Chiang Mai J. Sci. 2018; 45(2) 1073

Chiang Mai J. Sci. 2018; 45(2) : 1073-1086http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Topoisomerase I Inhibitory Activity and 3D QSARStudies of Chromone DerivativesChirattikan Maicheen [a], Narumol Phosrithong [b], Jiraphun Jittikoon [c] and

Jiraporn Ungwitayatorn* [c]

[a] Faculty of Pharmacy, Huachiew Chalermprakiet University, 18/18 Bang Na-Trad Road,

Samut Prakarn 10540, Thailand.

[b] Faculty of Pharmacy, Siam University, 38 Petkasem Road, Bangkok 10160, Thailand.

[c] Center of Excellence for Innovation in Drug Design and Discovery, Faculty of Pharmacy,

Mahidol University, 447 Sri-Ayudhya Road, Bangkok 10400, Thailand.

* Author for correspondence; e-mail: jiraporn.ung@mahidol.ac.th

Received: 8 December 2016

Accepted: 31 January 2017

ABSTRACT

Topoisomerase I (Top I) is the molecular target for a diverse set of anticancer agents.This study was a continuation of previous work examining the Top I inhibitory activity of aseries of chromone derivatives. Nine chromones were evaluated using eukaryotic DNA TOPI drug screening kit. The most potent inhibitor, chromone 20 showed greater inhibitoryactivity (IC

50 = 0.83 μM) than the previously reported chromone compounds as well as the

known Top I inhibitor, camptothecin. To develop the structure-Top I inhibitory activityrelationship, the 3 dimensional quantitative structure-activity relationship (3D QSAR)were performed using comparative molecular field analysis (CoMFA) and comparativemolecular similarity indices analysis (CoMSIA). The best CoMFA model gave cross-validatedr2 (q2) = 0.578 and non cross-validated r2 = 0.995 while CoMSIA gave q2 = 0.632, r2 = 0.996.The contour maps provide the fruitful structural features which are useful for designingnew compounds with higher activity.

Keywords: chromone derivatives, topoisomerase I inhibitory activity, CoMFA, CoMSIA

1. INTRODUCTION

DNA topoisomerases are importanttargets of approved and experimentalanticancer drugs. They are essential for anumber of cellular processes those involveDNA unwinding including DNA replication,transcription, recombination, and chromatinremodeling [1]. Their functions have beenreported to relieve the torsional stress in theDNA helix that is generated as a result of

replication, transcription, and other nuclearprocesses [2-3]. Topoisomerases affect thesupercoiling of closed circular DNA andlong strands of double-stranded DNAby introducing transient breaks in thephosphodiester backbone and form acovalent phosphotyrosine intermediate withthe DNA [4-6]. Based on the mechanism ofcleaving DNA, topoisomerases are classified

1074 Chiang Mai J. Sci. 2018; 45(2)

as type I and type II. Topoisomerase I(Top I), a monomeric enzyme, relaxessuperhelical DNA by cleaving one strand ofduplex DNA through transesterificationof Tyr723 and forms a 3′-phosphotyrosinelinkage to the DNA, followed by theunwinding of supercoiled DNA [7-8].Topoisomerase II (Top II) is dimeric,transiently breaks both strands of duplexDNA, and passes an intact DNA duplexthrough this transient double-strandedbreak in an ATP-dependent manner. Withouttopoisomerases, the DNA cannot replicatenormally, therefore the inhibitors of DNAtopoisomerases have been used as anticanceragents to stop the proliferation of malignantcells [9-11]. Top II inhibitors include anumber of compounds, e.g., etoposide,teniposide and amsacrine, all of which stabilizethe covalent binary complex formed betweenTop II and DNA [9]. There are fewer knowninhibitors of Top I, but camptothecin (CPT),an alkaloid extracted from Camptothecaacuminata initially discovered because of itspotent and broad spectrum antitumor activity[12] has been shown to target at Top I [13].CPT binds to the Top I-DNA covalentcomplex, resulting in a ternary complex,and thereby stabilizing it [14-16]. This preventsDNA relegation, therefore causes DNAdamage and leading to apoptosis.

Plant-derived flavonoids, low molecularweight polyphenolic compounds, have beenreported to inhibit DNA Top I [17-20].Quercetin and related natural flavonederivatives, such as acacetin, apigenin,kaempferol, and morin, stabilize the covalentDNA topoisomerase I-DNA post-cleavagecomplex by inhibiting the relegation process

[18]. In contrast to CPT, these compoundsdo not act directly on the catalytic intermediateand also do not interfere with DNA cleavage.Luteolin inhibits the catalytic activity ofeukaryotic DNA Top I with an IC50

of 5 μM[17]. Luteolin is similar to CPT, with respectto its ability to form the Top I-mediatedcleavable complex. But, unlike CPT, luteolininteracts with both free enzyme and substrateDNA. Thus, flavonoids have the potential tobe an ideal model for structural requirementsof Top I inhibitor and may serve for thedevelopment of new anticancer agents.

In our previous study, a series of 25chromone derivatives (structures as shown inTable 1) have been synthesized and 16compounds (chromones 1-16, Table 1) havebeen tested for their inhibitory activityagainst Top I. Most compounds showedmoderate to good Top I inhibitory activity[21]. Chromone 15 (or chromone 11b asnamed in the previously study) was foundto be the most potent inhibitor withIC

50 = 1.46 μM whereas CPT possessed

IC50

= 18.85 μM [22-24]. To continue thestudy, more 9 chromone derivatives(chromones 17-25) have been evaluated fortheir Top I inhibitory activity using eukaryoticTop I drug screening kit (TopoGen, Inc.,USA). To explore the relationships betweenthe structures of these compounds and theirTop I inhibitory activity, a three-dimensionalquantitative structure-activity relationship(3D QSAR) study using comparativemolecular field analysis (CoMFA) [25]and comparative molecular similarityindices analysis (CoMSIA) [26-27] were alsoperformed in this study.

Chiang Mai J. Sci. 2018; 45(2) 1075

Table 1. Structures of 25 synthesized chromone derivatives.

2. MATERIALS AND METHODS

2.1 Chemical SynthesisAll studied chromones were prepared by

one pot cyclization reaction modified fromthe method of Riva et al. [28] using phenolicketone and acid chloride. After the phenolicester and subsequent β-diketone intermediateswere formed, esterification of the freehydroxyl group followed by rearrangementof the acyl group to the β-diketoneintermediate, cyclization and dehydration

processes, the 2, 3-disubstitued chromoneester was obtained. Hydrolysis of thechromone esters yielded the requiredchromone derivatives. The chemical structuresof the synthesized chromones were elucidatedby Fourier transform infrared (FTIR) andproton nuclear magnetic resonances(1H NMR) spectra. Molecular weights ofthe compounds were determined by highresolution mass spectrometer. More detailsof the synthesis procedures and spectroscopic

Compd12345678910111213141516171819202122232425

R2

PhenylPhenylPhenylPhenylPhenyl3′-(OCH

3)-phenyl

3′-(OCH3)-phenyl

3′-(OCH3)-phenyl

3′-(OCH3)-phenyl

3′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(NO2)-phenyl

4′-(NO2)-phenyl

Phenyl3′-(OCH

3)-phenyl

4′-(OCH3)-phenyl

4′-(NO2)-phenyl

4′-(F)-phenyl4′-(t-butyl)-phenyl3′-(Cl)-phenyl4′-(NO

2)-phenyl

4′-(NO2)-phenyl

R3

BenzoylBenzoylBenzoylBenzoylBenzoyl3′′-(OCH

3)-benzoyl

3′′-(OCH3)-benzoyl

3′′-(OCH3) benzoyl

3′′-(OCH3)-benzoyl

3′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(NO2)-benzoyl

4′′-(NO2)-benzoyl

Benzoyl3′′-(OCH

3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(NO2)-benzoyl

4′′-(F)-benzoyl4′′-(t-butyl)-benzoyl3′′-(Cl)-benzoyl4′′-(NO

2)-benzoyl

4′′-(NO2)-benzoyl

R5

HHHHHHHHHHHHHHHHHHHHHHH

OHH

R6

HHHHHHHHHHHHHHHHHHHHHHHHH

R7

O-BenzylO-[3′ ′′-(OCH

3)-benzoyl]

O-[4′ ′′-(OCH3)-benzoyl]

O-[3′ ′′-(NO2)-benzoyl]

O-[4′ ′′-(NO2)-benzoyl]

O-BenzylO-[3′ ′′-(OCH

3)-benzyl]

O-BenzoylO-[3′ ′′-(OCH

3)-benzoyl]

O-[4′ ′′-(OCH3)-benzoyl]

O-[3′ ′′-(OCH3)-benzyl]

O-BenzoylO-[3′ ′′-(OCH

3)-benzoyl]

O-[4′ ′′-(OCH3)-benzoyl]

O-BenzoylO-[3′ ′′-(NO

2)-benzoyl]

OHOHOHOHOHOHOHOHOH

R8

HHHHHHHHHHHHHHHHHHHHHHHH

OH

1076 Chiang Mai J. Sci. 2018; 45(2)

data were reported in reference 29.

2.2 Topoisomerase I Inhibitory ActivityAssay

The ability of synthesized chromonecompounds to stabilize the formation ofthe Top I-DNA covalent binary complexwas determined using eukaryotic Top Idrug screening kit (TopoGen, Inc., USA)(www.topogen.com). Purified Top I waspurchased from TopoGen, Inc., USA.Supercoiled DNA (125 ng) was incubated inthe presence of Top I (2.5 units), Top I assaybuffer (1.5 μL) and synthesized compoundsat 37 °C. The concentrations used for thetest chromones were 0.1, 1, 10, 20, 40, and100 μM for chromones 19 and 20, the restof the compounds were tested at 100 μM.After 30 minutes, the reactions wereterminated by adding 10 % sodium dodecylsulfate (0.1 volume), and digested with0.5 mg/mL proteinase K at 37 °C for 30minutes. Gel loading buffer (1/10 volume)was added into the reaction mixture.Then DNA was separated by loading on1 % agarose gel. Relaxed DNA was includedin the electrophoresis run as markers forDNA topology. CPT (100 μM) was usedas positive control. Electrophoresis wasconducted at 100 V for 1.5 hours inTAE buffer (Tris-acetate, EDTA, pH 8.0).After electrophoresis, the gels were stainedwith 0.5 μg/mL ethidium bromide, andphotographed under UV light. The amountof nicked DNA was calculated usingImageQuant TL (Image analysis softwareversion 2003). All determinations wereperformed in triplicate. The percentage ofTop I inhibition was calculated using thefollowing equation:

% Inhibition = (Amount of nicked DNAsample

× 100) / Amount of nicked DNAcontrol

where amount of nicked DNAsample

wascalculated from the band produced bysynthesized compound and amount ofnicked DNA

control was calculated from the

band produced by 100 μM CPT.The IC

50 values (the concentration of

tested compound required to stabilize 50 %of nicked DNA) were determined byinterpolation from the calibration curveplotted between % inhibition (relative to theintensity of the band produced by 100 μMCPT) and sample concentrations in logscale. IC

50 of chromones 19 and 20 were

determined.

2.3 3D QSAR StudyThe inhibitory activity was converted to

log % inhibition and used as dependentvariables for 3D QSAR study.

The 3D structures of all studiedcompounds were modeled with SYBYL-X2.0 molecular modeling program (TriposAssociates, Saint Louis, MO) using sketchapproach. Each structure was first energyminimized using the standard Triposforce field with a distance-dependentdielectric function and the Powell conjugategradient algorithm. Convergence criteria of0.01 kcal/(mole. ) was used for energyminimization. The partial atomic chargeswere calculated using the Gasteiger-H ckelmethod. The minimized structures werefurther optimized by semi-empirical AM1using MOPAC. The fully geometricaloptimized structures were used in thefollowing 3D QSAR study.

2.3.1 Structural alignmentThe AM1 optimized molecules were

aligned using two criteria, i.e., field fit andalign database functions in SYBYL-X2.0 program. The most active compound,chromone 20, was used as a template molecule.

Chiang Mai J. Sci. 2018; 45(2) 1077

In align database command, chromonenucleus was used as substructure to evaluatethe best fit. Substructural overlap assumedthat the molecules shared a commoncore of atoms which were overlapped ineach molecule of the database. The fieldfit alignment of molecules was based ontrying to increase field similarity within aseries of studied molecules. The root-mean-square differences in the sum of steric andelectrostatic interaction energies averagedacross all (possibly weighted) lattice points,between that molecule and the template wasminimized to find the best fit.

2.3.2 CoMFA and CoMSIA setupCoMFA and CoMSIA were performed

using the QSAR option in SYBYL-X 2.0.In CoMFA, the cubic grid space was generatedaround molecules in the training set basedon the molecular volume of the structures.A sp3-carbon atom was probed with a +1.0unit charge, 2.0 grid spacing, and thedefault 30 kcal/mole energy cutoff for stericand electrostatic fields.

CoMSIA was performed using steric,electrostatic, hydrophobic, hydrogen bonddonor and hydrogen bond acceptor fields.These parameters were evaluated usingcommon probe atom with 1 radius, charge+1.0, hydrophobicity +1.0, hydrogen bonddonor and acceptor properties +1.0. Similarityindices were calculated using Gaussian-typedistance dependence between the probe andthe atoms of the molecules of the data set.The value of the attenuation factor a was setto 0.3.

2.3.3 Statistical analysisPartial least-squares (PLS) methodology

was used for all 3D QSAR analyses. The gridhad a resolution of 2.0 and extended beyondthe molecular dimensions by 4.0 in all

directions. Column filtering was set to2.0 kcal/mole. CoMFA and CoMSIA modelswere developed using the conventionalstepwise procedure. The optimum numberof components used to derive the non-validated model was defined as the numberof components leading to the highestcross-validated r2 (q2) and the lowest standarderror of prediction (SEP). The q2 valueswere derived after “leave-one-out” cross-validation. The non-cross-validated modelswere assessed by the explained variance r2,standard error of estimate (S) and F ratio.The non cross-validated analyses were usedto make prediction of the % inhibition ofthe chromone compounds from the test setand to display the coefficient contour maps.The actual versus predicted % inhibitions ofthe test chromone compounds were fittedby linear regression, and the “predictive” r2,S, and F ratio were determined.

2.3.4 QSAR coefficient contour mapsThe visualization of the results of the

best CoMFA and CoMSIA models havebeen performed using the “StDev*Coeff ”mapping option contoured by contribution.Favored and disfavored levels fixed at 80%and 20%, respectively. The contours of theCoMFA and CoMSIA steric maps are shownin green (more bulk is favored) and yellow(less bulk is favored). The electrostatic fieldsof both CoMFA and CoMSIA contours arecolored blue (positive charge is favored) andred (negative charge is favored). The contoursof the CoMSIA hydrophobic fields arecolored yellow (hydrophobic groups enhanceactivity) and white (hydrophilic groupsenhance activity). The hydrogen bond fieldcontours show regions where hydrogenbond acceptors (magenta) on the receptorenhance the activity and hydrogen bonddonors (cyan) increase the activity.

1078 Chiang Mai J. Sci. 2018; 45(2)

2.4 Docking StudyThe crystal structure of Top I complexed

with CPT (pdb code 1T8I) used in this studywas obtained from the BrookhavenProtein Database (http://www.rcsb.org/pdb). Docking was performed by AutoDockprogram version 4.2 (Scripps ResearchInstitute, USA) using an empirical free energyfunction and Larmarckian Genetic Algorithm,with an initial population of 150 randomlyplaced individuals, a maximum number of106 energy evaluations, a mutation rateof 0.02, and a crossover rate of 0.80. Onehundred independent docking runs wereperformed for each ligand. Results differingby <2.0 in positional root-mean-squaredeviation (RMSD) were clustered together andrepresented by the result with the mostfavorable free energy of binding.

3. RESULTS AND DISCUSSION

Topoisomerase I inhibitory activityIn this study, 9 compounds (chromones

17-25, structures as shown in Table 1) wereevaluated for their Top I inhibitory activityusing eukaryotic Top I drug screening kitwhich is a specific assay for Top I. Detectionof the transient DNA nicks requires trappingthe enzyme on DNA in a nicked intermediatecomplex using protein denaturants, e.g.,sodium dodecyl sulfate. The resulting covalentTop I-DNA complexes contain nicked opencircular DNA which can be detected byagarose gel electrophoresis (with ethidiumbromide). Normally, the process to trap thenicked intermediates is relatively difficultbecause the half-life of this cleavage complexis rather short. However, CPT can stabilizethe intermediate and increases in the nickedDNA product. Thus detection of agents thataffect Top I by stabilizing the cleavedintermediate complex can be performed.

The chromone compounds wereevaluated for percentage inhibition of Top I

at a concentration of 100 μM and results areshown in Table 2. The two most potentcompounds, chromones 19 and 20 with 86.89and 59.67 % inhibition, respectively, weresubjected to further determination of IC

50.

Chromone 20 was found to be more potent(IC

50 = 0.83 μM) than the previously reported

chromone 15 (IC50

= 1.46 μM). Chromone20 also showed higher potency than thestructurally related flavonoids, myricetin(IC

50 = 11.9 μg/mL, 37.39 μM), quercetin

(IC50

= 12.8 μg/mL, 42.35 μM) and fisetin(IC

50 = 20.6 μg/mL, 71.97 μM) [17].

Moreover, chromone 20 even exhibited higherpotency than irinotecan, the clinicallyapproved CPT analogue, which possessedTop I inhibitory activity for LoVo cells andHT-29 cells with IC

50 of 15.8 μM and 5.17

μM, respectively [30].Figure 1 shows results from the

ethidium-stained agarose gel. Typically,supercoiled DNA appeared to migrate morerapidly than the nicked DNA due to itssupercoiled nature, the DNA fragmentsbecame smaller in size and hence experiencedless frictional resistance from the gel. Thisresulted in the migration of supercoiledconformation to be faster than otherconformations of DNA. In the presence ofchromone compounds, Top I-DNA cleavagecomplexes, assayed as nicked DNA wereobserved. Lanes 1-2 in Figures 1a and 1brepresented the supercoiled DNA and Top Itreated with chromones 20, 17, 19 and 18,respectively. Lanes 5-7 in Figure 1c and lanes5-6 in Figure 1d were DNA and Top I treatedwith chromones 23, 22, 21, 25 and 24,respectively. Although the band intensity ofthe chromone compounds appeared to beweaker than the band caused by CPT, theresults indicated the ability of the testedchromones to stabilizing Top I-DNA cleavagecomplex.

Chiang Mai J. Sci. 2018; 45(2) 1079

Table 2. Top I inhibitory activity of 9 chromone derivatives.

Compd

171819202122232425

% Inhibition20 μM

ndnd

42.0692.00

ndndndndnd

100 μM55.0727.9386.8959.6744.6947.4349.0936.4735.63

IC50 (μM)

ndnd

15.240.83ndndndndnd

Figure 1. Stabilization of recombinant Top I-DNA complex by chromone compounds,measured after agarose gel electrophoresis. (a) Lanes 1-2 = supercoiled DNA + Top I +chromones 20 and 17, respectively; Lane 3 = relaxed plasmid DNA marker; Lane 4 =supercoiled DNA; Lane 5 = supercoiled DNA + Top I; Lanes 6 = supercoiled DNA + TopI + CPT. (b) Lanes 1-2 = supercoiled DNA + Top I + chromones 19 and 18, respectively;Lane 3 = supercoiled DNA + Top I; Lanes 4 = supercoiled DNA + Top I + CPT; Lane 5 =relaxed plasmid DNA marker; Lane 6 = supercoiled DNA. (c) Lane 1 = relaxed plasmidDNA marker; Lane 2 = supercoiled DNA; Lane 3 = supercoiled DNA + Top I; Lane 4 =supercoiled DNA + Top I + CPT; Lanes 5-7 = supercoiled DNA + Top I + chromones 23,22 and 21, respectively. (d) Lanes 1-4 same as (c); Lanes 5-6 = supercoiled DNA + Top I +chromones 25 and 24, respectively.

CoMFA studyIn order to investigate the structure-Top

I inhibitory activity relationship, 3D QSARCoMFA and CoMSIA were performed.Eighteen chromones (whose % inhibition weredetermined at 20 μM) were used as data set(Table 3), of which 5 compounds werechosen as test set while the remaining 13

compounds were treated as training set.The selected test set represented a range ofactivity similar to that of the training setand was used to evaluate the predictive powerof the CoMFA and CoMSIA models. Thepercentage inhibition was converted to log %inhibition and used as dependent variablesfor 3D QSAR study.

→nicked DNA

→supercoiled DNA

1 2 3 4 5 6 1 2 3 4 5 6

1 2 3 4 5 6 7 1 2 3 4 5 6

(a) (b)

(c) (d)

1080 Chiang Mai J. Sci. 2018; 45(2)

Field fit and align database methodswere the two alignment criteria used in the3D QSAR study. The CoMFA modelobtained from field fit alignment gavebetter statistical results than align databasealignment (data not shown). Any fieldcolumn the deviation of which was lessthan 2.0 kcal/mole in magnitude was excludedas well as any lattice point the energy ofwhich exceeded 30 kcal/mole was ignoredfrom the PLS analysis. The best CoMFAmodel gave cross-validated r2 (q2) = 0.578 andnon cross-validated r2 = 0.995 (Table 4), usingtraditional CoMFA steric and electrostaticfields. The steric and electrostatic fieldscontributed to the QSAR equation by 60.8 %and 39.2 0%, respectively, which suggested

that variation in the Top I inhibitory activitywas predominantly determined by stericproperty. The experimental % inhibitions,the predicted % inhibitions and the residualsof the predictions are shown in Table 5.The scattered plots of the experimentaland predicted activities of the compoundsin the training set and test set are shown inFigure 2a.

CoMSIA studyThe CoMSIA study was performed

using the same PLS protocol and stepwiseprocedure as in the CoMFA study. The bestCoMSIA model gave q2 = 0.632, r2 = 0.996and included electrostatic, steric, andhydrophobic fields. In this CoMSIA model,

Table 3. Structures and Top I inhibitory activity of chromones in the data set used in CoMFAand CoMSIA studies.

Compd

Trainingset23467811121314151620

Testset1591019

R2

PhenylPhenylPhenyl3′-(OCH

3)-phenyl

3′-(OCH3)-phenyl

3′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(OCH3)-phenyl

4′-(NO2)-phenyl

4′-(NO2)-phenyl

4′-(NO2)-phenyl

PhenylPhenyl3′-(OCH

3)-phenyl

3′-(OCH3)-phenyl

4′-(OCH3)-phenyl

R3

BenzoylBenzoylBenzoyl3′′-(OCH

3)-benzoyl

3′′-(OCH3)-benzoyl

3′′-(OCH3) benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

4′′-(NO2)-benzoyl

4′′-(NO2)-benzoyl

4′′-(NO2)-benzoyl

BenzoylBenzoyl3′′-(OCH

3)-benzoyl

3′′-(OCH3)-benzoyl

4′′-(OCH3)-benzoyl

R5

HHHHHHHHHHHHH

HHHHH

R6

HHHHHHHHHHHHH

HHHHH

R7

O-[3′′′-(OCH3)-benzoyl]

O-[4′′′-(OCH3)-benzoyl]

O-[3′′′-(NO2)-benzoyl]

O-BenzylO-[3′′′-(OCH

3)-benzyl]

O-BenzoylO-[3′′′-(OCH

3)-benzyl]

O-BenzoylO-[3′′′-(OCH

3)-benzoyl]

O-[4′′′-(OCH3)-benzoyl]

O-BenzoylO-[3′′′-(NO

2)-benzoyl]

OH

O-BenzylO-[4′′′-(NO

2)-benzoyl]

O-[3′′′-(OCH3)-benzoyl]

O-[4′′′-(OCH3)-benzoyl]

OH

R8

HHHHHHHHHHHHH

HHHHH

%inhibition

62.6352.3763.9644.5433.4148.6847.7655.7739.7842.6666.0364.7392.00

52.0350.6358.7650.2342.06

log %inhibition

1.7971.7191.8061.6491.5241.6871.6791.7461.6001.6301.8201.8111.964

1.7161.7041.7691.7011.624

Chiang Mai J. Sci. 2018; 45(2) 1081

Table 4. CoMFA and CoMSIA statistical results.

Cross-validation

Non cross-validation

Contributions

Number of molecules in training setNumber of molecules in test set

Optimal componentsq2

Sr2

SF

StericElectrostaticHydrophobic

CoMFA1355

0.5780.0980.9950.011

287.0130.6080.392

CoMSIA1356

0.6320.0990.9960.011

228.9970.1810.4620.357

Table 5. Experimental (observed), predicted activities and the residuals of CoMFA andCoMSIA models.

Compd.

Training set23467811121314151620

Test set1591019

log % inhibition

Experimental

1.7971.7191.8061.6491.5241.6871.6791.7461.6001.6301.8201.8111.964

1.7161.7041.7691.7011.624

Predicted

1.8061.7241.7991.6301.5361.6921.6821.7441.5931.6321.8191.8121.963

1.8161.8281.6201.6151.955

Residual

-0.009-0.0050.0070.019-0.012-0.005-0.0030.0020.007-0.0020.001-0.0010.001

-0.100-0.1240.1490.086-0.331

CoMFAPredicted

1.7951.7141.8091.6401.5401.6901.6761.7491.5861.6381.8171.8091.969

1.8071.7691.6041.6121.791

Residual

0.0020.005-0.0030.009-0.016-0.0030.003-0.0030.014-0.0080.0030.002-0.005

-0.091-0.0650.1650.089-0.167

CoMSIA

steric, electrostatic, and hydrophobic fieldscontributed to the QSAR equation by 18.1%, 46.2 %, and 35.7 %, respectively. Theexperimental and predicted % inhibition

and the scattered plots of both trainingset and test set are shown in Table 5 andFigure 2b, respectively.

1082 Chiang Mai J. Sci. 2018; 45(2)

Figure 2. Predicted and observed (experimental) activity for the training set (%) and the testset (Δ). (a) CoMFA model. (b) CoMSIA model.

log % inhibition (observed)

(a)

1.200 1.400 1.600 1.800 2.000 2.200

log % inhibition (observed)

(b)

1.200 1.400 1.600 1.800 2.000 2.200

log

% i

nhib

itio

n (p

redi

cted

) 2.400

2.200

2.000

1.800

1.600

1.400

1.200

log

% i

nhib

itio

n (p

redi

cted

) 2.400

2.200

2.000

1.800

1.600

1.400

1.200

R2 = 0.9954 R2 = 0.9957

CoMFA and CoMSIA contour mapsThe QSARs produced by CoMFA

and CoMSIA models, which are usuallyrepresented as 3D “coefficient contourmaps” are shown in Figures 3 and 4,

respectively. The molecular structure ofthe most potent chromone 20 and thesecond most potent chromone 15 weredisplayed inside the field as the referencestructures.

Figure 3. CoMFA contour map. (a) CoMFA steric contour map and (b) electrostatic contourmap. The green contours refer to sterically favored region; the yellow contours indicate disfavoredareas. The blue contours indicate region where electropositive substituent is favored and redcontours refer to region where electronegative substituent is favored.

(a) (b)

Chiang Mai J. Sci. 2018; 45(2) 1083

Figure 4. (a) CoMSIA setric contour map. (b) CoMSIA electrostatic contour map. (c) CoMSIAhydrophobic contour map, yellow contours indicate region where hydrophobic group isfavored.

(c)

(b)(a)

The CoMFA and CoMSIA steric contourmaps (Figures 3a and 4a, respectively) illustratecommon field features. The steric contourmaps indicate that bulky substituentsshould be located around C-4′ of ring B,and C-4′′ of ring D. This steric contours(corresponding to the hydrophobic contourmap from CoMSIA, Figure 4c) show theyellow regions at C-7, C-4′ and C-4′′.These yellow polyhedra indicate that thehydrophobic groups increase the activity.Based on the same substitutions at C-2 andC-3, these results explain the good inhibitoryactivity of chromones 15, 16, and 20(with exception of chromones 24 and 25

which exhibited only moderate activity).Although the electrostatic contour mapsfrom CoMFA (Figure 3b) and CoMSIA(Figure 4b) are not quite similar, they doshow a common field feature that theelectropositive substituent (the blue contours)should be located around C-7 of chromonering and the electronegative group at C-3′of ring B.

Figures 5a and 5b illustrate the hydrogenbonding interactions between the two mostpotent compounds, chromones 19 and 20,and Top I, respectively. The 7-OH group ofboth chromones formed hydrogen bondinginteraction with Asp533 of Top I.

1084 Chiang Mai J. Sci. 2018; 45(2)

4. CONCLUSION

In this study, chromone 20 showedhigher inhibitory activity against Top Ithan the previously reported chromonecompounds. The ligand-based 3D QSAR,CoMFA and CoMSIA have been applied toa set of chromone series. Statistical parametersindicate that the established CoMFA andCoMSIA models are reliable. The contourmaps provide relationships between structuralfeatures and Top I inhibitory activity.They suggest that steric groups at C-4′ (ringB) and C4′′ (ring D), electropositivesubstituent at C-7 (chromone ring), and

Figure 5. Hydrogen bonding interactions between DNA Top I (1T8I) and (a) chromone 19;(b) chromone 20.

electronegative substituent at C-3′ (ring B) arefavorable to activity. The 3D QSAR resultsgive the meaningful structural insightsinto possible modifications of chromonederivatives which could improve activity forthe future work.

ACKNOWLEDGEMENT

The authors thank High PerformanceComputer Center (HPCC), NationalElectronics and Computer TechnologyCenter (NECTEC) of Thailand for providingSYBYL-X 2.0 facilities.

(a)

(b)

Chiang Mai J. Sci. 2018; 45(2) 1085

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