Screening for In Vitro Antimycobacterial Activityand Three-Dimensional QuantitativeStructure–Activity Relationship (3D-QSAR) Studyof 4-(arylamino)coumarin Derivatives

Vijay Virsdoia1, Mushtaque S. Shaikh2,Atul Manvar3, Bhavik Desai4, AlpeshParecha5, Raju Loriya6, Kinnari Dholariya6,Gautam Patel7, Vipul Vora8, KuldipUpadhyay9, Karia Denish10, AnamikShah11,* and Evans C. Coutinho2,*

1Dishman Pharmaceuticals and Chemicals Ltd., API Division, Bavla,Ahmedabad-382220, India2Department of Pharmaceutical Chemistry, Bombay College ofPharmacy, Kalina, Santacruz (E), Mumbai-400 098, India3Jubilant Organosys, 1A, Sector 16-A, Noida-201 301,Uttar Pradesh, India4Beckman Coulter Inc,Webster, TX, USA5ATRI Pharma. Pvt. Ltd. Ankleshwar, India6Scifinder Scholar, Pune, India7US Vitamins, API Division, USA8Lupin Pharma, Ankleshwar, India9Torrent Research Center, Bhat, Gandhinagar-382428, Gujarat, India10H & H.B.Kotak Science College, Gujarat, India11Department of Chemistry, Saurashtra University, University Road,Rajkot-360 005, Gujarat India*Corresponding authors: Anamik Shah, anamik_shah@yahoo.com;Evans C. Coutinho, evans@bcpindia.org

The resurgence of tuberculosis and the emergenceof multidrug-resistant strains of Mycobacterianecessitate the search for new classes of antimyco-bacterial agents. We have synthesized a smalllibrary of 50 analogues of 4-(arylamino)coumarinswith various aromatic amines at the C4- position ofthe coumarin scaffold. The compounds were evalu-ated for antimycobacterial activity against Myco-bacterium tuberculosis H37Rv with rifampicin asthe standard. Of the molecules synthesized, com-pound 9 was found to be most potent with a mini-mum inhibitory concentration >6.25 lg ⁄ mL for100% inhibition. In an effort to develop new andmore effective molecules in this series, the rela-tionship between structure and activity was inves-tigated by comparative molecular field analysis.Various models were generated using comparativemolecular field analysis alone and comparativemolecular field analysis plus a hydropathy field

(HINT). In all, eight models were generated withatom-fit and field-fit alignment strategies. The com-parative molecular field analysis models (Models 3aand 4a) based on field-fit alignment were the bestwith statistically good correlation coefficients (r 2)and cross-validated q 2. The values of r 2

pred for thevalidation set were 0.469 and 0.516. Based on thecomparative molecular field analysis contours,some insights into the structure–activity relation-ship of the compounds could be gained.

Key words: 3D-QSAR, 4-(arylamino)coumarins, antitubercularagents, CoMFA, HINT

Abbreviations: 3D-QSAR, three-dimensional quantitative struc-ture–activity relationship; CoMFA, Comparative molecular field analysis;HINT, Hydropathic interactions; MIC, minimum inhibitory concentration;MDR-TB, multidrug-resistant tuberculosis; XDR-TB, extensively drug-resistance tuberculosis; TMPK, Thymidine monophosphate kinase.

Received 3 November 2009, revised 3 March 2010 and accepted forpublication 5 May 2010

Control and prevention of tuberculosis (TB) is a major challenge asone-third of the world's population is infected with Mycobacteriumtuberculosis. Nearly 2 million deaths because of TB occur every year(1–3). According to a WHO report by the year 2020 nearly one billionmore people will be infected, and 70 million will die from TB if propersteps are not taken to control it (4). TB is the world's second cause ofdeath from infectious diseases after acquired immune deficiency syn-drome (AIDS) (5). Moreover, the association of TB with AIDS and theemergence of drug-resistant TB strains have made available chemo-therapeutic strategies increasingly ineffective (6).

Unfortunately, no new drugs or antibiotics have been developed inthe last 30 years against TB. There are three frontline drugs viz.isoniazid, rifampicin, ethambutol, and several second-tier drugs thatinclude ethionamide, streptomycin, and p-aminosalicylic acid to treatTB. However, many of these drugs have disadvantages, such as pro-longed treatment schedules, host toxicity, ineffectiveness againstresistant strains. (5,7). The problem of TB is further complicated bythe emergence of multidrug-resistant TB (MDR-TB) (8–15) and'extensively drug-resistance tuberculosis' (XDR-TB) that threaten TBcontrol globally (16,17).

412

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Research Article

ª 2010 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2010.00997.x

The impact of ever-increasing drug resistance, the serious sideeffects of some current anti-TB drugs and the lack of efficacy ofcurrent treatments in immuno-suppressed patients combine tomake the development of new antimycobacterial agents anurgent priority (8,18). There are several reports in the literatureof new structural classes being developed as antitubercularagents. A series of C3 alkyl and arylalkyl-2,3-dideoxyhex-2-enopyr-anosides with minimum inhibitory concentration (MIC) valuesbetween 25.0 and 1.56 lg ⁄ mL against Mycobacterium tuberculo-sis H37Rv(7) has been reported. Similarly, 5-acetylenic derivativesof 2¢,3¢-dideoxyuridine and 3¢-fluoro-2¢,3¢-dideoxyuridine have beentested for antimycobacterial activity against M. bovis, M. tubercu-losis, and M. avium. The most promising compounds in thisseries, 5-dodecynyl-2¢,3¢-dideoxyuridine and 5-tetradecynyl-3¢-fluoro-2¢,3¢-dideoxyuridine showed excellent activity and inhibited thegrowth of M. bovis and M. tuberculosis with MIC90 in the rangeof 1–2 lg ⁄ mL, which is close to the reference drug rifampicin(19). The derivatives of 5¢-O-[(N-acyl)sulfamoyl]adenosine wereevaluated against M. avium and M. tuberculosis, under iron-defi-cient and iron-rich conditions. Of the various molecules in theseries, the 4-fluorosalicyl derivative was found to be the mostpotent (MIC99 of 0.098 lM), while the 4-aminosalicyl derivativewas the first analogue that exhibited improved selectivity againstM. tuberculosis under iron-rich conditions (MIC99 of 25 lM) (20).A series of 1-b-D-2¢-arabinofuranosyl and 1-(2¢-deoxy-2¢-fluoro-b-D-ribofuranosyl)pyrimidine nucleosides with a variety of alkynyl,alkenyl, alkyl, and halo substituents at the C5 position of theuracil moiety was investigated against M. tuberculosis, M. bovis,and M. avium. Among the designed nucleosides, 1-b-D-2¢-arabino-furanosyl-5-dodecynyluracil, 1-(2¢-deoxy-2¢-fluoro-b-D-ribofuranosyl)-5-dodecynyluracil, and 1-(2¢-deoxy-2¢-fluoro-b-D-ribofuranosyl)-5-tet-radecynyluracil were the most potent against M. bovis and M. tuber-culosis, respectively (21). Recently, thymidine monophosphate kinase(TMPK) has been proposed as an attractive target for the develop-ment of antitubercular agents. The newly developed (3-trifluorom-ethyl-4-chlorophenyl)thiourea has a Ki of 0.6 lM and a selectivityindex of 600 against human TMPK. Moreover, it represents the firstTMPK inhibitor with good inhibitory activity on growing M. bovis(MIC99 = 20 lg ⁄ mL) and M. tuberculosis (MIC50 = 6.25 lg ⁄ mL)strains (22).

Moreover, various structural classes, such as diarylmethanes (23),oxazolyl thiosemicarbazones (24), pyrazolines (25), amphiphilic galac-topyranosyl diamine and amino alcohols (26), isonicotinohydrazones(27), triazoles (28) have been identified as promising antitubercularleads. Calanolide A, a naturally occurring coumarin derivative andan inhibitor of HIV-1 reverse transcriptase also have shown promis-ing in vitro activity against M. tuberculosis with an MIC value of3.13 lg ⁄ mL (29,30).

We have been studying for quite some time heterocyclic com-pounds with a variety of scaffolds as novel antitubercular agents(31–37) and very recently, we have reported the in vitro antitu-bercular activity and 3D-QSAR studies of coumarin-4-acetic acidbenzylidene hydrazides (38). In continuation of this work, the cur-rent paper describes the syntheses of substituted 4-(arylami-no)coumarins and coumarins fused with a benzothiazipine nucleusand their in vitro antitubercular activity against M. tuberculosis

H37Rv strain. A three-dimensional quantitative structure–activityrelationship (3D-QSAR) study has been recently reported on 4-am-inocoumarins tested as antiplatelet agents (39,40). To understandthe structural requirements for antitubercular versus antiplateletactivity, the 3D-QSAR technique of comparative molecular fieldanalysis (CoMFA) (41,42) with and without inclusion of hydro-pathic interactions (HINT) hydropathy fields (43) was also carriedout.

Methods and Materials

ChemistryAll chemicals and solvents were purchased from Spectorchem(Mumbai, India) and SD Fine Chemicals (Mumbai, India) and usedwithout purification. For syntheses of the title compounds, amonomode, open-vessel microwave synthesizer (Q-ProM model;Questron Technologies Corporation, Mississauga, Ontario, Canada)was used. Melting points were determined in open glass capil-laries in a paraffin bath and are uncorrected. 1H NMR and 13CNMR spectra were recorded on a Bruker AC 300 MHz FT-NMRspectrophotometer using tetramethylsilane as the internal stan-dard. Mass spectra were recorded on a JEOL SX 102 ⁄ DA-6000by the FAB (fast atom bombardment) technique for ionization. FT-IR spectra were recorded on a Shimadzu-8400 using KBr disks.Thin layer chromatography (TLC) was carried out on Merck Kie-selgel 60F254 (Merck 5549, USA) plates. The structures of themolecules synthesized are given in Table 1, while all experimen-tal details are briefly given in the supplementary material(Table S1).

General method for the synthesis of 4-(arylamino)coumarin derivatives (2–45)Various 4 -hydroxycoumarins were prepared by earlier reportedmethod. Substituted 4-hydroxycoumarin (0.01 mol) and correspond-ing amines (0.015 mol) were taken in a reaction vessel. Thereaction mixture was subjected to microwave irradiation (400 W)at 140 �C (Scheme 1). The reaction was monitored by TLC (ethy-lacetate:hexane::3:2). After completion of the reaction as indi-cated by TLC, acetone was added to the reaction mixture andthe solid mass obtained was filtered. The crude reaction masswas finally purified by silica gel (60–100 mesh) column chroma-tography employing ethyl acetate and hexane (3:2) as the mobilephase (44–45).

General methods for the synthesis ofphenothiazine derivatives (46–51)Substituted 4-hydroxycoumarin (0.01 mol) and corresponding 2-ami-nothiophenols (0.012 mol) were taken in a reaction vessel and sub-jected to microwave irradiation (400 W) at 140 �C for anappropriate time (Schemes 2 and 3). The reaction was monitored byTLC (chloroform:methanol::9:1). On completion, the reaction mixturewas allowed to cool. The reaction mass was washed with acetonewhen the crude product separated out. This was purified by silicagel (60–100 mesh) column chromatography using chloroform andmethanol (9:1) as the mobile phase (46,47).

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 413

Table 1: The structures and activities of 4-aminoarylcoumarine derivatives

O O

HNR5

R6

R7

R8

*RAr

Series ICompounds R5 R6 R7 R8 Ar *Ro *Rm *Rp

bGI % (lg ⁄ mL) pALogit

2 -H -H -H -H -Ph -Ph -H -H 65 (>6.25) 4.973 -H -CH3 -H -H -Ph -Ph -H -H 14 (>6.25) 3.934 -H -Cl -CH3 -H -Ph -Ph -H -H 38 (>6.25) 4.556 -H -H -H -H -Napha - - - 81 (>6.25) 5.297 -H -CH3 -H -H -Napha - - - 10 (>6.25) 3.738 -H -H -H -CH3 -Napha - - - 54 (>6.25) 4.759 -H -Cl -CH3 -H -Napha - - - 100 (>6.25) ND

12 -H -H -CH3 -CH3 -Napha - - - 9 (>6.25) 3.7013 -CH3 -H -CH3 -H -Napha - - - 9 (>6.25) 3.7014 -H -H -H -H -Ph -H -H -COOH 18 (>6.25) 3.9915 -H -CH3 -H -H -Ph -H -H -COOH 15 (>6.25) 3.9216 -H -H -H -CH3 -Ph -H -H -COOH 33 (>6.25) 4.3717 -H -Cl -CH3 -H -Ph -H -H -COOH 8 (>6.25) 3.6619 -H -H -CH3 -CH3 -Ph -H -H -COOH 41 (>6.25) 4.5420 -CH3 -H -CH3 -H -Ph -H -H -COOH 20 (>6.25) 4.0921 -H -H -H -CH3 -Ph -H -H -Br 28 (>6.25) 4.3122 -H -H -H -CH3 -Ph di-Cl -H -H 11 (>6.25) 3.823 -H -CH3 -H -H -Ph -H -H -Br 1 (>6.25) 2.7324 -H -H -H -H -Ph -H -H -F 18 (>6.25) 3.9525 -H -H -H -CH3 -Ph -H -H -H 7 (>6.25) 3.5126 -H -CH3 -H -H -Ph -H -H -F 1 (>6.25) 2.6427 -H -CH3 -CH3 -H -Ph -H -H -F 31 (>6.25) 4.3128 -H -H -CH3 -CH3 -Ph -H -H -F 22 (>6.25) 4.1130 -H -H -H -H -Ph -H -Cl -F 15 (>6.25) 3.9131 -CH3 -H -CH3 -H -Ph -H -Cl -F 4 (>6.25) 3.4332 -H -H -H -H -Ph -Cl -H -Cl 28 (>6.25) 4.2833 -H -H -H -H -Ph -Cl -H -H 11 (>6.25) 3.7334 -H -H -H -H -Ph -H -H -Cl 10 (>6.25) 3.6835 -H -H -H -H -Ph -H -H -OCH3 15 (>6.25) 3.8836 -H -H -H -CH3 -Ph -H -H -OCH3 7 (>6.25) 3.5337 -H -H -H -CH3 -Ph -H -H -Cl 10 (>6.25) 3.7138 -CH3 -H -CH3 -H -Ph -H -H -Cl 9 (>6.25) 3.6839 -H -Ph -H -H -Ph -H -Cl -H 23 (>6.25) 4.2240 -H -H -H -H -Ph -H -NO2 -H 30 (>6.25) 4.2841 -H -H -H -H -Ph -CH3 -H -H 44 (>6.25) 4.542 -H -H -H -H -Ph -H -H -CH3 50 (>6.25) 4.643 -H -H -H -H -Ph -OCH3 -H -H 39 (>6.25) 4.4444 -H -H -H -H -Ph -CH3 -CH3 -H 51 (>6.25) 4.6545 -H -H -H -H -Ph -CH3 -H -CH3 49 (>6.25) 4.61

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414 Chem Biol Drug Des 2010; 76: 412–424

Table 1: (Continued )

O O

HNAr

R5

R6

Series II

Compounds R5 R6 Ar bGI % (lg ⁄ mL) pALogit

11 -H -H -Napha 11 (>6.25) 3.82

O O

HN

R7

R8

*RAr

Series III

Compounds R7 R8 Ar *Ro *Rm *RpbGI % (lg ⁄ mL) pALogit

5 -H -H -Ph -Ph -H -H 7 (>6.25) 3.6410 -H -H -Napha - - - 5 (>6.25) 3.4518 -H -H -Ph -H -H -COOH 26 (>6.25) 4.2729 -H -H -Ph -H -H -COOH 53 (>6.25) 4.74

Y

HN

S

O

R6

R5

R7

R8

*R

Series IV

Compounds Y R5 R6 R7 R8 Ar *Ro *Rm *RpbGI % (lg ⁄ mL) pALogit

46 O -H -H -H -H -Ph -H -Cl -H 29 (>6.25) 4.2947 O -CH3 -H -CH3 -H -Ph -H -Cl -H 36 (>6.25) 4.4748 NH -Cl -H -H -Cl -Ph -H -H -H 67 (>6.25) 5.0449 NH -H -Cl -H -H -Ph -H -H -H 8 (>6.25) 3.62

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 415

Antitubercular activityAntitubercular activity was determined using the BACTEC 460system modified as described below. Stock solutions of testcompounds were prepared in dimethylsulfoxide (DMSO) in1 mg ⁄ mL concentration and sterilized by passage through 0.22-lm PFTE filters (Millex-FG; Millipore, Bedford MA, USA). Volumesequivalent to 50 lL were added to 4 mL radiometric 7H12 broth

(BACTEC 12B; Bectron Dickinson Diagnostic Instrument system,Sparks, MD, USA) to achieve a final concentration of6.25 mg ⁄ mL. Controls received 50 mL DMSO. Rifampicin (SigmaChemical Co. St. Louis, MO, USA) was included as a positivedrug control. Rifampicin was solubilized and diluted in DMSOand added to BACTEC-12 broth to achieve MIC inhibiting 99% ofthe inoculum.

Table 1: (Continued )

O O OO

R9

S

HN

R10

R5

R8

*R

Series V

Compounds R5 R8 R9 R10 Ar *Ro *Rm *RpbGI % (lg ⁄ mL) pALogit

50 -H -H -CH3 - -Ph -H -H -H 78 (>6.25) 551 -H -H -CH3 -CH3 -Ph -H -H -H 54 (>12.5) 4.53

-Napha = a-naphthyl*Ro ⁄ m ⁄ p positions with respect to –NH.bGI = Growth inhibition of virulent strain of M. tuberculosisThe values in the parenthesis denote the MIC in lg ⁄ mL. MIC of Rifampin: 0.015–0.125 lg ⁄ mL against M. tuberculosis H37Rv (97% inhibition).

O O

OH

O O

NHAr

Microwave 400W, 140oC3-6 min

4-(arylamino)coumarins (2-45)4-Hydroxycoumarin 1

R R

Ar-NH2

Scheme 1: Microwave-assisted synthesis of substituted 4-arylaminocoumarins (2–45).

O O

OH

R

NH2

SH

R1

Microwave 400W, 140oC6-9 min

O

HN

S

O

R

R = Me or ClR1 = Cl

R1

Compounds (46-49)

Scheme 2: Microwave-assisted synthesis of phenothiazine derivatives (46–49).

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416 Chem Biol Drug Des 2010; 76: 412–424

Mycobacterium tuberculosis H37Rv strain (ATCC 27294; Americantype culture collection; Rockville, MD, USA) was cultured at 37 �Con a rotary shaker in middle brook 7H9 broth (Difco Laboratories,Detroit, MI, USA) supplemented with 0.2% v ⁄ v glycerol and 0.05%v ⁄ v Tween 80 until the culture turbidity achieved an optical densityof 0.45–0.55 at 550 nm. Bacteria were pelleted by centrifugation,washed twice, and resuspended in one-fifth of the original volumein Dulbecco's phosphate-buffered saline [PBS, Irvine Scientific,Santa Ana, (A)]. Large bacterial clumps were removed by passagethrough an 8 mm filter (Malgene, Rochester, NY, USA), and aliquotswere frozen at )80 �C. The cultures were thawed and an appropri-ate dilution was performed such that a BACTEC-12B vial inoculatedwith a 0.1 mL would reach a growth index (GI) of 999 in 5 days.One-tenth of the diluted inoculums were used to inoculate 4 mLfresh BACTEC 12B broth containing the test compounds. An addi-tional control vial was included, which received a further 1:100diluted inoculums (as well as 50 mL DMSO) for use in calculatingthe MIC of rifampicin, respectively by established procedures.

Cultures were incubated at 37 �C, and the growth of inhibition (GI)determined daily until control cultures achieved a GI of 999. Assayswere usually completed in 5–8 days. Percent inhibition was definedas 1-(GI of test sample ⁄ GI of control) 10. MIC of compound effect-ing a reduction in daily change in GI, which was less than thatobserved with a 1:100 diluted control culture on day the laterreached a GI of at least 30.

Computational detailsComparative molecular field analysis was carried out using Sybylv.7.1 (Tripos Inc., USA)a running on a Pentium IV computer underthe Linux RedHat Enterprise 2.3.1.

Ligand preparationThe antitubercular activity of the molecules considered in theQSAR studies span a 3 log unit range (Table 1). The moleculeswere built with the Sketch module in Sybyl. Figure 1 shows arepresentative structure for these molecules; for the phenothiazinederivatives, X represents a S-bridge between the coumarin andthe phenyl rings. To determine the correct geometry at the

4-amino position, ab initio calculations were run on molecule 21

with the Gaussian software.b Geometry optimization was carriedout at both the AM1 and HF-3 ⁄ 21G level of theories. Based onthese calculations, the atom type for the N-atom was determinedas 'N3' i.e., sp3-pyramidal. The conformation of the aromatic ringaround the N-CAr bond was scanned using Systematic Search forrepresentative molecules with different substituents in the orthoor meta-positions, and the global minimum conformation thusdetermined was adopted for these and other molecules with simi-lar substitutions.

The geometries of all molecules, prior to the QSAR study, wereoptimized by energy minimization by the conjugate gradient methodwith the Tripos force field and the Gasteiger H�ckel charges for allatoms, until a gradient 0.01 kcal ⁄ mol ⁄ � was reached. Gasteiger-H�ckel charges were used for the QSAR studies.

Atom-fit-based alignmentThe atoms of the 4-aminocoumarin ring are a common feature inall molecules and this set was used for superimposition onto refer-ence molecule 9. The alignment based on atom-fit strategy isshown in Figure 2.

Field-fit-based alignmentField-fit alignment was also utilized for generating 3D-QSAR mod-els. The steric and electrostatic fields were calculated around all

O OOO

OH

Microwave 400 W/140oC8 min

O OOO

S

NH

NH2

SH

+

Scheme 3: Microwave-assisted synthesis of pyrano-coumaro-phenothiazine derivative (50–51).

Y O

HN

X

12

3

456

78

R1

1′

4′

5′

6′7′

8′R′

Figure 1: A general representation of the structure of molecules(X = S, Y = NH ⁄ O).

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 417

molecules with a grid spacing of 0.5 �. The molecules were thenaligned using the field-fit methodology with the fields around mole-cule 9 as the reference. The alignment based on field-fit strategyis shown in Figure 3.

Statically rotated alignmentsThe molecules previously aligned by atom-fit and field-fit method-ologies were rotated in 3D space to generate additional orienta-tion sets for QSAR studies. The STATIC ROTATE command inSybyl was used for generating such differently oriented sets. Theorientation that yielded statistically significant results was theone obtained by rotating the molecules by 90� in the X, 180� inthe Y, and 270� in the Z directions (models 1a, 2a, 3a and 4ain Table 2).

DatasetThe set of 36 diverse molecules (Table S2 in supplementary infor-mation) was used in the CoMFA study. The dataset was divided

into a training set (27 molecules) and a test set (9 molecules) bymeans of chemical as well as biological diversity. Daylight finger-prints of the molecules along with the pAlogit data were used toseparate the molecules into training and test sets based on theTanimoto similarity coefficient. Later on, few molecules wereswapped among the two datasets to get statistically sound models.The final models were generated on a training set of 27 moleculesand a test set of six molecules.

Biological DataFor the QSAR study, the activity values were transformed asfollowsc

Log activity (pALogit Þ ¼ �logc þ logit ð1Þ

where 'c' is molar concentration = concentration (lg ⁄ mL) * 0.001 ⁄(molecular weight); and

logit ¼ log½% inhibition=ð100�% inhibition)�: ð2Þ

CoMFA studiesIn all CoMFA studies, default settings for the 3D cubic lattice, thegrid spacing, the probe atom, and the energy cut-offs were used.

The CoMFA descriptors (steric and electrostatic fields) were usedas independent variables and the activity (as the negative loga-rithm) values as the dependent variable in a partial least squares(PLS) regression analysis (48) to derive 3D-QSAR models. The CoM-FA models were generated from molecular alignments generated byatom fit and field fit and their orientation in the 3D-grid optimizedwith the static rotation command in Sybyl. The hydrophobic fieldcalculated using HINT (43) was used in conjunction with the CoMFAfields to derive additional models.

The hydropathic fields were calculated with the HINT module inSybyl. The region defined for calculation of the CoMFA fieldswas also used to describe the space in which the HINT fieldswere generated. HINT calculates the hydrophobic interactionbetween all atom pairs in a molecule using the following equa-tion

B ¼XX

bij ð3Þ

where, bij = aiaj SiSj Rij Tij

bij = micro-interaction constant representing the attraction ⁄ interac-tion between atoms i and j

ai = the hydrophobic atom constant for atom i

Si = the solvent accessible surface area for atom i

Rij = the functional distance behavior for the interaction betweenatoms i and j

Figure 2: 4-Aminocoumarins aligned by the Atom-fit method.

Figure 3: 4-Aminocoumarin derivatives aligned by the Field-fittechnique.

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418 Chem Biol Drug Des 2010; 76: 412–424

Tij = a discriminant function designed to keep the signs of interac-tions consistent with the HINT convention that favorable interac-tions are positive and unfavorable interactions are negative.

The models were internally evaluated by leave-one-out (LOO) cross-validation. The optimal number of components was determined bythe SAMPLS (49) method and this was subsequently used to derivethe final QSAR models. In addition to the q2, the conventional cor-relation coefficient r2 and standard errors (SE) were also computed,the statistics of the models generated are shown in Table 2. Themodels obtained were internally validated by bootstrapping (50–52),Y-scrambling (53,54), and externally validated by calculating ther2

pred for the validation set (test set).

Design of new moleculesNew molecules were designed based on a protocol publishedrecently (55). A library of 913 molecules was generated using theCombiChem I utility in Cerius2 (Accelrys Inc., San Diego, CA, USA).The generation of this library of molecules was guided by the CoM-FA contours. Appropriate side chains were attached to molecules 6,26 and 50. Molecules generated in this way were optimized withthe same protocol of energy minimization and then aligned asdescribed earlier. The activities of these molecules were predictedusing the best models 3a and 4a, respectively (Table 3). The ADMEand toxicity properties of the molecules were predicted with Quick-Propd and the web-based PreADMET (56) tools to identify drugablecompounds.

Results and Discussion

ChemistryThe synthesis of 4-(arylamino)coumarins as reported in the literatureinvolves two steps. 4-Hydroxycoumarin is first converted to 4-chloro-

coumarin and this is then converted to the respective amines (57).The 4-chlorocoumarins are strong skin irritants with several toxiceffects, so even a small-scale preparation is environmentally haz-ardous. With the use of microwaves, it is possible to carry out thisreaction in a single step with a drastic reduction in reaction time.This facile method has been optimized to prepare a small library of50 compounds reported in this paper. The use of microwaves repre-sents a far simpler and quicker mode of synthesis of 4-(arylami-no)coumarins over methods reported in the literature. The methodinvolves irradiation of the reactants, the 4-hydroxycoumarin deriva-tives, and the aryl amines in appropriate proportion, with 400 Wmicrowave power at 140 �C in the absence of a solvent for anappropriate time (Schemes 1, 2 and 3). All reactions were carriedout in a monomode open-vessel microwave synthesizer. The struc-tures of the synthesized molecules were confirmed by 1H NMR, IRspectra and mass spectra; details are given in supplementary data(Table S1).

Antitubercular activityThe compounds were screened against M. tuberculosis H37Rvstrain using the BECTEC 460 radiometric system. The results aresummarized in Table 1 with rifampin, as the standard. Of thefifty compounds, compound 9 is the most potent showing 100%inhibition of M. tuberculosis at 6.25 lg ⁄ mL, while compound 6 ismoderately active with 81% inhibition at the same concentration.It is interesting to note that both bear a bulky naphthyl group atthe 4-position of the coumarin ring. The benzenoid part of thecoumarin skeleton in 9 is substituted with a 6-chloro and a 7-methyl group, while in case of 6, it is unsubstituted. In contrast,compounds with methyl substitutions at both the 5- and 8-posi-tions on the coumarin ring (e.g., compounds 7, 12 and 13) arenearly inactive. Compound 2 with a biaryl functionality at the 4-position shows 65% inhibition and is more potent than closelyrelated analogues. When the biphenyl derivatives are additionally

Table 2: PLS statistics of CoMFA and CoMSIA 3D-QSAR models derived by atom-fit-based and field-fit alignments

PLS statistics

Atom-fit alignment Field-fit alignment

CoMFA CoMFA + HINT CoMFA CoMFA + HINT

Model 1 Model 1a Model 2 Model 2a Model 3 Model 3a Model 4 Model 4a

n 27 27 27 27 27 27 27 27PLS components 6 5 6 5 6 6 6 6q2 (LOO) 0.41 0.47 0.38 0.35 0.46 0.58 0.40 0.56r2 0.88 0.93 0.94 0.90 0.98 0.99 0.97 0.98r2

pred 0.50 0.41 0.38 0.51 0.49 0.47 0.52 0.52r2

BS 0.97 0.98 0.98 0.97 0.99 0.99 0.99 0.99r2

YS – – – – – 0.377 – 0.305F 40.2 56.9 51.8 39.1 151.8 245.4 121.4 187.6SE 0.219 0.169 0.163 0.201 0.097 0.077 0.108 0.087Field contribution

Steric 0.611 0.649 0.505 0.502 0.576 0.625 0.448 0.501Electrostatic 0.389 0.351 0.248 0.235 0.424 0.375 0.276 0.250HINT – – 0.248 0.263 – – 0.276 0.250

The models designated by letter 'a' refer to those deduced from datasets obtained with static rotation of particular alignments in 3D space.CoMFA = Comparative molecular field analysis, HINT = Hydropathic interactions, n = number of molecules in training set, r2 = correlation coefficient, q2

(LOO) = cross-validated r2 with Leave-one-out method, r2pred = Correlation coefficient for prediction of external datasets, r2

BS = Bootstrap Correlation coefficient,F = F statistics, SE = standard error, PLS = partial least square analysis.

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 419

substituted with methyl groups on the coumarin ring, the activityfalls drastically e.g., compounds 3 and 4. No worthwhile activitywas observed in the series possessing an electron withdrawing –COOH group (molecules 14–20). The activity of compounds bear-ing a halogen substituent in the aromatic ring joined at the4-position of the coumarin ring is variable with no clear-cutpattern. In the phenothiazine series of compounds (molecules46–50), compounds 50 and 48 show 78% and 67% inhibition,respectively. From the activity range, it can be concluded that thepresence of the naphthyl group enhances potency, while methylsubstituents on the coumarin ring diminishes potency. Becauseof the very interesting pattern of activity shown by these novelcompounds, the molecules were subjected to CoMFA studies to

probe the fine relationship between structure and activity thatexists for these compounds.

3D-QSAR studyThe most interesting feature in these molecules is the geometry ofthe 4-amino group. In an ideal situation, it must have a planar tri-gonal geometry as it is backed by resonance to an aromatic systemas well as to the coumarin carbonyl group. However, preliminarymolecular mechanics calculations showed lack of planarity as thehydrogen atom attached to the 4-amino group experiences severesteric clash with the C5 hydrogen. To gain a better perspective ofthe correct geometry, ab initio calculations were run on molecule

Table 3: The predicted pALogit values for the designed molecules (A) Derivatives of molecules 6 (52–64) (B) Derivatives of molecules26 (65–67) and (C) Derivatives of molecules 50 (68–79)

OOO O

S

NR2

R3

O O

N

R1

R2

R3

O O

N

R1

R2

A B C

Molecules Series

Predicted activity Substitution

Model 3a Model 4a R1 R2 R3

52 A 5.004 5.069 -COCH3 -CH2Ph -53 A 5.136 5.209 -H -CH2CH3 -54 A 5.546 5.713 -COCH3 -CH2CH3 -55 A 4.955 5.032 -CH2Ph -CH2CH3 -56 A 5.427 5.587 -COOH -CH2CH3 -57 A 5.272 5.288 -CH2CH3 -CH2CH3 -58 A 5.188 5.204 -CH=CH2 -CH2CH3 -59 A 5.266 5.263 -Ph -CH2CH3 -60 A 5.244 5.273 -OPh -CH2CH3 -61 A 5.095 5.146 -CH3 -CH2CH3 -62 A 5.237 5.167 -OCH3 -CH2CH3 -63 A 5.151 5.306 -OH -CH2CH3 -64 A 4.998 5.02 -tBu -CH2CH3 -65 B 5.009 5.171 -COCH3 -CH2CH3 -CN66 B 5.065 5.268 -COCH3 -CH2CH3 -H67 B 5.327 5.472 -COCH3 -CH=CH3 -OCH3

68 C 5.076 5.198 - -H -H69 C 5.017 5.069 - -CH2Ph -NH2

70 C 5.243 5.355 - -CH2CH3 -NH2

71 C 5.217 5.313 - -CH=CH2 -NH2

72 C 5.286 5.383 - -Ph -NH2

73 C 5.141 5.207 - -COCH3 -OH74 C 5.042 5.099 - -CH2CH3 -F75 C 5.17 5.252 - -CH2CH3 -H76 C 5.085 5.149 - -CH2CH3 -OH77 C 5.042 5.108 - -H -OCH3

78 C 4.99 5.031 - -CH2CH3 -SO2NH2

79 C 5.018 5.053 - -CH=CH2 -SO2NH2

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420 Chem Biol Drug Des 2010; 76: 412–424

21 with the Gaussian software.b Calculations were run on struc-tures with both pyramidal and planar trigonal geometries for the 4-amino group. The calculations revealed that the N-atom adopts astate that is between these two ideal geometries. As no such atomtype has been defined in the Tripos force field, the atom type forthe N-atom was set to be 'N3' i.e., sp3-pyramidal.

The next issue was to fix the geometry at the N-C4 bond betweenthe amine N and the C4 atom on the coumarin ring (Figure 1). Asmentioned previously, certain molecules have an S-bridge betweenthe aromatic ring and the 3-coumarin position. The activities ofthese constrained cyclic amines (e.g., molecule 50) are higher thanthe corresponding non-cyclic amines; this indicates that the confor-mation attained by such systems must be considered in buildingthe conformation of the other molecules in the series. This factwas used to fix the conformation around the N-C4 bond for theopen chain molecules. The constrained arylaminocoumarins werefirst subjected to complete minimization, and the final conforma-tions achieved for these molecules were adopted for the other non-cyclic arylaminocoumarins. Further, Systematic conformationalsearch was executed on representative molecules to derive theoptimal conformation to be used for the CoMFA studies.

Comparative molecular field analysis alone and in conjunction withHINT hydropathy fields was carried out. CoMFA models 3a and 4awith additional HINT fields were the most robust, with correlationcoefficients (r2) of 0.987 and 0.983 and cross-validated r2 (q2) of0.578 and 0.556, respectively (Table 2). Both these models weretested for their predictive ability on the validation set. The r2

pred forthe various models are presented in Table 2 and the best value is0.522, indicating that most models have decent predictivepowers. Further the robustness of the models was gauged by cross-validation using bootstrapping (50–52) carried out with 100 runs.The values of r2

BS are close to the related r2 values. To check theredundancy and chance correlation, the activity values were ran-domized (Y-scrambling) (53–55) and the regression coefficients werecalculated for models 3a and 4a. These r2

YS calculated on random-ized activity were significantly smaller (0.377 and 0.305 for models3a and 4a, respectively) indicating low degree of redundancy in thetraining set.

Molecules 8, 21 and 39 were found to be outliers; their activitieswere not well predicted by any of the models. One of the reasonsfor the poor prediction may be an inadequate consideration of theconformation of these molecules in the development of the models.

Incorporation of the HINT hydropathy field to the CoMFA modelsgave a significant improvement in the quality of the models. Thestatistical data in Table 2 indicate that the hydrophobic propertyalong with steric and electrostatic fields is necessary to explain theactivity of these molecules. The contribution of the HINT hydropathyfield was seen to be equal to the electrostatic field.

Graphical interpretation of the resultsThe CoMFA contour maps were generated using scalar products ofcoefficients and standard deviation (STDEV*COEFF) set at 80% and20% for favored and disfavored levels, respectively.

Analysis of the contours for CoMFA modelsThe CoMFA contours (Figures 4, 5, and 6) were analyzed aroundthe most active, moderately active, and the least active molecules50, 3 and 26, respectively. The steric contours indicate bulkygroups attached to the 7-position on the aromatic ring of the cou-marin ring are strongly favored. The C6-position on the coumarinring should be unsubstituted as indicated by the small yellow con-tour near this position (Figure 6). This is aptly borne by the factthat the alkyl and aryl groups decrease the activity of molecules 3

and 26, respectively compared to molecule 50, which does nothave a substituent at the C6-position (Figure 5).

The electrostatic contours (Figure 4) depict that electronegativegroups on the aromatic ring will improve activity. For moleculeswith a phenothiazine ring (e.g., molecule 50), electronegativegroups are favored at the meta-positions to the amino functionalityi.e., 5¢ and 7¢-positions, while the para-position i.e., 6¢-positionneeds an electropositive group to improve activity. The same pat-tern is followed in the case of molecules with N-phenyl groups. Itis interesting to find that electronegative group para to the aminogroup have a detrimental effect on the activity and indeed thisposition needs to be filled with electropositive groups. This is themain reason why molecules 3 and 26 with electronegative carboxyland fluoro groups have greatly diminished activities.

As mentioned earlier, some 3D-QSAR studies have been carriedout on 4-aminocoumarins with regard to antiplatelet activity(39,40). One of these studies was carried out on a diverse set of2- ⁄ 4-aminocoumarins, coumarins, and benzocoumarins substitutedwith 1-piperazine, and pyrimidine, pyrido[1,2-a]pyrimidine and thia-zo[1,2-a]pyrimidines analogues. The difference between these mol-ecules and the present series is the presence of an aromaticgroup on 4-amino functionality. For antiplatelet activity, bulky sub-stituents are required at the 7- and 8-positions of the coumarinring but are disfavored in the 4¢-position of the 4-piperazinegroup. In strong contrast, for antitubercular activity, bulky groupsare preferred at the 7-position, and further, this site seems to beencompassed by a large hydrophobic pocket. Similarly, comparison

Figure 4: Comparative molecular field analysis electrostatic con-tours displayed around molecule 50.

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 421

of the electrostatic contours reveals a favor for electronegativegroups at the 4-position for antitubercular activity. Thus, there areclear distinctions between the steric and electrostatic attributes in4-aminocoumarins that separate antiplatelet from antitubercularactivity.

Design of new moleculesThe end purpose of all medicinal chemistry endeavors is to designand synthesize better molecules. The molecules should not only bepotent but also have a good safety profile. Thus, it is necessary toconsider ADMET properties right at the design stage itself. Withthis idea, around 913 molecules were designed and their activitypredicted using the best CoMFA models. The 135 molecules pre-dicted to be highly active were then screened for various ADMETproperties like aqueous solubility (QPlogS), partition coefficient indifferent solvents ⁄ media (QPlogPoct, QPlogPw, QPlogPo ⁄ w, CIQPlogS),

partition coefficient in biological fluids, and membranes like skinand Blood Brain Barrier (BBB), apparent Caco-2 cell permeability,MDCK cell permeability, binding to human serum albumin, metabo-lism, human oral absorption, IC50 value for blockage of HERG K+

channels, and central nervous system activity. For details of theessential ADMET properties, the reader is referred to the supple-mentary material. A few molecules with the best activity and thesatisfactory ADMET profile are enlisted in Table 3. Their respectiveADMET data (Table S3 in supplementary data) show that most mol-ecules have satisfactory drug-like attributes. Toxicity data show thatonly 28 molecules are predicted to be non-carcinogenic and sixwere devoid of mutagenic effects.

Conclusions

A small library of 4-(arylamino)coumarins was synthesized usingmicrowave-assisted synthesis in good yields and short reactiontimes. All compounds were screened for activity against M. tuber-culosis H37Rv strain. A 3D-QSAR analysis of this library of com-pounds was carried out using CoMFA alone and CoMFA inconjunction with a hydrophobic field evaluated with HINT to mapthe structural features responsible for antitubercular activity. Inclu-sion of the HINT hydropathy field to the CoMFA fields improved thequality of the models. Models obtained by field-fit alignment werebetter in terms of statistics than those with database ⁄ atom-fitalignment. Analysis of the CoMFA contours provided details on thefine relationship linking structure and activity and offered ideas forstructural modifications that can improve the activity. There is stilla good scope for optimization of these molecules.

Acknowledgments

The authors are thankful to DST (program FIST, Sanction No.SR ⁄ FST ⁄ CSI-072 ⁄ 2003, Dt. 24 ⁄ 12 ⁄ 2003) and to UGC (SAP program,Sanction No. 540 ⁄ 6 ⁄ DRS ⁄ 2004, SAP-I, Dt. 26 ⁄ 03 ⁄ 2004) for theirgenerous financial and instrumentation support. Special thanks aredue to 'National Facility for Drug Discovery through New Chemicalentities (NCE's) Development & Instrumentation Support to SmallManufacturing Pharma Enterprises' Program under Drug & PharmaResearch Support (DPRS) jointly funded by Department of Science& Technology (DST, sanction letter no. VI ⁄ D&P ⁄ 188 ⁄ 06-07 ⁄ , TDT,dated 30 ⁄ 03 ⁄ 07) New Delhi, Government of Gujarat Industries Com-missionerate (Sanction Letter No. IC ⁄ SSI ⁄ R&D ⁄ SU ⁄ 07 ⁄ 3279 ⁄ 168,dated 17 ⁄ 01 ⁄ 07) & Saurashtra University (Sanction Letter No.PL-G ⁄ UGC ⁄ MS ⁄ 238 ⁄ 20006, dated 19 ⁄ 09 ⁄ 06), Rajkot. The authors arealso grateful to Government of Gujarat for PG Diploma's for Instru-mentation & Patentization Financial Grant (Sanction Letter KVT ⁄ U-NI ⁄ CHABA ⁄ 2008 ⁄ 20690-207).

Authors are also thankful to the Dr. Cecil Kwong, Tuberculosis Anti-microbial Acquisition and Coordinating Facility (TAACF), USA, for an-titubercular screening. The computational facilities at BCP wereprovided by the All India Council for Technical Education throughgrant F. No. 8022 ⁄ RID ⁄ NPROJ ⁄ RPS-5 ⁄ 2003-04 ⁄ . The Department ofScience and Technology is also thanked for infrastructure facilitiesthrough the FIST program (SR ⁄ FST ⁄ LS1-163 ⁄ 2003).

Figure 5: Comparative molecular field analysis contours dis-played around molecules 3 and 26.

Figure 6: Comparative molecular field analysis steric contoursdisplayed around molecule 50.

Virsdoia et al.

422 Chem Biol Drug Des 2010; 76: 412–424

References

1. Dye C., Scheele S., Dolin P., Pathania V., Raviglione M.C. (1999)Consensus statement. Global burden of tuberculosis: estimatedincidence, prevalence, and mortality by country. WHO GlobalSurveillance and Monitoring Project. J Amm Med Assoc;282:677–686.

2. Frieden T.R., Sterling T.R., Munsiff S.S., Watt C.J., Dye C. (2003)Tuberculosis. Lancet;362:887–899.

3. WHO. (2006) Tuberculosis Facts. In: Tuberculosis Facts.4. WHO. (2004) Tuberculosis Fact Sheet. In: Tuberculosis Fact

Sheet.5. Guerrin-Tran E., Thiolet J.M., Rousseau C., Henry S., Poirier C.,

Che D. et al. (2006) An evaluation of data quality in a networkfor surveillance of Mycobacterium tuberculosis resistance toantituberculosis drugs in Ile-de-France region-2001–2002. Eur JEpidemiol;21:783–785.

6. Somoskovi A., Parsons L.M., Salfinger M. (2001) The molecularbasis of resistance to isoniazid, rifampin, and pyrazinamide inMycobacterium tuberculosis. Respir Res;2:164–168.

7. Saquib M., Gupta M.K., Sagar R., Prabhakar Y.S., Shaw A.K., Ku-mar R. et al. (2007) C-3 alkyl ⁄ arylalkyl-2,3-dideoxyhex-2-enopyr-anosides as antitubercular agents: synthesis, biologicalevaluation, and QSAR study. J Med Chem;50:2942–2950.

8. Pilheu J.A. (1998) Tuberculosis 2000: problems and solutions. IntJ Tuberc Lung Dis;2:696–703.

9. Espinal M.A. (2003) The global situation of MDR-TB. Tuberculo-sis (Edinb);83:44–51.

10. Peloquin C.A., Berning S.E. (1994) Infection caused by Mycobac-terium tuberculosis. Ann Pharmacother;28:72–84.

11. Murray J.F. (1998) Tuberculosis and HIV infection: a global per-spective. Respiration;65:335–342.

12. Gordin F.M., Nelson E.T., Matts J.P., Cohn D.L., Ernst J., BenatorD. et al. (1996) The impact of human immunodeficiency virusinfection on drug-resistant tuberculosis. Am J Respir Crit CareMed;154:1478–1483.

13. Moss A.R., Alland D., Telzak E., Hewlett D. Jr, Sharp V., ChiliadeP. et al. (1997) A city-wide outbreak of a multiple-drug-resistantstrain of Mycobacterium tuberculosis in New York. Int J TubercLung Dis;1:115–121.

14. Maher D., Raviglione M. (2005) Global epidemiology of tubercu-losis. Clin Chest Med;26:167–182, v.

15. Frieden T.R., Munsiff S.S. (2005) The DOTS strategy for controllingthe global tuberculosis epidemic. Clin Chest Med;26:197–205.

16. No authors listed (2006) Editorial:XDR-TB-a Global Threat.Lancet; 368:964.

17. Wright A., Bai G., Barrera L., Boulahbal F., Mart�n-Casabona N.,Gilpin C. et al. (2006) Emergence of Mycobacterium tuberculosiswith Extensive Resistance to Second-Line Drugs–Worldwide,2000–2004. Morbidity and Mortality Weekly Report (MMWR);55:301–5.

18. He X., Alian A., Stroud R., de Montellano P.R.O. (2006) Pyrroli-dine carboxamides as a novel class of inhibitors of enoyl acylcarrier protein reductase from Mycobacterium tuberculosis. JMed Chem;49:6308–6323.

19. Rai D., Johar M., Srivastav N.C., Manning T., Agrawal B., Kun-imoto D.Y. et al. (2007) Inhibition of Mycobacterium tuberculosis,

Mycobacterium bovis, and Mycobacterium avium by novel dide-oxy nucleosides. J Med Chem;50:4766–4774.

20. Qiao C., Gupte A., Boshoff H.I., Wilson D.J., Bennett E.M., SomuR.V. et al. (2007) 5¢-O-[(N-acyl)sulfamoyl]adenosines as antituber-cular agents that inhibit MbtA: an adenylation enzyme requiredfor siderophore biosynthesis of the mycobactins. J MedChem;50:6080–6094.

21. Johar M., Manning T., Tse C., Desroches N., Agrawal B., Kunim-oto D.Y. et al. (2007) Growth inhibition of Mycobacterium bovis,Mycobacterium tuberculosis and Mycobacterium avium in vitro:effect of 1-beta-D-2¢-arabinofuranosyl and 1-(2¢-deoxy-2¢-fluoro-beta-D-2¢-ribofuranosyl) pyrimidine nucleoside analogs. J MedChem;50:3696–3705.

22. Van Daele I., Munier-Lehmann H., Froeyen M., Balzarini J., VanCalenbergh S. (2007) Rational design of 5¢-thiourea-substitutedalpha-thymidine analogues as thymidine monophosphate kinaseinhibitors capable of inhibiting mycobacterial growth. J MedChem;50:5281–5292.

23. Panda G., Parai M.K., Das S.K., Shagufta, Sinha M., ChaturvediV. et al. (2007) Effect of substituents on diarylmethanes for an-titubercular activity. Eur J Med Chem;42:410–419.

24. Gupta R.A., Gupta A.K., Soni L.K., Kaskhedikar S.G. (2007) Ratio-nalization of physicochemical characters of oxazolyl thiosemicar-bazone analogs towards multi-drug resistant tuberculosis: aQSAR approach. Eur J Med Chem;42:1109–1116.

25. Ali M.A., Shaharyar M., Siddiqui A.A. (2007) Synthesis, struc-tural activity relationship and anti-tubercular activity of novelpyrazoline derivatives. Eur J Med Chem;42:268–275.

26. de Almeida M.V., Le Hyaric M., Amarante G.W., Silva LourencoM.C., Lima Brandao M.L. (2007) Synthesis of amphiphilic galac-topyranosyl diamines and amino alcohols as antitubercularagents. Eur J Med Chem;42:1076–1083.

27. Swamy B.N., Suma T.K., Rao G.V., Reddy G.C. (2007) Synthesisof isonicotinoylhydrazones from anacardic acid and their in vitroactivity against Mycobacterium smegmatis. Eur J MedChem;42:420–424.

28. Shiradkar M., Suresh Kumar G.V., Dasari V., Tatikonda S., AkulaK.C., Shah R. (2007) Clubbed triazoles: a novel approach to an-titubercular drugs. Eur J Med Chem;42:807–816.

29. McKee T.C., Covington C.D., Fuller R.W., Bokesch H.R., Young S.,Cardellina I.J. et al. (1998) Pyranocoumarins from tropical spe-cies of the genus Calophyllum: a chemotaxonomic study ofextracts in the National Cancer Institute collection. J NatProd;61:1252–1256.

30. Spino C., Dodier M., Sotheeswaran S. (1998) Anti-HIV coumarinsfrom Calophyllum seed oil. Bioorg Med Chem Lett;8:3475–3478.

31. Dodia N., Shah A. (1999) Single step synthesis and antimicro-bial, antitubercular screening of substituted2,4-dihydroquinolines.Ind J Heterocyl Chem;9:139–142.

32. Dodia N., Shah A. (2000) Synthesis and antitubercular activity ofsome substituted 3-nitro and 3-bromo-4-hydroxy-2-quinolones.Ind J Hterocyl Chem;10:69–70.

33. Desai B., Sureja D., Naliapara Y., Shah A., Saxena A.K. (2001)Synthesis and QSAR studies of 4-substitutedphenyl-2,6-dimethyl-3,5-bis-N-(substitutedphenyl)carbamoyl-1,4-dihydropyridines aspotential antitubercular agents. Bioorg Med Chem;9:1993–1998.

4-(arylamino)coumarins: New Anti T.B. agents

Chem Biol Drug Des 2010; 76: 412–424 423

34. Gaveriya H., Desai B., Vora V., Shah A. (2002) Synthesis and an-titubercular activity studies of some unsymmetrical 1,4-dihydro-pyridines. Ind J Pharm Sci;64:59–62.

35. Kharkar P.S., Desai B., Gaveria H., Varu B., Loriya R., NaliaparaY. et al. (2002) Three-dimensional quantitative structure-activityrelationship of 1,4-dihydropyridines as antitubercular agents. JMed Chem;45:4858–4867.

36. Virsodia V., Pissurlenkar R.R., Manvar D., Dholakia C., AdlakhaP., Shah A. et al. (2008) Synthesis, screening for antitubercularactivity and 3D-QSAR studies of substituted N-phenyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxami des. EurJ Med Chem;43:2103–2115.

37. Virsodia V., Manvar A., Upadhyay K., Loriya R., Karia D., JaggiM. et al. (2009) Synthesis of 1-(2,6-dichlorophenyl)-3-methylene-1,3-dihydroindol-2-one derivatives and in vitro anticancer evalua-tion against SW620 colon cancer cell line. Eur J MedChem;44:1355–1362.

38. Manvar A., Malde A., Verma J., Virsodia V., Mishra A., Upadhy-ay K. et al. (2008) Synthesis, anti-tubercular activity and 3D-QSAR study of coumarin-4-acetic acid benzylidene hydrazides.Eur J Med Chem;43:2395–2403.

39. Roma G., Cinone N., Di Braccio M., Grossi G., Leoncini G., Si-gnorello M.G. et al. (2000) Synthesis, antiplatelet activity andcomparative molecular field analysis of substituted 2-amino-4H-pyrido[1,2-a]pyrimidin-4-ones, their congeners and isosteric ana-logues. Bioorg Med Chem;8:751–768.

40. Roma G., Braccio M.D., Carrieri A., Grossi G., Leoncini G., GraziaSignorello M. et al. (2003) Coumarin, chromone, and 4(3H)-pyri-midinone novel bicyclic and tricyclic derivatives as antiplateletagents: synthesis, biological evaluation, and comparative molec-ular field analysis. Bioorg Med Chem;11:123–138.

41. Cramer R.D., Patterson D.E., Bunce J.D. (1988) Comparativemolecular field analysis (CoMFA). 1 Effect of shape on bindingof steroids to carrier proteins. J Am Chem Soc;110:5959–5967.

42. Cramer R.D. 3rd, Patterson D.E., Bunce J.D. (1989) Recentadvances in comparative molecular field analysis (CoMFA). ProgClin Biol Res;291:161–165.

43. Kellogg G.E., Semus S.F., Abraham D.J. (1991) HINT: a newmethod of empirical hydrophobic field calculation for CoMFA. JComput-Aid Mol Des;5:545–552.

44. Chavda M., Shah A., Bhatt S., Deo K., Kundu P. (2003) Antimi-crobial screening and single step synthesis of some arylaminocoumarins. Ind J Chem;42B:1502–1507.

45. Shah A.K., Bhatt N.S., Raval R.V., Thakor V.M. (1984) Synthesisof 4-hydroxycoumarins. Current Science;53:1241–1242.

46. Shah A., Naliapara Y., Sureja D., Motohashi N., Kurihara T.,Kawase M. et al. (1998) Biological activity of 6,12-dihydro-1-benzopyrano [3,4-b][1,4] benzothiazin-6-ones. AnticancerRes;18:61–63.

47. Shah A., Naliapara Y., Sureja D., Motohashi N., Kawase M.,Miskolci C. et al. (1998) 6,12-Dihydro-1-benzopyrano [3, 4-b] [1,4] benzothiazin-6-ones: Synthesis and MDR reversal in tumorcells. Anticancer Res;18:3001–3004.

48. Wold S., Johansson E., Cocchi M. (1993) PLS-partial leastsquares projections to latent structures in 3D-QSAR in drugdesign; theory methods and applications. In: Kubinyi M.H., edi-tor. PLS-partial least squares projections to latent structures in

3D-QSAR in drug design; theory methods and applications. TheNetherlands: ESCOM Lieden; p. 523–550.

49. Bush B.L., Nachbar R.B. Jr (1993) Sample-distance partial leastsquares: PLS optimized for many variables, with application toCoMFA. J Comput Aided Mol Des;7:587–619.

50. Richard D., Cramer R.D. III, Bunce J.D., Patterson D.E., Frank I.E.(1988) Crossvalidation, bootstrapping, and partial least squarescompared with multiple regression in conventional QSAR stud-ies. Quant Struct-Act Relat;7:18–25.

51. Gunter B. (1994) Bootstrapping. Infect Control Hosp Epidemi-ol;15:543–547.

52. Shao J. (1996) Bootstrap Model Selection. J Am Stat As-soc;91:655–665.

53. Tropsha A., Gramatica P., Gombar V.K. (2003) The Importance ofBeing Earnest: validation is the Absolute Essential for Success-ful Application and Interpretation of QSPR Models. QSAR CombSci;22:69–77.

54. Rucker C., Rucker G., Meringer M. (2007) y-Randomizationand its variants in QSPR ⁄ QSAR. J Chem Inf Model;47:2345–2357.

55. Manvar A.T., Pissurlenkar R.R., Virsodia V.R., Upadhyay K.D.,Manvar D.R., Mishra A.K. et al. (2010) Synthesis, in vitro antitu-bercular activity and 3D-QSAR study of 1,4-dihydropyridines. MolDivers;14:285–305.

56. Lee S.K., Lee I.H., Kim H.J., Chang G.S., Chung J.E., No K.T.(2003) The PreADME approach: Web-based program for rapidprediction of physico-chemical, drug absorption and drug-likeproperties. Massachusetts, USA: Blackwell Publishing.

57. Zagorevskii V.A., Savelev V.L., Meshcheryakova L.M. (1970) Onsynthesis of 4-aminocoumarins and on mechanism of halogensubstitution in 4- chlorocoumarin. Khim Geterotsikl Soed;8:1019–1023.

Notes

aSybyl version 7.1 Tripos Inc. USA.bGaussian 03, Gaussian Inc., Wallingford CT, USA.cCoMFA and QSAR Manual S, Associates Inc., 1699 S Hanley Rd.,St. Louis, MO 631444, USA.dQuickProp. Schrodinger LLC.

Supporting Information

Additional Supporting Information may be found in the online ver-sion of this article:

Table S1. Synthetic and spectral data of the compounds.

Table S2. The experimental and predicted pALogit values for thetraining and test set molecules used in 3D-QSAR studies.

Table S3. ADMET data for the designed compounds.

Please note: Wiley-Blackwell is not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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