Combinatorial Synthesis, Lead Identification, and Antitumor Study of a Chalcone-Based...

Combinatorial Synthesis, Lead Identification, and Antitumor Study of aChalcone-Based Positional-Scanning Library

by Ahsan Ullaha), Farzana Latif Ansari*a), Ihsan-ul-Haqb), Samina Nazira), and Bushra Mirzab)

a) Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan(e-mail: [email protected])

b) Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan

A 175-member chalcone library was designed and synthesized from seven differently substitutedacetophenones (A1–A7) and 25 differently substituted aryl or heteroaryl aldehydes (B1–B25). Potentiallead compounds were identified by deconvolution of a two-dimensional library matrix via positionalscanning, and the members of the most-active sub-libraries were synthesized and screened against crown-gall tumors with the aid of the potato-disc assay. The resulting hits gave rise to significant antitumoractivities, with no antibacterial effect on the tumor-producing bacterium Agrobacterium tumefaciens. Twoidentified lead structures, (2E)-3-(2-chlorophenyl)-1-phenylprop-2-en-1-one (A1B9) and the hydroxyanalogue (2E)-3-(2-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (A2B9), are promising candi-dates to be developed into highly effective anticancer chemotherapeutics.

Introduction. – Chalcones are medicinally important a,b-unsaturated ketones.Though structurally simple, they have displayed an impressive array of biologicalactivities including antimalarial, antileishmanial, anti-asthmatic, anti-oxidant, antibac-terial, antifungal, anti-inflammatory, antitubercular, immunomodulatory, tyrosinase-inhibition, cholinesterase-inhibition, cytotoxic, antimitotic, anticancer, antiulcer, andhepatoprotective properties [1 –15]. Some chalcones are used as medicines, whileothers are being investigated as drug candidates [16]. Despite the wide range ofbiological activities, relatively little has been reported so far on the antitumorproperties of chalcones, with no reports on the inhibition of crown-gall tumor.

Crown gall is a neoplastic disease of plants, which occurs in more than 60 families ofdicotyledons and many gymnosperms. The causative agents of this disease are specificstrains of the Gram-negative Agrobacterium tumefaciens. The relevance of the crown-gall-tumor system to the general cancer problem has been thoroughly reviewed [17 –23]. The inhibition of crown-gall tumor induced by A. tumefaciens in potato-disc tissueis an assay based on antimitotic activity; this assay is capable of detecting a broad rangeof known and novel antitumor effects. Crown gall causes the bulging of a mass of tissuesfrom stems and roots of woody and herbaceous plants. These tumors may be spongy orhard, and may or may not have deleterious effects on the plant. Histologically, crown-gall tumors are similar to those found in humans and animals. During infection of plantmaterial with A. tumefaciens, a tumor-producing plasmid, found in bacterial DNA, isincorporated in the chromosomal plant DNA. When plant tissue is wounded, it releasesphenols and other compounds that activate the Ti-plasmid in A. tumefaciens. The Ti-plasmid causes the plant:s cells to multiply rapidly without going through apoptosis,

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C 2007 Verlag Helvetica Chimica Acta AG, ZFrich

resulting in tumor formation similar in nucleic acid content and histology to human andanimal cancers.

The validity of this bioassay is based on the observation that certain tumorigenicmechanisms are similar in plants and animals. It has been shown that the inhibition ofcrown-gall tumor on potato discs and their subsequent growth shows good correlationwith compounds and extracts active in the 3PS leukemic mouse assay [21]. Ferrigniet al. showed that the potato-disc tumor assay is statistically more predictive in terms of3PS activity than either the 9KB or the 9PS cytotoxicity assays [22], and McLaughlinconcluded that the potato-disc assay can be used as a fairly rapid, inexpensive, andreliable prescreen for antitumor activity [23].

The generation and use of combinatorial libraries for the identification of novelchemical lead compounds or for the optimization of promising lead candidates hasemerged as a powerful method for drug discovery [24 – 27]. Initially explored withpeptide or oligonucleotide libraries and related oligomeric structures [25] [28 – 36],more-recent efforts have been directed at exploiting the greater diversity and range ofuseful properties embodied in small molecular libraries [34 – 40]. A wide range ofapproaches to the generation of chemical libraries have been disclosed, including splitor mixed [41], encoded [42], indexed [43], or parallel and spatially addressed synthesison pins [28] [38], beads [44], chips [45], and other solid supports [46].

The synthesis of positional-scanning libraries represents one of the most-usefulprotocols for mixture synthesis. Not only is it much less time intensive as compared toparallel synthesis of individual compounds or small mixtures, but also producesdepository libraries for use in multiple screens with immediate deconvolution[30] [47] [48]. Thus, unlike other deconvolution protocols [49] [50], positional-scanninglibraries provide lead identities in a single round of testing. These libraries, being lessdemanding to prepare, allow an accurate detection of significant activities, but more-subtle discoveries and less-distinguishable activities are not detected. This is a naturalconsequence of testing the larger compound mixtures and the relative insensitivity ofthe assays to the contribution of any single, uniquely acting compound in the mixture.Thus, the disadvantages associated with the loss of some information contained withinthe library must be balanced against the advantages of the ease of library synthesis, andjudged in the light of library-screening objectives [51].

The current library synthesis of chalcones has been carried out in solution, beingtechnically non-demanding. In fact, the synthesis of positional-scanning libraries[30] [47] [52] [53] represents one of the most-useful protocols for mixture synthesis, butcan only be conducted by means of solution-phase techniques, and is not easily adaptedto solid-phase synthesis [54].

We have earlier reported the synthesis and antibacterial screening of a 120-memberlibrary of chalcones in solution [55], from which we identified lead antibacterial agentsby deconvolution using the positional-scanning protocol. Following the same strategy,we herein report the synthesis of a 175-member library of aryl and heteroaryl chalconesand its screening by means of the crown-gall potato-disc assay, with a view to find novellead structures that may be further developed into potential antitumor agents.

Results and Discussions. – 1. Library Setup. The 175-member chalcone library wasdesigned as a two-dimensional (2D) matrix made up of 7 columns and 25 rows,

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corresponding to seven acetophenones, A1–A7, and 25 substituted aldehydes, B1–B25,respectively, as building blocks (Fig. 1). The choice of the reagents reflects chemicaldiversity in terms of hydrophobic and lipophilic substituents, and of aromatic residues.All library components share a common a,b-unsaturated carbonyl skeleton.

The designed library was synthesized as two sets of sub-libraries with the two typesof building blocks shown in Fig. 1. Each of the sub-libraries of Set 1 (AL1–AL7),corresponding to columns of the 2D matrix, was synthesized by the reaction of eachindividual acetophenone (A1–A7) with a mixture of all 25 aldehydes (B1–B25). Thelibraries of Set 2, corresponding to rows in the 2D matrix, were likewise synthesized byreaction of each individual aldehyde (B1–B25) with a mixture of all seven acetophe-nones (A1–A7). The synthesis of these chalcone libraries was carried out by classicalClaisen–Schmidt condensation under standard conditions.

A conceptual matrix showing all 175 members of the library is presented in Table 1.Such a library to be tested can be represented as a 2D matrix, wherein the x-axis hasseven structural variants (acetophenones), and the y-axis has 25 structural variants(aldehydes), which leads to a 7�25 grid. The assay value of each cell is contained in thecombination AxBy (x¼1 – 7, y¼1 – 25). Obviously, the examination of all the purecompounds would require 175 experiments. Since only one cell out of 175 may possessthe maximum response function, the next step involves its identification withoutlooking at all 175 cells. The best way of doing this is to choose any B for testing with allA-type compounds and, vice versa, any A for testing with all B-type members. Byscreening seven columns and 25 rows, which are indices to the cells at theirintersections, as mixtures, only 32 reactions (instead of 175) need to be carried outto find the maximum response. Therefore, a combinatorial synthesis of 32 sub-librarieswas carried out. This method has the advantages over the parallel approach in that it is

Fig. 1. Building blocks for library synthesis


fast, relatively inexpensive, and the number of compounds prepared is much greaterthan the number of chemical steps required.

To eliminate kinetics effects, all reactions were forced to completion by conductingthem with a stoichiometric quantity of the unitary reagent relative to the total of themixed reagents. The presence of all the components of the mixture in libraries wasconfirmed by means of thin-layer chromatography (TLC). The synthesis of the sub-library BL7 was carried out both by regular heating as well as under microwaveirradiation. The composition of the library was then analyzed and found to be the sameunder both conditions.

2. Screening and Deconvolution. All indexed libraries (AL1–AL7 and BL1–BL25)were tested for their activity in terms of crown-gall-tumor inhibition (potato-discassay). The screening of two sets of libraries enabled the testing of each compoundtwice, once as a component of the sub-libraries of Set 1, and then as a member of thesub-libraries of Set 2. Thus, a total of 32 assays were required to reveal the antitumoractivity of 175 compounds.

The sub-libraries of two sets, AL1–AL7 and BL1–BL25, were screened initially at aconcentration of 1000 ppm, and their antitumor activities were determined as shown inTable 2. All the libraries showed significant antitumor activity. LibrariesAL1,AL2,BL4,


Table 1. Conceptual Matrix for the Design of a 175-Member Chalcone Library

Set 1Set 2

AL1 AL2 AL3 AL4 AL5 AL6 AL7

BL1 A1 B1 A2 B1 A3 B1 A4 B1 A5 B1 A6 B1 A7 B1









BL10 A1 B10 A2 B10 A3 B10 A4 B10 A5 B10 A6 B10 A7B10







BL17 A1 B17 A2 B17 A3B17 A4 B17 A5 B17 A6 B17 A7 B17









BL5,BL7–BL9,BL11, and BL12 showed 100% tumor inhibition. Further short listing wasdone by screening these nine specific libraries at lower concentration, i.e., at 100 and10 ppm, as shown in Table 3. As a result, the libraries AL1 and BL9, corresponding tocolumn 1 and row 9, respectively, were found to be the most active, both at 10 and100 ppm.

Having synthesized and screened all the sub-libraries, identification of the lead(s)may be carried out by deconvolution. This requires the calculation of activities of all themembers of the library using the experimentally determined values at 1000 ppm asindices to the cells of columns and rows of the 2D matrix. The data were expanded in175 cells of chalcones on the matrix by taking the average of the activity of therespective column and row, which were used as indices to the particular cell. Thisresulted in calculated antitumor activities for all 175 chalcones in the 2D matrix, asshown in Fig. 2 and Table 4.

As is evident from Table 4, the calculated percent inhibition was 100% for 14chalcones at a concentration of 1000 ppm. So, the activities of nine short-listed sub-libraries (at 10 ppm concentration) were used as indices to the cells of the 2D matrixcomprising two columns and seven rows, and the data were spread over 14 cells, asshown in Table 5.

From Table 5, compound A1B9, belonging to both the AL1 and BL9 sub-libraries,was predicted to be the most-active lead. Thus, for real identification, parallel


Table 2. Tumor Inhibition (in %) of the Sub-Libraries of Set 1 and Set 2 at a Concentration of 1000 ppm

Library Inhibition Library Inhibition Library Inhibition

AL1 100 BL5 100 BL16 65AL2 100 BL6 96 BL17 64AL3 98 BL7 100 BL18 69AL4 95 BL8 100 BL19 79AL5 71 BL9 100 BL20 64AL6 78 BL10 77 BL21 80AL7 48 BL11 100 BL22 66BL1 40 BL12 100 BL23 95BL2 85 BL13 79 BL24 33BL3 95 BL14 57 BL25 74BL4 100 BL15 66

Table 3. Tumor Inhibition (in %) of the Sub-Libraries of Set 1 and Set 2 at a Concentration of 10 ppm

Library Inhibitiona) Library Inhibition

AL1 64 (88) BL8 23 (45)AL2 60 (84) BL9 73 (95)BL4 11 (78) BL11 14 (35)BL5 33 (54) BL12 22 (41)BL7 15 (29)

a) In parentheses, inhibition (in %) at 100 ppm is given.

preparation of the members of the most active column, AL1, and the most active row,BL9, was carried out by means of microwave-assisted organic synthesis. The resultingproducts A1B1–A1B25 are shown in Fig. 3. These compounds were isolated, purified,and tested at concentrations of 10, 100, and 1000 ppm. The resulting antitumoractivities are collected in Table 6.

As can be seen from Table 6, the most-active members of the library wereA1B9 andA2B9, with 100% tumor inhibition at a concentration of 10 ppm, and, thus, can beregarded as hits of the designed library. It may be recalled that the calculateddeconvolution led to the identification of the same chalcone, A1B9, as lead structure(see above). However, the experimental data show that A2B9, also exhibiting 100%tumor inhibition, may be an equally important candidate to be developed into highlyeffective chemotherapeutic agents. Therefore, as mentioned above, library deconvo-lution by means of positional scanning is capable of identifying a lead compound, butsubtle differences in activity within the library cannot be detected. This disadvantage,however, is compensated by the advantage of the ease of library synthesis.

3. Antibacterial Studies. Since the initial step in the formation of crown-gall tumorsinvolves the attachment of A. tumefaciens to a binding site [56], it seemed necessary to


Fig. 2. Graphical representation of the calculated overall antitumor activity of the chalcone library at aconcentration of 1000 ppm. For numeric data, see Table 4.

confirm that the observed antitumor activities were not due to potential antibacterialproperties of the tested array of compounds. Therefore, all the individual compounds ofthe active column and row were tested for their antibacterial activity against A.tumefaciens. However, none of the compounds were found to show any effect on


Table 4. Calculated Antitumor Activities of the Members of Designed Sub-Libraries at a Concentration of1000 ppm

Set 1Set 2

AL1 AL2 AL3 AL4 AL5 AL6 AL7

BL1 70 70 69 67 56 59 44BL2 92 92 92 90 78 82 67BL3 97 97 96 95 83 86 71BL4 100 100 99 97 85 89 74BL5 100 100 99 97 85 89 74BL6 98 98 97 96 84 87 72BL7 100 100 99 97 85 89 74BL8 100 100 99 97 85 89 74BL9 100 100 99 97 85 89 74BL10 88 88 87 86 74 77 62BL11 100 100 99 97 85 89 74BL12 100 100 99 97 85 89 74BL13 89 89 89 87 75 78 64BL14 78 78 78 76 64 67 53BL15 83 83 82 80 68 72 57BL16 82 82 82 80 68 71 57BL17 82 82 81 79 68 71 56BL18 84 84 83 82 70 73 58BL19 89 89 88 87 75 78 63BL20 82 82 81 79 67 71 56BL21 90 90 89 87 76 79 64BL22 83 83 82 80 68 72 57BL23 97 97 96 95 83 86 71BL24 66 66 66 64 52 55 41BL25 87 87 86 84 73 76 61

Table 5. Calculated Antitumor Activities of the Most-Active Library Members at 10 ppm

Set 1Set 2

AL1 AL2

BL4 37.5 35.5BL5 48.5 46.5BL7 39.5 37.5BL8 43.5 41.5BL9 50.5 48.5BL11 39.0 37.0BL12 43.0 41.0

bacterial viability when added to bacterial-growth medium. Therefore, tumorinhibition seems to be solely due to the intrinsic antitumor activities of the testedcompounds.

4. Structure–Activity Relationship. Following the identification of hits of thedesigned library by deconvolution, it is essential to highlight the structural features that


Fig. 3. Members of the Active Column AL1 and the Active Row BL9

Table 6. Experimental Antitumor Activities of the Individually Synthesized Chalcones of the Most-ActiveSub-Libraries AL1 and BL9

Chalcone Inhibitiona) Chalcone Inhibition Chalcone Inhibition

A1B1 17.0, 34.1, 62.1 A1B12 64.6, 74.4, 78.2 A1B23 67.2, 80.3, 83.6A1B2 35.3, 40.2, 80.5 A1B13 32.9, 53.6, 93.9 A1B24 63.9, 90.2, 98.4A1B3 53.6, 58.5, 100 A1B14 37.8, 58.5, 69.5 A1B25 42.6, 49.2, 68.8A1B4 34.1, 56.1, 64.6 A1B15 53.6, 54.9, 74.4 A2B9 100, 100, 100A1B5 19.5, 52.6, 57.5 A1B16 1.6, 29.5, 31.1 A3B9 61.0, 78.6, 98.0A1B6 34.1, 48.8, 54.8 A1B17 14.7, 14.7, 36.1 A4B9 70.7, 77.9, 100A1B7 35.4, 47.7, 53.6 A1B18 11.5, 32.7, 49.2 A5B9 49.4, 59.1, 100A1B8 56.1, 68.7, 70.7 A1B19 26.2, 37.7, 45.9 A6B9 55.8, 70.1, 100A1B9 100, 100, 100 A1B20 54.1, 56.4, 78.7 A7B9 52.6, 66.8, 86.4A1B10 43.1, 47.5, 76.8 A1B21 44.3, 54. 1, 54.1A1B11 71.9, 75.7, 96.3 A1B22 40.9, 75.4, 80.3

a) Percent Inhibition at 10, 100, and 1000 ppm, resp.

contribute to tumor inhibition. Correlation may be performed at the library level aswell as at the level of individual compounds. When screening the library, one candifferentiate between Set 1 (AL1–AL7) and Set 2 (BL1–BL25). The sub-libraries ofSet 1 are derived from seven acetophenones: acetophenone proper and three hydroxy-and three amino-substituted congeners. The order of activity within these seven sub-libraries, in terms of substituent, is H>OH>NH2, or more specifically, 2-OH>3-OH>4-OH>3-NH2>2-NH2>4-NH2. Evidently, the library derived from unsubsti-tuted acetophenone (AL1) shows 100% tumor inhibition, while all the three librariesderived from isomeric amino acetophenones (AL4–AL7) possess poor tumor-inhibitory potential (Table 2). Moreover, substituents in para position lower theactivity to a greater extent as compared to those in ortho or meta positions.

Among the libraries of Set 2 (BL1–BL25), seven sub-libraries (BL4, BL5, BL7, BL8,BL9, BL11, and BL12) showed 100% tumor inhibition at a concentration of 1000 ppm.All the sub-libraries of various analogues of methoxy benzaldehydes (BL5, BL7, andBL8) showed 100% activity, reflecting the significance of a MeO substituent at thearomatic ring.

Let us now look at the tumor-inhibitory potential of the individual chalcones. First,it is interesting to note that A1B9 was found to be the lead structure, since this furtherconfirms the above conclusion that an unsubstituted B-ring is important for tumorinhibition. Moreover, chalcones substituted with an electron donor on ring A, e.g., aMe2N (A1B16) or Me (A1B17) group, were found to be the least active. Further, a varietyof chalcones was derived from heteroaryl aldehydes, but only the pyridyl chalcones(A1B23 and A1B24) were found to be significantly active.

F. L. A., A. U., and S. N. kindly acknowledge financial support provided by the Higher EducationCommission (HEC), Pakistan.

Experimental Part

General. All the libraries and individual chalcones were synthesized by microwave irradiation using ahousehold microwave oven. All reactions were monitored by TLC on precoated silica-gel glass plates(Merck HF254, 0.25 mm).

General Procedure for the Synthesis of Libraries of Set 1 (AL1–AL7).All the molecular libraries were prepared according to a modified literature procedure. To an EtOH

soln. (40 ml) of the corresponding acetophenone (A1–A7; 1 mmol each) and a mixture of the 25aldehydes (B1–B25 ; 1 mmol each, 25 mmol in total) was added a 4m soln. of aq. NaOH (30 ml, 4.8 mmol).The mixture was exposed to microwave irradiation for 3 min, and then left at 0–48 overnight. The massobtained was cooled and neutralized with cold dil. aq. HCl. The chalcones were successively extractedwith AcOEt (5�10 ml) and CH2Cl2 (5�10 ml). All org. phases were combined and concentrated invacuo, which led to a dark-brown solid mass.

General Procedure for the Synthesis of Libraries of Set 2 (BL1–BL25).All the libraries of Set 2 were synthesized according to the procedure described for Set 1, by reacting

an EtOH soln. (10 ml) of the corresponding aldehyde (B1–B25 ; 7 mmol) with a mixture of the sevenacetophenones (A1–A7; 1 mmol each, 7 mmol in total) in the presence of 4m aq. NaOH soln. (10 ml).

Synthesis of Individual Chalcone Members (A1B1–A1B25 and A1B9–A7B9).To an EtOH soln. of an acetophenone (5 mmol) and an aldehyde (5 mmol) was added 4.0m aq.

NaOH soln., and the mixture was irradiated in a microwave oven till the reaction was found to becomplete, as confirmed by TLC. The mixture was kept at 0–48 overnight, cooled, and neutralized withice-cold dilute aq. HCl. The resulting precipitate was filtered off and purified either by recrystallization


from EtOH or by flash chromatography (SiO2; hexane/AcOEt). The purity of the compounds waschecked by multiple TLC (SiO2; hexane/AcOEt 2 : 1, 3 : 1, and 1 : 1). The yields of the individualchalcones varied from 61 to 85%.

Potato-Disc Antitumor Assay. The assay was performed according to a standard procedure describedin [23] using a 48-h-old bacterial culture of the AT-10 strain of Agrobacterium tumefaciens. Inoculumswith three concentrations of each test sample (10, 100, and 1000 ppm) containing bacterial culture wereprepared. Red-skinned potatoes were surface-sterilized in 0.1% HgCl2 soln. A borer of 8-mm diameterwas used to create potato cylinders, which were cut into 5-mm discs. Autoclaved agar soln. (1.5%) waspoured in Petri plates and allowed to solidify. The potato discs were placed on agar, and inoculum (50 ml)was poured on each disc. The plates were sealed with Parafilm to avoid contamination and moisture loss.The plates were then incubated at 288 for 21 d, the potato discs were stained with Lugol:s soln. (10% KI/5% I2), and tumors were counted under a dissecting microscope. All the tests were performed inreplicates, and the percent tumor inhibition was calculated by dividing the average number of tumors persample by the average number of tumors of a control, and then multiplied by a factor of 100.

Antibacterial Assay. All the synthesized compounds were tested against Agrobacterium tumefaciensusing the agar-well diffusion method [57]. Briefly, broth culture (0.75 ml) containing ca. 106 colony-forming units (CFU) per milliliter of testing strain was added to nutrient agar medium (75 ml) at 458. Thesample was mixed, and then poured into a 14-cm sterile metallic Petri plate. The medium was allowed tosolidify, and 8-mm wells were drug with a sterile metallic borer. An aliquot (100 ml) of the test sample(1 mg/ml, in DMSO) was added to the respective well. DMSO served as neg. control, and the standardantibacterial drugs roxithromycin (1 mg/ml) and cefixime (1 mg/ml) were used as pos. controls. Triplicateplates of each bacterial strain were prepared and incubated aerobically at 378 for 24 h. Antibacterialactivities were determined by measuring the diameter of zones showing complete bacterial-growthinhibition, using a Vernier caliper with a precision of up to 0.1 mm. Growth inhibition was then calculatedwith reference to the pos. controls.

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Received October 16, 2006


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