DRUG DEVELOPMENT RESEARCH 71 : 188–196 (2010)

Research Article

Synthesis and Screening of Substituted1,4-Naphthoquinones (NPQs) as Antifilarial Agents

Nisha Mathew,� Twinkle Karunan, Lakshmy Srinivasan,and Kalyanasundaram Muthuswamy

Vector Control Research Centre, Indian Council of Medical Research, Indira Nagar,Pondicherry 605006, India

Strategy, Management and Health Policy

Enabling

Technology,

Genomics,

Proteomics

Preclinical

Research

Preclinical Development

Toxicology, Formu-

lation Drug Delivery,

Pharmacokinetics

Clinical Development

Phases I-III

Regulatory, Quality,

Manufacturing

Postmarketing

Phase IV

ABSTRACT Eleven amino-substituted 1,4-naphthoquinones were synthesized via the reaction of 1,4-naphthoquinone with different primary and secondary mono- and diamines in the presence ofdichloromethane ethanol (1:2) solvent at room temperature. All compounds were purified by flashcolumn chromatography, characterized by TLC, HPLC, 13C-NMR, 1H-NMR, and FT-IR spectral analysisand were evaluated in vitro for antifilarial activity using adult bovine filarial worm Setaria digitata byassessing worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduc-tion. Seven of the 11 compounds had macrofilaricidal activity with compounds 9 (2-[(1,3-dimethylbutyl)amino] naphthalene-1,4-dione) and 11 (2-(4-methylpiperazin-1-yl) naphthalene-1,4-dione) havingmaximum activity (ED50 values of 0.91 and 1.2mM, respectively, at 48 h). The effect of differentsubstitutions on antifilarial activity is discussed. Drug Dev Res 71:188–196, 2010. r 2009 Wiley-Liss, Inc.

Key words: 1, 4-naphthoquinone; antifilarial; macrofilaricide; filariasis; Setaria digitata

INTRODUCTION

Lymphatic filariasis (LF) and river blindness(onchocerciasis) are important tropical diseases causedby filarial nematodes that result in considerablemorbidity. Onchocerciasis is caused by infection withthe nematode worm, Onchocerca volvulus, transmittedby black flies. LF results from infection with theparasitic nematodes Wuchereria bancrofti, Brugiamalayi, and B. timori, which are transmitted bymosquito vectors. LF is present in 83 countries atendemic proportions with greater than 1.2 billionindividuals at risk globally, greater than 120 millionpeople estimated to be infected, and greater than 40million individuals with clinical signs of filariasis[www.filariasis.org]. Onchocerciasis is presently treatedwith ivermectin (IVM), while the drugs used forlymphatic filariasis are diethylcarbamazine (DEC)and IVM in combination with albendazole (ALB).

None of these treatments is effective in completelykilling the long-lived adult worms (macrofilariae) and,therefore, treatments are aimed at reducing transmis-sion and pathology. The development of new antifilarialdrugs that affect new molecular targets with effects onadult worms is a challenging task but is essential to

DDR

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ddr.20357

Received 13 September 2009; Accepted 23 November2009

Grant sponsors: Department of Science and Technology;Government of India; Grant number: SR/SO/HS-83/2005.

�Correspondence to: Dr. Nisha Mathew, Vector ControlResearch Centre, Indian Council of Medical Research, IndiraNagar, Pondicherry 605006, India.E-mail: nishamathew@yahoo.com

�c 2009 Wiley-Liss, Inc.

improve treatment and control by killing the adultfilarial worms [Nisha and Kalyanasundaram, 2007].

Aminoquinones are used as medicines [Elslageret al., 1970; Kallmayer and Tappe, 1987] and herbicides[William and Anja, 2001] and have interesting redoxswitching properties [Tucker and Collinson, 2002].Aminoquinones are formed by the reaction of differentamines with quinones [Acheson and Sansom, 1955;Hakobu et al., 1979; Roushdi et al., 1976]. Similarreactions of 1,4-naphthoquinone with primary aminesresults in the formation of 2-amino-1,4-naphthoqui-nones [Bayen et al., 2007]. The synthesis of simplealkylamino derivatives of naphthoquinones and relatedcompounds is of considerable interest, since theyexhibit potent antitumor [Konoshima et al., 1989; Linet al., 1989] and antimalarial [Lin et al., 1991] activities.In addition, due to their functional properties they arewidely used in color chemistry [Takagi et al., 1984] andphotostabilizers [Esscolastico et al., 1994]. Further-more, the aminonaphthoquinone moiety is a compo-nent of the molecular framework of several naturalproducts (e.g., rifamycins [Nagaoka and Kishi, 1981],kinamycins [Kumamoto et al., 2007; Omura et al.,1973], rifampicins [Lancini and Zanichelli, 1977], andstreptovaricins [Lanz et al., 1991]) and has been usedas a synthetic intermediate for several biologicallyimportant compounds [Kita et al., 1992].

Antiparasitic effects of naphthoquinones againstTrypanosoma cruzi [Uchiyama et al., 2006], Toxoplasmagondii, Leishmania sp. [Touraire et al., 1996], andPlasmodium sp. have been reported [Bullock et al.,1970]. In pneumonia caused by Pneumocystis carinii,atovaquone [Williams and Clark, 1998] (2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoqui-none) is approved as the drug of choice by the FDA.Antibacterial activity has been described for isoxazolylnaphthoquinones [Bogdanov et al., 1993], CribrarioneA [Naoe et al., 2003], and some hydroxyquinones andtheir metal complexes [Bakola-Christianopoulou et al.,1986]. The fungal toxicity of naphthoquinones, particu-larly against several species of Candida [Gafner et al.,1996; Gershon and Shanks, 1975; Gupta et al., 1981], andthe inhibitory activity of some naphthoquinones on HIV-1protease, has also been described [Brinworth and Fairlie,1995; Mazumder et al., 1996]. Anticancer, antiviral,antifilarial activity of naphthoquinones isolated frommedicinal plants has been reported [Lakshmy et al.,2009; Nisha et al., 2002; Rastogi and Dhawan, 1990].

Polyamines have macrofilaricidal activity and maythus represent a nucleus around which highly effectivecompounds can be synthesized [Kinnamon et al.,1999]. N-alkyl amines have been reported as thepharmacophore for antifilarials [Srivastava et al., 2000].The product formed from the simple reaction of amine

with various quinones has considerable scope forexploration. The search for new macrofilaricidal agentsis an important line of research because of thecomplexities of the filarial disease and the absence ofan effective adulticidal drug against the filarial parasite,making elimination of the disease from the affectedcommunity more challenging. The present work pro-vides a preliminary account of results obtained from thereaction of 1,4-naphthoquinone with primary andsecondary mono- and diamines and their screeningagainst bovine filarial worm Setaria digitata (Nematoda:Filariodea) for antifilarial activity.

MATERIALS AND METHODS

General

All reactions were carried out in a solvent mix ofdichloromethane (DCM)–ethanol (1:2) at room tem-perature. Purification of the compounds was selective,depending on the nature of the amino substitution, asthe mobility in TLC was very much influenced. Basedon the TLC pattern, the mobile phase for the flashchromatography was selected for each compound. Thediamine compounds were more polar; hence a polarsolvent like methanol was added to the mobile phaseduring its purification. For 1–4, purification wascarried out by flash chromatography using petroleumether/chloroform/ethyl acetate in a ratio of 4:10:1 as themobile phase. For 5, petroleum ether/chloroform/ethylacetate was used in a ratio of 4:3:1. For 6 and 7,petroleum ether/chloroform/ethyl acetate/methanol ina ratio of 2:5:1:2 was used, while for 8, petroleumether/chloroform/ethyl acetate/methanol in a ratio of2:5:1:1 was used. For 9, 10, and 11, petroleum ether/chloroform/ethyl acetate was used in ratios of 2:3:1,2:4:1, and 8:1:14, respectively.

Corrected melting points were taken in an A.Kruss Optronic GmbH melting point apparatus (modelKSP II). 1H-NMR and 13C-NMR spectra wererecorded on a 400-MHz Bruker NMR spectrometer.All chemical shifts are reported as d values (ppm)relative to TMS or the central peak of deuteriochloro-form (dC 77.0). The FT-IR spectrum was recorded inaccordance with the KBr disk technique on a ShimadzuFT-IR model 8300 (Shimadzu, Kyoto, Japan). Solventsand reagents were of analytical grade and columnchromatography was performed with silica gel 60(mesh size 230–400; E. Merck, Germany). All com-pounds were purified by subjecting them to 5-g IsoluteSPE Flash Si II columns connected to a FlashmasterPersonal flash chromatography system (ArgonautTechnologies Ltd., Hengoed, Mid Glamorgan, UnitedKingdom) with appropriate mobile phases. Thin layerchromatography (TLC) was performed with silica gel

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F254 (Merck, Germany) coated aluminum sheets.Synthesis was carried out in a combinatorial librarysynthesizer, Miniblock XT (Mettler-Toledo Bohdan).Absorbance measurements were made using Spectra-Max Plus (Molecular Devices, Sunnyvale, CA) withSoftmaxPro software.

HPLC analysis was carried out using a Thermo-Finnigan HPLC system (ThermoFinnigan, CA) com-posed of Spectra System P4000 solvent delivery system,Spectra System AS3000 autosampler, and Photodiodearray (PDA) detector SN4000. Output signals weremonitored via a ChromQuest 4.0 Chromatographyworkstation. A 3-mm Supelcosil ABZ plus analyticalcolumn (150� 4.6 mm) and a mobile phase combina-tion of acetonitrile-water (70:30) at a flow rate of1 ml min�1 at 401C and detection at 280 nm were usedfor the analysis. All compounds were synthesized bythe reaction between 1,4-naphthoquinone and sub-stituted amine by modifications of the method ofSalmon-Chemin et al. [2001].

Synthesis of 2-(Ethylamino)Naphthalene-1,4-Dione (1)

To a stirred solution of ethylamine (3.46 ml,6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone(367 mg, 2.32 mM) was added slowly in 2 ml ofdichloromethane (DCM). Stirring was continued for5–6 h at room temperature. The reaction was mon-itored by TLC. The color of the reaction mixture waschanged from yellow to deep black. Purification wascarried out by flash chromatography in silica columnwith petroleum ether/chloroform/ethyl acetate 4:10:1mobile phase to yield orange yellow crystals: yield184 mg (39.4%); mp 141.41C; TLC (petroleum ether/chloroform/ethyl acetate 4:10:1) Rf 0.78; HPLC Rt 2.11min. 13C-NMR (400 MHz, CDCl3): d 182.98, 181.92,147.87, 134.75, 133.68, 131.94, 130.50, 126.26, 126.18,100.749, 37.29, 13.52. 1H-NMR (400 MHz, CDCl3): d8.08 (d, 1H), 8.03 (d, 1H), 7.75 (t, 1H), 7.60 (t, 1H),5.86 (bs, 1H), 5.72 (s, 1H), 3.25 (m, 2H), 1.34 (t, 3H).IR (KBr) n 3354, 1676, 1643 cm�1.

Synthesis of 2-(Propylamino)Naphthalene-1,4-Dione (2)

To a stirred solution of propylamine (0.572 ml,6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone (367 mg,2.32 mM) was added slowly in 2 ml of dichloromethane(DCM). Stirring was continued for 5–6 h at roomtemperature. The reaction was monitored by TLC. Thecolor of the reaction mixture was changed from yellowto deep black. Purification was carried out by flashchromatography with a petroleum ether/chloroform/ethyl acetate 4:10:1 mobile phase to yield orangeyellow crystals: yield 254 mg (50.9%); mp 116.71C;

TLC (petroleum ether/chloroform/ethyl acetate 5 4:10:1)Rf 0.91; HPLC Rt 2.29 min. 13C-NMR (400 MHz,CDCl3): d 182.95, 181.97, 148.0, 134.75, 133.70,131.92, 130.52, 126.25, 126.18, 100.74, 44.26, 21.57,11.52. 1H-NMR (400 MHz, CDCl3): d 8.10 (d, 1H),8.01 (d, 1H), 7.70 (t, 1H), 7.63 (t, 1H), 5.92 (bs, 1H),5.73 (s, 1H), 3.16 (m, 2H), 1.72 (m, 2H), 1.03 (t, 3H).IR (KBr) n: 3246, 1683, 1631 cm�1.

Synthesis of 2-[(1-Methylethyl)Amino]Naphthalene-1,4-Dione (3)

To a stirred solution of isopropylamine (0.597 ml,6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone(367 mg, 2.32 mM) was added slowly in 2 ml of DCM.Stirring was continued for 5–6 h at room temperature.The reaction was monitored by TLC. The color of thereaction mixture was changed from yellow to deep black.Purification was carried out by flash chromatography ina silica column with a petroleum ether/chloroform/ethylacetate 4:10:1 mobile phase to yield orange yellowcrystals: yield 238 mg (47.7%); mp 87.71C; TLC (petro-leum ether/chloroform/ethyl acetate 4:10:1) Rf 0.89;HPLC Rt 2.27 min. 13C-NMR (400 MHz, CDCl3):d 182.96, 182.09, 146.84, 134.75, 133.64, 131.90, 130.53,126.27, 126.14, 100.89, 43.98, 21.76, 21.76. 1H-NMR(400 MHz, CDCl3): d 8.1 (d, 1H), 8.03 (d, 1H),7.72 (t, 1H), 7.61 (t, 1H), 5.79 (bs, 1H), 5.74 (s, 1H),3.66 (m, 1H), 1.31 (d, 6H). IR (KBr) n: 3263, 1678,1602 cm�1.

Synthesis of 2-(Butylamino)Naphthalene-1,4-Dione (4)

To a stirred solution of butylamine (0.684 ml,6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone(367 mg, 2.32 mM) was added slowly in 2 ml of DCM.Stirring was continued for 5–6 h at room temperature.The reaction was monitored by TLC. The color of thereaction mixture was changed from yellow to deepblack. Purification was carried out by flash chromato-graphy in silica column with a petroleum ether/chloro-form/ethyl acetate 4:10:1 mobile phase to yield orangeyellow crystals: yield 308 mg (57.9%); mp 125.01C; TLC(petroleum ether/chloroform/ethyl acetate 4:10:1) Rf

0.87; HPLC Rt 2.58 min. 13C-NMR (400 MHz, CDCl3):d 182.93, 181.96, 147.99, 134.75, 133.70, 131.91, 130.51,126.25, 126.17, 100.68, 42.29, 30.25, 20.21, 13.71.1H-NMR (400 MHz, CDCl3): d 8.11 (d, 1H), 8.05 (d,1H), 7.75 (t, 1H), 7.63 (t, 1H), 5.91 (bs, 1H), 5.73 (s, 1H),3.19 (m, 2H), 1.65 (n: 3352, 1674, 1629 cm�1.

Synthesis of 2-[(1-Methylpropyl)Amino]Naphthalene-1,4-Dione (5)

To a stirred solution of 2-aminobutane (0.699 ml,6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone (367 mg,

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2.32 mM) was added slowly in 2 ml of DCM. Stirring wascontinued for 5–6 h at room temperature. The reactionwas monitored by TLC. The color of the reaction mixturechanged from yellow to deep black. Purification wascarried out by flash chromatography in a silica column withpetroleum ether/chloroform/ethyl acetate 4:3:1 mobilephase to yild orange yellow crystals: yield 261 mg(49.1%); mp 60.21C; TLC (petroleum ether/chloroform/ethyl acetate 5 4:3:1) Rf 0.89; HPLC Rt 2.48 min. 13C-NMR (400 MHz, CDCl3): d 182.96, 182.11, 147.15,134.76, 133.68, 131.88, 130.54, 126.28, 126.14, 100.71,49.58, 28.84, 19.23, 10.31. 1H-NMR (400 MHz, CDCl3):d 8.05 (d, 1H), 8.04 (d, 1H), 7.71 (t, 1H), 7.61 (t, 1H),5.79 (bs, 1H), 5.74 (s, 1H), 3.52 (m, 1H), 1.64 (m, 2H),1.26 (d, 3H), 0.99 (t, 3H). IR (KBr) n: 3338, 1676,1627 cm�1.

Synthesis of 2-f[3-(Dimethylamino)Propyl]AminogNaphthalene-1,4-Dione (6)

To a stirred solution of 3-(dimethylamino)-1-propylamine (0.871 ml, 6.96 mM) in ethanol (4 ml),1,4-naphthoquinone (367 mg, 2.32 mM) was addedslowly in 2 ml of DCM. Stirring was continued for5–6 h at room temperature. The reaction was mon-itored by TLC. The color of the reaction mixturechanged from yellow to deep black. Purification wascarried out by flash chromatography in silica columnwith a petroleum ether/chloroform/ethyl acetate/methanol 2:5:1:2 mobile phase to yield orange yellowcrystals: yield 57 mg (9.5%); mp 71.31C; TLC (petro-leum ether/chloroform/ethyl acetate/methanol (2:5:1:2)Rf 0.37; HPLC Rt 0.96 min. 13C-NMR (400 MHz,CDCl3): d 182, 181, 147, 133.62, 132.50, 130.86,125.50, 125.20, 125.12, 99.39, 58, 51, 43.93, 43.93,23.71. 1H-NMR (400 MHz, CDCl3): d 8.016 (d, 1H),7.98 (d, 1H), 7.67 (t, 1H), 7.56 (t, 1H), 5.85 (bs, 1H),5.64 (s, 1H), 3.22 (t, 2H), 2.49 (t, 2H), 2.31 (s, 6H), 1.85(m, 2H). IR (KBr) n: 3348, 1680, 1597 cm�1.

Synthesis of 2-f[3-(Diethylamino)Propyl]AminogNaphthalene-1,4-Dione (7)

To a stirred solution of 3-(diethylamino)-1-pro-pylamine (1.09 ml, 6.96 mM) 3 in ethanol (4 ml), 1,4-naphthoquinone (367 mg, 2.32 mM) was added slowlyin 2 ml of DCM. Stirring was continued for 5–6 h atroom temperature. The reaction was monitored byTLC. The color of the reaction mixture was changedfrom yellow to deep black. Purification was carried outby flash chromatography in silica column with petro-leum ether/chloroform/ethyl acetate/methanol 2:5:1:2mobile phase to yield a dark red liquid: yield 306 mg(46.1%); TLC (petroleum ether/chloroform/ethylacetate/methanol) 2:5:1:2 Rf 0.52; HPLC Rt 0.98 min.

13C-NMR (400 MHz, CDCl3): d 182.87, 180, 148.74,134.50, 133.92, 131.71, 130.77, 126.16, 126.08, 100.06,52.25, 46.87, 46.87, 42.98, 24.51, 11.53, 11.53. 1H-NMR (400 MHz, CDCl3): d 8.02 (d, 1H), 7.92 (d, 1H),7.71 (t, 1H), 7.61 (t, 1H), 5.85 (bs, 1H), 5.68 (s, 1H),3.29 (t, 2H), 2.60 (m, 4H), 1.85 (t, 2H), 1.28 (m, 2H),1.09 (t, 6H). IR (KBr) n: 3257, 1685, 1631 cm�1.

Synthesis of 2-f[3-(Dibutylamino)Propyl]AminogNaphthalene-1,4-Dione (8)

To a stirred solution of 3-(dibutylamino)-1-propyl-amine (1.56 ml, 6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone (367 mg, 2.32 mM) was added slowlyin 2 ml of DCM. Stirring was continued for 5–6 h atroom temperature. The reaction was monitored by TLC.The color of the reaction mixture was changed fromyellow to deep black. Purification was carried out byflash chromatography in silica column with petroleumether/chloroform/ethyl acetate/methanol 2:5:1:1 mobilephase to yield orange yellow crystals: 287 mg (36.1%);mp 76.71C; TLC (petroleum ether/chloroform/ethylacetate/methanol) 5 2: 5: 1: 1 Rf 0.76; HPLC Rt0.99 min. 13C-NMR (400 MHz, CDCl3): d 181.86,180.89, 147.66, 133.47, 132.88, 130.70, 129.73, 125.15,125.06, 99.11, 53.81, 52.22, 52.22, 41.79, 27.89, 27.89,23.85, 19.73, 19.73, 13.06, 13.06. 1H-NMR (400 MHz,CDCl3): d 8.04 (d, 1H), 7.98 (d, 1H), 7.64 (t, 1H), 7.52(t, 1H), 5.82 (bs, 1H), 5.61(s, 1H), 3.19 (t, 2H), 2.50(t, 4H), 2.49 (t, 2H), 1.76 (m, 2H), 1.41 (m, 4H),1.26 (m, 4H), 0.84 (t, 6H). IR (KBr) n: 3257, 1683,1629 cm�1.

Synthesis of 2-[(1,3-Dimethylbutyl)Amino]Naphthalene-1,4-Dione (9)

To a stirred solution of 1,3-dimethylbutylamine(0.977 ml, 6.96 mM) in ethanol (4 ml), 1,4-naphthoqui-none (367 mg, 2.32 mM) was added slowly in 2 ml ofDCM. Stirring was continued for 5–6 h at roomtemperature. The reaction was monitored by TLC.The color of the reaction mixture changed from yellowto deep black. Purification was carried out by flashchromatography in silica column with a petroleumether/chloroform/ethyl acetate 2:3:1 mobile phase toyield orange yellow crystals: 254 mg (42.5%); mp81.51C; TLC (petroleum ether: chloroform: ethylacetate): 2:3:1 Rf 0.96; HPLC Rt 3.16 min. 13C-NMR(400 MHz, CDCl3): d 181.93, 181.11, 146.01, 133.74,132.68, 130.83, 129.52, 125.25, 125.12, 99.42, 45.31,44.52, 24.03, 21.55, 21.55, 18.90. 1H-NMR (400 MHz,CDCl3): d 8.04 (d, 1H), 7.96 (d, 1H), 7.66 (t, 1H), 7.55(t,1H), 5.95 (bs, 1H), 5.77 (s, 1H), 3.48 (m, 1H), 1.49(m, 1H), 1.34 (m, 2H), 1.17 (d, 3H), 0.87 (dd, 6H). IR(KBr) n: 3315, 1678, 1618 cm�1.

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Synthesis of 2-f[2-(4-Chlorophenyl)Ethyl]AminogNaphthalene-1,4-Dione (10)

To a stirred solution of 2-(4-chlorophenyl)-ethylamine (0.97 ml, 6.96 mM) in ethanol (4 ml), 1,4-naphthoquinone (367 mg, 2.32 mM) was added slowlyin 2 ml of DCM. Stirring was continued for 5–6 h atroom temperature. The reaction was monitored byTLC. The color of the reaction mixture changed fromyellow to deep black. Purification was carried out byflash chromatography in silica column with a petroleumether: chloroform: ethyl acetate 2:4:1 mobile phase toyield orange yellow crystals: 549 mg (75.9%); mp171.01C; TLC (petroleum ether/chloroform/ethyl acet-ate) 2:4:1 Rf 0.91; HPLC Rt 3.03 min. 13C-NMR(400 MHz, CDCl3): d 181.98, 180.72, 146.60, 135.24,133.80,132.53, 131.83, 131.05, 129.44, 129.44, 128.94,128.01, 128.01, 125.29, 125.21, 100.11, 42.43, 32.63.1H-NMR (400 MHz, CDCl3): d 8.01 (d, 1H), 7.97(d, 1H), 7.68 (t, 1H), 7.56 (t, 1H), 7.24 (d, 2H), 7.09(d, 2H), 5.84 (bs, 1H), 5.70 (s, 1H), 3.38 (t, 2H), 2.89(t, 2H). IR (KBr) n: 3340, 1670, 1622 cm�1.

Synthesis of 2-(4-Methylpiperazin-1-yl)Naphthalene-1,4-Dione (11)

To a stirred solution of 1-methylpiperazine(0.772 ml, 6.96 mM) in ethanol (4 ml), 1,4-naphthoqui-none (367 mg, 2.32 mM) was added slowly in 2 ml ofDCM. Stirring was continued for 5–6 h at roomtemperature. The reaction was monitored by TLC.The color of the reaction mixture changed from yellowto deep black. Purification was carried out by flashchromatography in silica column with petroleum ether:chloroform: ethyl acetate 8:1:14 mobile phase to yieldorange yellow crystals: 217 mg (36.5%); mp 131.61C;TLC (petroleum ether: chloroform: ethyl acetate):8:1:14 Rf 0.46; HPLC Rt 1.25 min. 13C-NMR(400 MHz, CDCl3): d 183.69, 183.07, 153.75, 133.87,132.82, 132.48, 132.36, 126.68, 125.56, 111.80, 54.57,54.57, 48.81, 48.81, 45.97. 1H-NMR (400 MHz,CDCl3): d 8.07 (d, 1H), 7.99 (d, 1H), 7.71 (t, 1H),7.64 (t, 1H), 6.03 (s, 1H), 3.54 (t, 4H), 2.59 (t, 4H), 2.35(s, 3H).IR (KBr) n: 3354, 1676, 1643 cm�1.

In Vitro Screening for AntifilarialActivity Against S. digitata

Adults of the cattle filarial parasite of S. digitatawere used to screen macrofilaricidal activity. AdultS. digitata worms collected from the peritoneal cavityof freshly slaughtered cattle were washed with normalsaline (0.85%) to free them of extraneous material andtransferred to Dulbecco’s modified Eagle medium(DMEM) containing 0.01% streptopenicillin andsupplemented with 10% heat-inactivated fetal calf

serum (FCS) and were used within an hour as reportedearlier [Lakshmy et al., 2009; Nisha et al., 2002, 2008].

Worm motility assayStock drug solutions were prepared in ethanol or

dimethylsulfoxide (DMSO) depending upon the solu-bility of the compound at 1 mg ml�1. After use thestocks were stored at �201C. For the assays, thecompounds were further diluted to the appropriateconcentration using complete assay medium. TheDMSO/ethanol concentration in the medium with thehighest compound concentration was below 1%.Preliminary screening was done at a concentration of0.1 mg ml�1. A simultaneous control with an equalvolume of the vehicle in the DMEM was included. Twoadult female S. digitata worms were introduced intoeach Petri dish with three replicates for both test andcontrol. Worms were incubated at 371C for 24 and 48 hin an incubator. After the incubation period, thenumber of immobilized worms was counted. Immedi-ately after counting, the worms were washed twice withfresh medium and transferred to another set of Petridishes containing fresh medium, without the testsolution, to assess whether any of the immobile wormsregained motility. If the worms did not revive, thecondition was considered irreversible and the concen-tration lethal. Each experiment was repeated twice.

MTT-formazan colorimetric assayfor viability of worms

Substituted naphthoquinones were furtherscreened for viability of adult S. digitata through aMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide) reduction assay [Comley et al., 1989].Yellow MTT is reduced to purple formazan bymitochondrial enzymes present in living cells. Thisreduction takes place only when mitochondrial reduc-tase enzymes are active, and therefore conversion isused as a measure of viable (living) cells. During theassay the formazan formed is extracted with DMSOand is quantified. As the values of absorption correlatewith formazan formation, worm viability was estimatedas percentage inhibition in formazan formation relativeto control worms. Adult female worms were used forthis assay. After the exposure of the worms tosubstituted naphthoquinones (0.1 mg/ml) in DMEMat a 24- and 48-h incubation period, the parasites werefurther incubated for 30 min individually in phosphate-buffered saline (pH 7.4, 0.5 ml) containing MTT(0.25 mg ml�1). A control was set up with untreatedadult females but exposed to ethanol or DMSO asdescribed above. All the test concentrations andcontrols were in 12 replicates. At the end of the MTTincubation, worms were transferred to a microtiter

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plate containing 400 ml of spectroscopic-grade DMSOand equilibrated at room temperature for 1 h, withoccasional gentle shaking to extract the color devel-oped. The absorbance of the resulting formazansolution was then determined at 492 nm in a microplatespectrophotometer relative to DMSO blank. As thevalues of absorption correlate with formazan formation,worm viability was estimated as percentage inhibition informazan formation relative to control worms. Com-pounds showing greater than 50% inhibition in formazanformation with respect to control at 0.1 mg/ml wereconsidered effective and were further screened at lowerconcentrations to generate an ED50 (dose that gives a50% response, determined by a sigmoid plot obtained byplotting the logarithm of the dose on the x-axis and thepercentage response on the y-axis. ED50 was calculatedusing the ED50 plus program (version 1.0) developed byM.H. Vargas [www.iner.gob.mx/docs/ed50.htm] and thestructure-activity relationship was studied.

RESULTS

The results of preliminary screening of com-pounds 1–11 at 0.1 mg/ml for antifilarial activity in vitroagainst the adult bovine filarial worm S. digitata byworm motility assay and visual observation are shownin Table 1. Worms treated with 1 and 4 treated showedsluggish movement after 24-h incubation and thosetreated with 2, 3, 5, 9, and 11 were either completely

paralyzed or dead. Worms treated with 6, 7, 8, and 10were highly active and not different from control worms.After 48-h incubation, all the treated worms wereimmobile except for worms treated with 6, 7, 8, and 10.

The results of in vitro macrofilaricidal screeningat 0.1 mg/ml in the MTT reduction assay are also shownin Table 1. Seven of the 11 compounds investigatedfor antifilarial activity had promising macrofilaricidalactivity against the adult S. digitata with greater than50% inhibition of formazan formation. No macrofilar-icidal activity was observed for 6, 7, 8, and 10. Themacrofilaricidal activity in terms of ED50 values for theseven effective compounds are shown in Figure 1. 9and 11 had maximal macrofilaricidal activity with ED50

values of 2.6 and 3.5mM at 24-h incubation and 0.91and 1.2mM at 48-h incubation, respectively, followedby 5, 3, 2, 4, and 1. The observed retention times inHPLC analysis were 0.96, 0.98, and 0.99 min for 6, 7,and 8, respectively. For 1–5 this ranged from 2.11 to2.58 min. For 9, 10, and 11 the respective values were3.16, 3.03, and 1.25 min.

DISCUSSION

As no vaccine is available and vector control doesnot result in sustained effect except in a small numberof locations, the strategy of a Global Program toeliminate lymphatic filariasis is to interrupt transmission

TABLE 1. In Vitro Macrofilaricidal Activity of Substituted Amino Naphthoquinones Against Adult Setaria digitata Worms at 0.1 mg ml�1

Substitution (R)

O

O

R% MTT reduction7SE (n 5 12) Worm motility

Compound 24 h 48 h 24 h 48 h

1 �NHCH2CH3 58.3474.34 84.6971.67 Sluggish Immotile2 �NHCH2CH2CH3 87.8171.43 91.2570.71 Immotile Immotile3 �NHCH (CH3)2 84.070.77 85.1673.04 Immotile Immotile4 �NHCH2CH2CH2CH3 45.6876.19 74.1074.94 Sluggish Immotile5 �NHCH (CH3)CH2CH3 92.9970.39 93.7270.34 Immotile Immotile6 �NHCH2CH2CH2N(CH3)2 13.9870.90 44.4270.71 Active Active7 �NHCH2CH2CH2N(CH2CH3)2 38.5272.70 47.8973.38 Active Active8 �NHCH2CH2CH2N(CH2CH2CH2CH3)2 12.3674.26 49.0471.34 Active Active9 �NHCH(CH3)CH2CH(CH3)2 90.3971.52 92.471.04 Immotile Immotile10

Cl-NHCH2CH2

27.8572.27 39.7872.57 Active Active

11

N-N CH3

88.9070.76 94.1170.48 Immotile Immotile

SE, standard error; n, number of worms.

193SUBSTITUTED NAPHTHOQUINONES AS MACROFILARICIDES

Drug Dev. Res.

through annual mass drug administration using acombination of two drugs, either DEC plus ALB orIVM plus ALB [www.filariasis.org/resources/prevent_eliminatelf.htm]. While the programs have been verysuccessful in general there are drawbacks, such ascoverage being too low within a population, targeting astage (the microfilariae) that does not induce pathologyin LF and thus lowers compliance and raises thepotential development of drug resistance the firstindications of which have been observed in onchocer-ciasis [Awadzi et al., 2004]. Therefore, there is a criticalneed for the development of efficient, complementarychemotherapeutic approaches that produce a long-lasting reduction of the pathology-inducing wormstages, e.g., microfilaria in onchocerciasis and adultworms in LF or to a macrofilaricidal effect. Consider-able efforts have been focused on developing aneffective and safe drug that could kill or permanentlysterilize adult filarial worms and include DEC analo-gues, benzimidazoles, thiazoles, selenazoles, pyrazoles,triazines, nitroimidazoles, indoles, phenylamines, ben-zazoles, phenylimidazo pyridine, coumarins, epoxy andethyne sulfonamides, cyanine dyes, thiazine, acylhy-drazones, semicarbazones, phenylguanidines, ami-noalkanes, organophosphorus compounds, quinolines,antimycine analogues, moxidectin, organometallic com-plexes, antibiotics, and lymphotropic agents [Nisha andKalyanasundaram, 2007]. Although many of thesecompounds showed promising activity, none reachedthe final stage as an adulticidal drug either due to

toxicity or poor absorption and other practical reasons.Many plant based compounds are also being studiedfor macrofilaricidal activity [Chatterjee et al., 1992;Nisha et al., 2002, 2007, 2008; Singh et al., 1994].

Seven out of 11 compounds investigated forantifilarial activity showed promising macrofilaricidalactivity against the adult S. digitata with greater than50% inhibition of formazan formation at 0.1 mg/ml. Nomacrofilaricidal activity was observed for 6, 7, 8, and10 by motility assay and MTT reduction assay whentested at 0.1 mg ml�1. ED50 values were determinedfor the seven active compounds.

The importance of lipophilicity for pharmacologicaland toxicological potency of xenobiotics has long beenrecognized. The reference lipophilicity scale is defined bythe logarithm of partition coefficient, log P, determined inthe octanol–water partition system. The tediousness ofdeterminations and limited inter-laboratory reproducibilityof log P, on one hand, and the observations of linearrelationship between log P and chromatographic retentionparameters, on the other, gave rise to the substitutionof the former by the readily available chromatographicdata [Nasal et al., 2003]. Here HPLC retention time hasbeen considered in comparing the lipophilicity of thecompound.

Comparison of macrofilaricidal activity of theeffective compounds showed that 9 is promising withan ED50 value of 2.6mM at 24-h incubation and0.91mM at 48-h incubation followed by 11, 5, 3, 2, 4,and 1; 6, 7, and 8 were ineffective in killing adultworms and due to the presence of diamino groupsresulting in unfavorable polarity or low lipophilicity(evidenced by the reduced retention times (Rts) 0.96,0.98, and 0.99 min, respectively, in HPLC analysis asshown in Fig. 2) preventing the membrane transport orthe presence of additional methylene groups leading tobulkiness that inhibits binding to targets. Incorporation

Fig. 2. Comparison of HPLC retention times of compounds 1–11.

Fig. 1. In vitro macrofilaricidal activity of compounds againstS. digitata worms.

194 MATHEW ET AL.

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of a halogen improves lipophilicity [Thomas, 2000].In the case of 10, however, the 4-chlorophenylsubstitution reduced activity due to its bulkinessand high lipophilicity (a higher HPLC Rt of3.03 min). In 1–5, where alkyl chains with and withoutbranching were substituted, activity was dependent onnumber of carbons in the chain. When the substitutionchanged from ethyl (1) to propyl (2) activity wasincreased 8-fold, while with isopropyl (3) branchingenhanced activity by 83-fold. Lengthening the chainbeyond certain limit results in a rapid decrease inactivity [Thomas, 2000]. In the case of a 4 carbon chain(4) activity was reduced compared with 2 but increasedconsiderably with branching 5, i.e., 96 times that of 1.The lipophilicity of compounds showed an increasingtrend with increasing chain length, while a reductionwith branching. The HPLC Rts of ethylamine 1 was2.11 min and propylamino 2 and butyl amino 4 were2.29 and 2.58 min, respectively, where the increasingtrend in Rt in lipophilicity was observed. The Rt valuesof isopropyl 3 and isobutyl 5 were 2.27 and 2.48 min,respectively, showing a reduction in Rt with branchingcompared with unbranched compounds. Heterocyclicaromatic systems can also introduce extra functionalgroups that may also affect the potency and activity[Thomas, 2000]. Substitution with a N-methylpiper-azine moiety (11) showed a good enhancement inmacrofilaricidal activity, 248 times that of 1. Although11 is a diamino compound, the observed activity maybe due to the presence of a closed ring with an extratertiary amine moiety compared with 6, 7, and 8. Thelipophilicity of 11 is also higher than that of 6, 7, and 8as seen by the higher retention time of 1.25 min inHPLC analysis. 9 was 327 times more macrofilaricidalthan 1, which has an ethylamine substitution, 46 timesmore effective than 4, which has a butyl aminosubstitution and 3.4 times effective than 5, whichhas an isobutylamino substitution. In the case of 9, the1,3-dimethyl substitution in the butylamino groupfavors a higher lipophilicity with potentially betterbinding to the active site resulting in higher macro-filaricidal activity.

New antifilarial agents effective against adultfilarial worms are required due to a dearth of newchemotherapeutic agents against filarial infections[Townson et al., 2007]. Of the 11 NPQs described inthe present study, 7 showed macrofilaricidal activitywith 9 (2-[(1,3-dimethylbutyl) amino] naphthalene-1,4-dione) and 11 2-(4-methylpiperazin-1-yl) naphtha-lene-1,4-dione) showing maximum activity with ED50

values of 0.91 and 1.2mM, respectively, at 48-hincubation. Further detailed study of these effectivecompounds could be promising for the development ofan effective antifilarial drug.

ACKNOWLEDGMENTS

Dr. Nisha Mathew expresses sincere gratitude tothe Department of Science and Technology (DST),Government of India for funding (grant SR/SO/HS-83/2005) to conduct this study. The authors are grateful toDr. P. Jambulingam, Director, Vector Control ResearchCentre, Pondicherry, for his encouragement during thestudy. The help rendered by Chemistry Department,Pondicherry University is gratefully acknowledged. Thevaluable suggestions by Mr. A. Elango and technicalassistance by Mrs. R. Anilakumari and Mr. S. Srinivasanare also acknowledged.

REFERENCES

Acheson RM, Sansom BF. 1955. The reaction between quinonesand anthranilic acids. J Chem Soc 4440–4443.

Awadzi K, Attah SK, Addy ET, Opoku NO, Quartey BT, Lazdins-Helds JK, Ahmed K, Boatin BA, Boakye DA, Edwards G. 2004.Thirty-month follow-up of sub-optimal responders to multipletreatments with ivermectin, in two onchocerciasis-endemic foci inGhana. Ann Trop Med Parasitol 98:359–370.

Bakola-Christianopoulou MN, Ecateriniadou LB, Sarris KJ. 1986.Evaluation of the antimicrobial activity of a new series of hydroxy-quinone chelates of some transition metals. Eur J Med Chem 21:385–390.

Bayen S, Barooah N, Sarma RJ, Sen T, Karmakar A, Baruah JB.2007. Synthesis, structure and electrochemical properties of 2,5-bis(alkyl/arylamino) 1,4-benzoquinones and 2-aryl 1,4-naphtho-quinones. Dyes Pigment 75:770–775.

Bogdanov PM, Albesa I, Sperandeo NR, De Bertorello MM. 1993.Actividad antibacteriana in vitro de isoxazolilnaftoquinonas. RevArg Microbiol 25:119–128.

Brinworth RI, Fairlie DP. 1995. Hydroxyquinones are competitivenon-peptide inhibitors of HIV-1 proteinase. Biochim BiophysActa 1253:5–8.

Bullock FJ, Tweedie JF, McRitchie DD, Tucker MA. 1970. Anti-protozoal quinones: 2-amino-1,4-naphthoquinoneimines potentialantimalarials. Eur J Med Chem 13:550–552.

Chatterjee RK, Fatma N, Murthy PK, Sinha P, Kulshrestha DK,Dhawan BN. 1992. Macrofilaricidal activity of the stembark ofStreblus asper and its major active constituents. Drug Dev Res 26:67–78.

Comley JCW, Rees MJ, Turner CH, Jenkins DC. 1989. Colorimetricquantitation of filarial viability. Int J Parasitol 19:77–83.

Elslager EF, Werbel ML, Worth DF. 1970. Synthetic schistosomi-cides. XIV. 1,4-naphthoquinone mono(O-acyloximes), 4-amino-1,2-naphthoquinones, 2-amino-3-chloro-1,4-naphthoquinones, andother naphthoquinones. J Med Chem 13:104–109.

Esscolastico C, Santa Maria MD, Claramunt RM, Jimeno ML,Alkorta I, Foces-Foces C, Cano FH, Elguero J. 1994. Imidazoleand benzimidazole addition to quinines. Formation of meso andd,l Isomers and crystal structure of the d,l isomer of 2,3-bis(benzimidazol-1’-yl)-1,4-dihydroxybenzene. Tetrahedron 50:12489–12510.

Gafner S, Wolfender JL, Nianga M, Stoceckli-Evans H,Hostettmann K. 1996. Antifungal and antibacterial naphthoqui-nones from Newbouldia leavis roots. Phytochemistry 42:1315–1320.

195SUBSTITUTED NAPHTHOQUINONES AS MACROFILARICIDES

Drug Dev. Res.

Gershon H, Shanks L. 1975. Fungitoxicity of 1,4-naphthoquinonesto Candida albicans and Trichophyton menthagrophytes. Can JMicrobiol 21:1317–1320.

Gupta HP, Singh R, Srivastava OP, Khana JM, Mathur IS,Gupta SK. 1981. Anti-fungal property of some substitutednaphthoquinones, anthraquinone and imidazolquinones. IndianJ Microbiol 21:57–59.

Hakobu S, Yojiro H, Masazumi S, Yoshio K, Seiji T, Toru H. 1979.Naphthoquinone herbicides. Tokyo, Koho: Jpn Kokai. p 6.

Kallmayer HJ, Tappe C. 1987. Quinone-amine reactions. Part 23:4-Amino-1,2-naphthoquinone derivatives of the desipramine-typepsychodrugs. Pharm Acta Helv 62:2–6.

Kinnamon KE, Engle RR, Poon BT, Ellis WY, McCall JW,Dzimianski MT. 1999. Polyamines: agents with macrofilaricidalactivity. Ann Trop Med Parasitol 93:851–858.

Kita Y, Tohma H, Inagaki M, Hatanaka K, Yakura T. 1992. Totalsynthesis of discorhabdin C: a general aza spiro dienone formationfrom O-silylated phenol derivatives using a hypervalent iodinereagent. J Am Chem Soc 114:2175–2180.

Konoshima T, Kozuka N, Koyama J, Okatani T, Tagahara K, Tokuda H.1989. Studies on inhibitors of skin tumor promotion. VI. Inhibitoryeffects of quinones on Epstein-Barr virus activation. J Nat Prod 52:987–995.

Kumamoto T, Kitani Y, Tsuchiya H, Yamaguchi K, Seki H, Ishikawa T.2007. Total synthesis of (7)-methyl-kinamycin C. Tetrahedron 63:5189–5199.

Lakshmy S, Nisha M, Kalyanasundaram M. 2009. In vitro antifilarialactivity of Glutathione S transferase inhibitors. Parasitol Res 105:1179–1182.

Lancini G, Zanichelli W. 1977. Antibiotics. Perlman D, editor.New York: Academic Press. p 531–600.

Lanz T, Tropf S, Marner F-J, Schroder J, Schroder G. 1991. The roleof cysteines in polyketide synthases. Site-directed mutagenesis ofresveratrol and chalcone synthases, two key enzymes in differentplant-specific pathways. J Biol Chem 266:9971–9976.

Lin T-S, Xu S-P, Zhu L-Y, Cosby L, Sartonelli A. 1989. Synthesisof 2,3-diaziridinyl-1,4-naphthoquinone sulfonate derivatives aspotential antineoplastic agents. J Med Chem 32:1467–1471.

Lin T-S, Xu S-P, Zhu L-Y, Divo A, Sartonelli A. 1991. Synthesis andantimalarial activity of 2-aziridinyl- and 2,3-bis(aziridinyl)-1,4-naphthoquinonyl sulfonate and acylate derivatives. J Med Chem34:1634–1639.

Mazumder A, Shaomeng W, Neamati N, Nicklaus M, Sunder S,Chen J, Milne G, Rice W, Pommier Y. 1996. Antiretroviral agentsas inhibitors of both human immunodeficiency virus type 1integrase and protease. J Med Chem 39:2472–2481.

Nagaoka H, Kishi Y. 1981. Further synthetic studies on rifamycins.Tetrahedron 37:3873–3888.

Naoe A, Ishibashi M, Yamamoto Y. 2003. Cribrarione A, a newantimicrobial naphthoquinone pigment from a myxomyceteCribraria purpurea. Tetrahedron 59:3433–3435.

Nasal A, Siluk D, Kaliszan R. 2003. Chromatographic retentionparameters in medicinal chemistry and molecular pharmacology.Curr Med Chem 10:381–426.

Nisha M, Kalyanasundaram M. 2007. Antifilarial agents. ExpertOpin Ther Patents 17:767–789.

Nisha M, Paily KP, Vanamail P, Abidha, Kalyanasundaram M,Balaraman K. 2002. Macrofilaricidal activity of the plantplumbago indica/rosea in vitro. Drug Dev Res 56:33–39.

Nisha M, Kalyanasundaram M, Paily KP, Abidha, Vanamail P,Balaraman K. 2007. In vitro screening of medicinal plant extractsfor macrofilaricidal activity. Parasitol Res 100:575–579.

Nisha M, Misra-Bhattacharya S, Vanamail P, Kalyanasundaram M.2008. Antifilarial lead molecules isolated from Trachyspermumammi. Molecules 13:2156–2168.

Omura S, Nakagawa A, Yamada H, Hata T, Furusaki A, Watanabe T.1973. Structures and biological properties of Kinamycin A, B, C,and D. Chem Pharm Bull 21:931–940.

Rastogi RP, Dhawan BN. 1990. Anticancer and antiviral activities inIndian medicinal plants: a review. Drug Dev Res 19:1–12.

Roushdi IM, Mikhail AA, Ahmed IC. 1976. Synthesis of some quinonesof potential therapeutic interest. Acta Pharm Jugosl 26:287–294.

Salmon-Chemin L, Buisine E, Yardley V, Kohler S, Marie-Ange D,Landry V, Sergheraert C, Croft SL, Krauth-Siegel L, Davioud-Charvet E. 2001. 2- and 3-substituted 1,4-naphthoquinonederivatives as subversive substrates of trypanothione reductaseand lipoamide dehydrogenase from Trypanosoma cruzi: synthesisand correlation between redox cycling activities and in vitrocytotoxicity. J Med Chem 44:548–565.

Singh SN, Chatterjee RK, Srivastava AK. 1994. Effect of glycosidesof Streblus asper on motility, glucose uptake, and certain enzymesof carbohydrate metabolism of Setaria cervi. Drug Dev Res 32:191–195.

Srivastava SK, Chauhan PMS, Bhaduri AP, Murthy PK,Chatterjee RK. 2000. Secondary amines as new pharmacophoresas for macrofilaricidal drug. Bioorg Med Chem Lett 10:313–314.

Takagi K, Matsuoka M, Hamano K, Kitao T. 1984. Amination of 5,8-dihydroxy-1,4-naphthoquinone. Dyes Pigments 5:241–251.

Thomas G. 2000. Medicinal chemistry. An introduction. New York:John Wiley & Sons.

Townson S, Ramirez R, Fakorede F, Mouries MW, Nwaka S. 2007.Challenges in drug discovery for novel antifilarials. Expert OpinDrug Discov 2(Suppl 1):S63–S73.

Touraire C, Caupole R, Payard M, Commenges G, Bessieres MH,Bories C, Loiseal PM, Gayral P. 1996. Synthesis and protozoocidalactivities of quinines. Eur J Med Chem 31:507–511.

Tucker JHR, Collinson SR. 2002. Recent developments in theredox-switched binding of organic compounds. Chem So Rev 31:147–156.

Uchiyama N, Kiuchi F, Ito M, Honda G, Takeda Y, Khodzhimatov OK,Ashurmetov OA. 2006. Trypanocidal constituents of Dracocephalumkomarovi. Tetrahedron 62:4355–4359.

Williams DR, Clark MP. 1998. Synthesis of Atovaquone. Tetrahe-dron Lett 39:7629–7632.

William RA, Anja KL. 2001. Herbicide-induced oxidative stress inphotosystem II. Trends Biochem Sci 26:648–653.

196 MATHEW ET AL.

Drug Dev. Res.