. In a comparison between compounds 2 and 7,compound 7 showed more potent antimalarial activitythan compound 2 although both contained three nitrogens.We assumed that in triazole substituted chalcone, spacing ofnitrogen and the orientation of the molecule on active site ofenzyme are good enough to provide stronger and effectivehydrogen bonding with His 67 of cysteine protease. Therefore,we propose a possible interaction of compound 7 with the en-zyme as shown in Fig. 4. Moreover, in the case of benzotria-zole substituted chalcone, bulky nature or orientation ofbenzene ring on enzyme active site gives some kind of sterichindrance which prevents accessibility of nitrogens for effec-tive hydrogen bonding or protonation on enzyme active site,thus making the compound to be less potent than compound 7.However, the exact reason remains elusive.

Nitrogen containing heterocyclic compounds are generallyknown to possess antimalarial activity as seen in the case ofquinolynyl (pKa 4.9) as they tend to concentrate in the acidicfood vacuole (pH 5) of the parasite. The explanation is basedon the crystal structure of cysteine protease showing His 67 atthe active site of the enzyme [20]. We assumed that proton-ation of triazolyl chalcone under weakly acidic conditionsmay enhance their interaction with His 67 in the active sitesof enzyme or vice versa (free His pKa of 6).

4. Conclusion

In vitro antiplasmodial results of 4-chloro, 4-methoxy and3,4,5-trimethoxy series suggested that small or medium sizedbut highly lipophilic groups containing multiple nitrogen or

CH2

CH2

SH..

:

Enzyme

N:

NH

His

Cys

Fig. 4. Proposed mechanism of compound 2 binding to His 67 in the site S2 of

enzyme.

1533N. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 1530e1535

amine in acetophenone moiety impart antiplasmodial poten-tial. Such compounds may provide additional hydrogen bond-ing with histidine residue present at the active site of theenzyme, cysteine protease. This is the first report in whichchalcones containing small highly polar, lipophilic cyclicamines are showing antimalarial potential.

More work is needed to obtain second generation version ofthese interesting compounds to establish meaningful SAR.Compounds 1, 2 and 7 could be used as standard precursorsfor this purpose. In the meanwhile these compounds are beingevaluated as partners in combination with artemisinin to lookfor synergistic action.

5. Experimental protocol

Melting points are determined in an open capillary and areuncorrected. 1H NMR was recorded on Brucker (AMX-300)using CDCl3 as solvent. TMS was used as internal referencefor 1H NMR. Mass spectra were recorded on a MacromassG spectrophotometer. Elemental analysis was performed onPerkineElmerSeries-II CHN analyzer.

5.1. Procedure for the synthesis ofsubstituted acetophenone

A solution of 4-fluoroacetophenone (5.1 ml, 40 mmol), pyr-rolidine (40 mmol) and K2CO3 (11.1 g, 50 mmol) in dry DMF(20 ml) was refluxed for 18 h at 110 �C. The solvent was re-moved in vacuo and H2O (50 ml) was added to the resultantresidue. The aqueous phase was extracted with diethyl ether(2� 100 ml), dried over anhydrous Na2SO4 and evaporatedin vacuo to yield the desired product. The intermediate was

characterized by mass spectra and used in the next step forthe preparation of chalcones without purification. The remain-ing intermediates were prepared in the similar way.

5.2. General procedure for the synthesis of substituted1,3-diaryl propenone derivatives

The title compounds were prepared as follows. Asubstituted methyl ketone (3 mmol) and a substituted aldehyde(3 mmol) were dissolved in a minimum amount of methanol(normally 5 ml) with stirring. NaOH pellets (300 mg) wereadded into it. In most cases off-white to yellow solids wereformed within a few minutes to 18 h. The solid was filteredand washed three times with cold water. The product was re-crystalized from appropriate solvents. Column chromatogra-phy was used to purify the products.

5.2.1. 1-(4-(1H-Pyrrol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 68%, Rf 0.67, off-white crystals, m.p. 132e134 �C,MS m/z 309.15 (Mþ 1), 1H NMR (CDCl3, 300 MHz):d 7.19e8.0 (m, 14H, Ar).

5.2.2. 1-(4-(1H-Benzo[d][1,2,3]triazol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 60%, Rf 0.68, off-white crystals, m.p. 154e155 �C,MS m/z 360 (Mþ 1), 1H NMR (CDCl3, 300 MHz): d 6.52e8.15 (m, 14H, Ar).

5.2.3. 1-(4-(1H-Pyrazol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 74%, Rf 0.67, off-white crystals, m.p. 152e153 �C,MS m/z 309.5 (Mþ 1), 1H NMR (CDCl3, 300 MHz):d 6.53e8.15 (m, 13H, Ar).

5.2.4. 1-(4-(1H-Pyrrolidinl-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 72%, Rf 0.58, light yellow crystals, m.p. 208e210 �C,MS m/z 312 (Mþ 1), 1H NMR (CDCl3, 300 MHz): d 3.40 (t,4H, CH2eNeCH2), d 2.50 (t, 4H, CH2eCH2e), d 7.3e8.0 (m,8H, Ar H).

5.2.5. 1-(4-(1H-Morpholin-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 68%, Rf 0.56, yellow crystals, m.p. 162e164 �C, MSm/z 349.04 (MþNa), 1H NMR (CDCl3, 300 MHz): d 3.08 (s,6H, morpholinyl), d 3.05 (s, 2H, CHeCHe), d 6.68e8.0 (m,10H, Ar H).

5.2.6. 1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 70%, Rf 0.68, yellow crystals, m.p. 160e162 �C, MSm/z 309.12 (Mþ), 1H NMR (CDCl3, 300 MHz): d 7.50 (s, 1H,imidazolyl eNeCHeNe), d 8.05 (d, 2H, imidazolyl CHeNeCHe), d 7.18e7.79 (m, 10H, Ar).

1534 N. Mishra et al. / European Journal of Medicinal Chemistry 43 (2008) 1530e1535

5.2.7. 1-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 79%, Rf 0.55, yellow crystals, m.p. 160e162 �C, MSm/z 309.12 (Mþ), 1H NMR (CDCl3, 300 MHz): d 7.31e7.90(m, 12H, Ar).

5.2.8. 1-(4-(1H-Piperidin-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one

Yield 73%, Rf 0.66, light yellow crystals, m.p. 108e110 �C, MS m/z 326 (Mþ), 1H NMR (CDCl3, 300 MHz):d 3.39 (t, 5H, piperidine), d 2.04 (t, 5H, piperidine),d 6.54e8 (m, 10H, Ar).

5.2.9. 1-(4-(1H-Pyrazol-1-yl)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one

Yield 44%, Rf 0.46, yellow crystals, m.p. 100e102 �C, MSm/z 305.07 (Mþ), 1H NMR (CDCl3, 300 MHz): d 3.86 (s, 3H,OCH3), 6.52 (t, 1H, pyrazolyl eCHe), d 7.7 (d, 2H, pyrazolyleCHeNeNCH), d 6.93e8.15 (m, 10H, Ar).

5.2.10. 1-(4-(1H-Benzo[d][1,2,3]triazol-1-yl)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one

Yield 54%, Rf 0.63, yellowish white crystals, m.p. 124e128 �C, MS m/z 356 (Mþ 1), 1H NMR (CDCl3, 300 MHz):d 3.86 (s, 3H, OCH3), 6.9e7.9 (m, 12H, Ar).

5.2.11. 3-(3,4,5-Trimethoxyphenyl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one

Yield 76%, Rf 0.39, orange crystals, m.p. 160e162 �C, MSm/z 381 (Mþ), 1H NMR (CDCl3, 300 MHz): d 3.93 (s, 9H,OCH3), d 1.67 (s, 6H, CH2eCH2eCH2 of piperidine),d 3.39 (s, 4H, CH2eNeCH2e), d 6.86e7.98 (m, 12H, Ar).

5.2.12. 1-(4-(1H-Pyrazol-1-yl)phenyl)-3(3,4,5-trimethoxyphenyl)prop-2-en-1-one

Yield 70%, Rf 0.31, yellow crystals, m.p. 168e170 �C, MSm/z 365 (Mþ 1), 1H NMR (CDCl3, 300 MHz): d 3.90 (s, 9H,OCH3), d 6.54 (s, 1H, pyrazolyl), d 7.45 (d, 2H, pyrazolyl eCHeNe), d 7.73e8.15 (m, 10H, Ar).

5.2.13. 3-(3,4,5-Trimethoxyphenyl)-1-(4-morpholinophenyl)prop-2-en-1-one

Yield 76%, Rf 0.34, bright yellow crystals, m.p. 115e116 �C, MS m/z 384 (Mþ 1), 1H NMR (CDCl3, 300 MHz):d 3.92 (s, 9H, OCH3), d 3.09 (s, 6H, morpholinyl), d 3.04 (s,2H, morpholinyl), d 6.67e8.02 (m, 10H, Ar).

5.2.14. 1-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one

Yield 68%, Rf 0.56, yellow crystals, m.p. 55e57 �C, MS m/z 365.44 (Mþ), 1H NMR (CDCl3, 300 MHz): d 3.90 (s, 3H,OCH3), d 3.92 (s, 6H, OCH3), d 6.86e7.94 (m, 10H, Ar),d 7.97 (s, 2H, triazolyl).

5.3. Measurement of in vitro antiplasmodial activity

Antiplasmodial activity of chalcones was essentially car-ried out using candle jar method [26]. Briefly, asexual bloodstage parasites of P. falciparum strain 3D7 were challengedto graded concentration of each compound in multiwell tis-sue culture plates in triplicates for 48 h at 37 �C. The para-sites were grown in Bþ human erythrocytes in RPMI-1640medium supplemented with 10% heat inactivated pooledcompatible serum. The percent inhibition of parasitemia inrelation to controls was determined from Giemsa stainedthin blood films. Concentrationeresponse curve was pre-pared to compute IC50 value of each compound. Stock solu-tion of each compound at a concentration of 1 mg/ml wasprepared in DMSO. The final concentration of DMSOused in the culture media was not affecting the parasitegrowth. In vitro sensitivity of 3D7 P. falciparum line tochloroquine was 0.0272 mM.

Acknowledgements

S.K.A. is thankful to Department of Science and Technol-ogy, India for the financial support. This work was supportedin part by the Department of Biotechnology, Ministry of Sci-ence and Technology, Government of India. L.C.M. andA.B. received fellowships from the Council of Scientific andIndustrial Research, New Delhi.

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