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Current Drug Targets, 2012, 13, 000-000 1

From Body Art to Anticancer Activities: Perspectives on Medicinal Properties of Henna

Rohan Pradhan1,, Prasad Dandawate1,, Alok Vyas1, Subhash Padhye1,2,*, Bernhard Biersack4, Rainer Schobert4, Aamir Ahmad2 and Fazlul H. Sarkar2,3,*
ISTRA, Department of Chemistry, Abeda Inamdar Senior College, University of Pune, Pune 411001, India; Department of Pathology and 3Oncology, Barbara Ann Karmanos Cancer Institute and Wayne State University School of Medicine, Detroit, MI 48201, USA; 4Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30, 95440, Bayreuth, Germany
2 1

Abstract: Nature has been a rich source of therapeutic agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources based on the uses of these plants in traditional medicine. Henna is one such plant commonly known as Persian Henna or Lawsonia inermis, a bushy, flowering tree, commonly found in Australia, Asia and along the Mediterranean coasts of Africa. Paste made from the leaves of Henna plant has been used since the Bronze Age to dye skin, hairs and fingernails especially at the times of festivals. In recent times henna paste has been used for body art paintings and designs in western countries. Despite such widespread use in dyeing and body art painting, Henna extracts and constituents possess numerous biological activities including antioxidant, anti-inflammatory, antibacterial and anticancer activities. The active coloring and biologically active principle of Henna is found to be Lawsone (2hydroxy-1, 4-naphthoquinone) which can serve as a starting building block for synthesizing large number of therapeutically useful compounds including Atovaquone, Lapechol and Dichloroallyl lawsone which have been shown to possess potent anticancer activities. Some other analogs of Lawsone have been found to exhibit other beneficial biological properties such as antioxidant, anti-inflammatory, antitubercular and antimalarial. The ability of Lawsone to undergo the redox cycling and chelation of trace metal ions has been thought to be partially responsible for some of its biological activities. Despite such diverse biological properties and potent anticancer activities the compound has remained largely unexplored and hence in the present review we have summarized the chemistry and biological activities of Lawsone along with its analogs and metal complexes.

Keywords: Anticancer, antioxidant, anti-inflammatory, chemical reactivity, henna, lawsone, metal complexes. INTRODUCTION Henna is the Indian name for the plant commonly known as Persian Henna or Lawsonia inermis, a bushy, flowering tree, which was originally found in Australia, Asia and along the Mediterranean coasts of Africa. Intricate designs are usually painted on new brides and as part of the marriage ceremony in India using the paste made from Henna plant. According to Indian mythology the paste was worn by Parvati, Lord Shiva's wife, to charm her difficult-to-please husband. In Morocco henna is considered magical and is used in the transformation of man and woman into husband and wife. Henna ceremonial painting is considered sacred work and a form of worship in many diverse cultures in India, Africa, and the Middle East. More recently this form of body art has become popular in western countries including celebrities,
*Address correspondence to these authors at the ISTRA, Department of Chemistry, Abeda Inamdar Snior College, University of Pune, Pune 411001 India; Tel/Fax: ???????????????; E-mail: sbpadhye@hotmail.com and Departments of Pathology & Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, 740 HWCRC Bldg, 4100 John R. Street, Detroit, MI 48201 USA; Tel: 313-576-8327; Fax: 313-576-8389; E-mail: fsarkar@med.wayne.edu Contributed Equally. 1389-4501/12 $58.00+.00

which has helped make it hip and bring it into the mainstream of cosmetic clinics and beauty parlors [1]. Since Bronze Age henna has been used widely in different cultures across the world for ceremonial and celebratory purposes [2]. The earliest recorded evidence of the cosmetic use of henna is from ancient Egypt. It was common practice among the Egyptians to dye their fingernails a reddish hue with henna, and was considered ill-mannered not to do so, while traces of henna have been found on the hands of Egyptian mummies up to five thousand years old [1]. The prophet Mohammed is said to have dyed his beard with Henna [3]. The use of different words for henna in ancient languages implies that henna had more than one point of discovery and origin. Relatively little is known about the medicinal uses of henna although the extract of the plant has been prescribed in Indian System of Medicine, viz. Ayurved, mainly for skin and hair diseases. The plant is also mentioned in the treatment of intestinal infections, arthritis and leprosy in combination with other plants. In Unani medicine it is prescribed for treating ulcers, wounds, burns, headaches, muscular rigidity, menorrhagia, and as blood purifier respectively [4, 5]. Lawsone (2-hydroxy-1, 4-naphthoquinone) is the major constituent of henna, which is known for its staining ability Fig.
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(1) and many biological activities. In current review, we have summarized the biological activities of lawsone and its structural analogs along with a brief account of its chemical reactivity, reactions and metal complexing ability. PLANT SOURCES OF LAWSONE Henna is a tall shrub with about 8 to 9 feet height. It is glabrous, multi-branched with spine tipped branchlets. Leaves are placed opposite, glabrous, sub-sessile, elliptical, and broadly lanceolate, acuminate, having depressed veins on the dorsal surface. Henna flowers have four sepals and a calyx tube with spread lobes. Petals are obviate, white or red stamens inserted in pairs on the rim of the calyx tube. Ovary is four-celled, style up to 5 mm long and erect. Fruits are small, brownish capsules, with 3249 seeds per fruit, and open irregularly into four splits Fig. (1) [6]. Although the plant is native to tropical and sub-tropical regions of Africa, southern Asia, and northern Australasia in semi-arid zones its indigenous zone is the tropical savannah and tropical arid zone in which highest dye content is produced in the plant. During the onset of rains, the plant grows rapidly; putting out new shoots after which growth slows down. The leaves gradually turn yellow and fall during prolonged dry or cool intervals. The plant does not thrive where minimum temperatures are below 11C. The plant is commercially cultivated in UAE, Morocco, Algeria, Yemen, Tunisia, Libya, Saudi Arabia, Egypt, India, Iraq, Iran, Pakistan, Bangladesh, Afghanistan, Turkey, Somalia and Sudan [6]. The active ingredient of henna has been shown to be lawsone (2-hydroxy1,4-naphthoquinone). In the leaves it occurs in a colorless reduced glycosidic form. On the Indian subcontinent the henna plant is also known as Henna in Hindi, Rakigarbha in Sanskrit, Mailanchi in Malayalam, Muruthani in Tamil, Benjati in Oriya and Mehedi in Bengali [7]. Different parts of plant are used cosmetically and medicinally for over 9000 years. The ethno-medical uses of the plant are documented in ancient texts of Ayurved and Tibetean Medicine [8-11]. EXTRACTION, ISOLATION AND SPECTROSCOPIC CHARACTERIZATION Lawsonia inermis is one of the chief sources of lawsone (1) which is a red-orange compound primarily concentrated in the leaves, especially in the petioles of the leaf. Whole, unbroken leaves do not stain the skin unless lawsone molecule is released from the leaf. Fresh henna leaves stain the skin red orange if they are smashed with a mildly acidic liquid. The compound gradually migrates into outer layer of the skin and binds to the proteins creating a fast stain. Varieties of methods have been described for the isolation of lawsone by different researchers, which are the modified versions of a procedure originally described by Lal and Dutt in 1933 [1218]. Typically it involves extraction of the dried leaves of L. inermis with water or some organic solvent. Aqueous extraction is generally followed by treatment with solid NaHCO3, filtration, re-acidification and extraction with diethyl ether. Separation of the final product by column chromatography over silica gel using ethanol-ethyl acetate (1:2 v/v) as the eluent yields lawsone although with a low yield of about 0.25 % [12].

Successive soxhlet extraction in solvents of increasing polarity followed by column chromatographic separations and combining the appropriate fractions containing lawsone has been found to improve the yield but only slightly. Tomassis procedure involved acidification of the extracts to pH 3 by addition of 0.12 M HCl followed by extraction with diethyl ether and stripping of the organic solvent on a rotary evaporator. The reddish product obtained after re-crystalli zation from ethanol-water mixture generally gives higher yields [13]. Several HPLC studies have been carried out dealing with quantification of lawsone in the L. impatiens species [14], in mixtures of related naphthoquinones like juglone and plumbagin [15], in henna powders sold in the markets [16], in natural colorants [17] and in henna tattoos containing p-phenyldiamine [18]. Recently, Gevrenova has described HPLC procedure without involving any sample clean-up method using Hypersil ODS RP18 column and employing linear gradient elution program which can quantitatively determine lawsone in plant materials as well as cosmetic products in the range 0.22 to 1.44 mg/mL [17]. CHEMISTRY OF LAWSONE The electronic spectra of numerous simple 1, 4naphthoquinones have been compiled and analyzed by Singh and co-workers [19]. The spectrum of the parent 1, 4naphthoquinone comprises of intense benzenoid and quinonoidal electron-transfer absorptions in the region 240-290 nm and a medium intensity benzenoid band at 335 nm. In lawsone, these bands are observed at 242.5, 248, 274 and 334 nm in ethanol respectively. The carbonyl frequencies of quinone in their IR-spectra are useful diagnostic aids in their structure determination. The carbonyl bands observed for lawsone at 1674 cm-1(free carbonyl) and 1640 cm-1 (Hbonded) are typical for this compound. However, the broad and displaced hydroxyl stretch at 3150 cm-1 has been shown to be due to intermolecular hydrogen bonding interaction between C(3)H O(1) of the adjacent lawsone molecule [20]. An apparently rigid molecule of lawsone is known to exist in three polymorphic modifications due to the differences in the intermolecular interactions brought about essentially by the solvent used for crystallization. For example, bifurcated hydrogen bonding was observed for 3-methyl lawsone (2) and 3-chlorolawsone (3) by Gaultier and Hauw when the compounds were crystallized from acetonitrile [21, 22]. On the other hand inter-molecular hydrogen bonding was observed between C(3)H O(1) by Salunke-Gawali and co-workers [23]. In 2006 the structure of lawsone was solved by Todkary and coworkers which showed the molecule exhibiting intra-molecular O(2) H(2) O(1) hydrogen bonding, with OHO angle of 115
[24]. The compound can be efficiently synthesized in the laboratory following the method described by Fieser and Martin [25] from ammonium 1,2-naphthoquinone-4-sulfonate (4) and treating it with concentrated H2SO4 to isolate an intermediate methoxynaphthoquinone (5) compound and then hydrolyzing it with NaOH Fig. (2). 1. Reactivity of Lawsone Being a hydroxyquinone, the reactivity of lawsone in general is related to presence of electron-donor substituents

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Fig. (1). Leaves (A ) and Flowers (B) of Lawsonia inermis, Commercial powder preparation of Lawsone powder for hair dying (C), Henna Tattoos kept for drying (D) and stained tattoos (E ).

Fig. (2).

and keto-enol equilibrium which offers interesting synthetic opportunities as summarized below. a. C-C Bond Formation The substitution at C-3 position of hydroxynaphthoquinone compounds has shown to exert interesting biological activities. In case of lawsone, three general types of C-C bond formation reactions have been reported in the literature Fig. (3).

Fig. (3).

i)

The reactions involving C-3 substitution without disturbing free hydroxyl group.

ii) The reactions where free hydroxyl group takes part in reaction to yield 5-or 6-membered cyclized products. iii) The reactions involving tautomerism where hydroxyl group enolizes to keto form leading to formation of 5- or 6-membered ring at C-4 position.

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(i) Alkyl Derivatives of Lawsone Although reaction of lawsone with diacetyl peroxides produces a large number of alkylated hydroxynaphthoquinones (6) through a radical mechanism possessing antimalarial activity, their yields are not satisfactory Fig. (4) [26]. Better results are obtained from the reaction of the acylated Lawsone (7) with carboxylic acid in the presence of peroxysulfate which is a radical decarboxylation reaction. Several lawsone analogs Fig. (5) have been prepared following this method, which exhibit potential pesticidal activities [27]. Bieber and co-workers have shown that lawsone as well as alkoxy quinones, can be directly alkylated at C-3 position by using alkylboranes as catalyst Fig. (6) [28]. Kobayashi and co-workers have reported the reaction of benzenoid substituted Lawsone with o-fluoronitrobenzene (8) yielding 2hydroxy-3-(2-nitroaryl-1,4-naphthoquinone) (9) in reasonable yields Fig. (7) [29]. Dabiri et al. have reported an efficient one-pot synthesis of fluorescent hydroxyl naphthalene1,4-dione derivatives by a simple, atom-economical and three-component reaction involving 2- hydroxynaphthalene1,4-dione, aromatic aldehydes (10) and heterocyclic or aromatic amines (11) in the presence of a catalytic amount of InCl3 in refluxing water. The reaction provides operational simplicity, easy work-up procedures and high yields of products. The products (12) are fluorescent in solution emitting green light (546-560 nm) Fig. (8) [30]. Baramee et al. have synthesized ferrocenylamine (13)-substituted hydroxynaphthoquinones following Mannich base condensation reaction and have evaluated them for their in vitro activity along with standard inhibitors like atovaquone and chloroquine against two apicomplexan parasites of medical importance, viz. Toxoplasma gondii and Plasmodium falciparum, respectively. The compounds with an amino-ferrocene moiety and an aliphatic chain with 68 carbon atoms (14-16) Fig. (9 ) were found to be significantly active against T. gondii [31]. Tisseh and Bazgir have devised a simple and efficient method for the synthesis of 3, 3-(arylmethylene)-bis-(2hydroxynaphthalene-1,4-dione) derivatives (17-20) using catalytic amount of LiCl in aqueous media Fig. (10) [32].

ing lawsone. Thus, reaction of lawsone with 2-bromo propanol afforded the ortho-quinone furo-derivative (21) through initial alkylation of C-2 Fig. (11) [33]. Analogous reaction in case of 3,4-dibromo-2-butanone (22) leads to furan (23) and dehydrofuran (24) derivatives in presence of DBU(1,8-Diazabicyclo[5.4.0]undec-7-ene) Fig. (12) where the former exhibits significant anticancer activity [34]. Lawsone and its derivatives Fig. (13) can react with alkenes in the presence of two equivalents of cerium ammonium nitrate to give mixtures of furo-derivatives of ortho-(25) and paraquinone (26) [35]. The para-quinone isomer was usually found to predominate and acetonitrile solvent was found to be the best solvent for this reaction. In these cyclization reactions initially formation of free radicals is involved. On the other hand, Kobayashi and coworkers have reported on the reaction of lawsone (27a-c) with enamines (28a-c), which start with the nucleophilic attack of quinone with enamine yielding furoparaquinones (29a-c) in satisfactory yields without the formation of ortho-quinones Fig. (14) [36]. Cyclization of hydroxynaphthoquinones to the corresponding six-member pyran derivatives is also important and is usually achieved by the oxidative cyclization of the 3-alkenyl derivative of lawsone. In this reaction both ortho- and paraquinone compounds are obtained. For example, alkylation of lawsone can yield lapachol (30), which can then be converted to
-lapachone (31a) at room temperature as shown in Fig. (15) [37]. Thus, it is observed that lawsone can serve as an attractive synthetic building block for generating compounds of therapeutic importance.

Fig. (6).

b. C-N and C-S Bond Formation Another reaction which takes place at C-3 position or C-4 carbonyl atom of lawsone deals with compounds like amines, hydrazides and mercaptans leading to formation of C-N or C-S bonds. The compounds arising from such reactions were also found to be of biological importance. Thus, Tandon et al. have synthesized a series of (S)-N-(1,4naphthoquinon-2-yl)-
-amino acid methyl esters (32-34), 2N,N-dialkylamino-1,4-naphthoquinones (35-36) and 2-hyd roxy-3-(2-mercaptoimidazolyl)-1,4-naphthoquinones (3738) following such reactions Fig. (16) which have resulted in compounds having antifungal and antibacterial activities [38]. Oliveira et al. have synthesized several 1,4-naphthoq uinone derivatives having hydrazinic side chain (39-41) which were synthesized from 3-diazo-naphthalene-1,2,4trione (42) and are found to be potential antimicrobial agents Fig. (17) [39]. Granero et al. have reported on the preparation of isoxazolylnaphthoquinones with an ethyl group on the isoxazole ring by condensing sodium 1,2-naphthoq uinone-4-sulfonate (42) with 3-methyl-5-ethyl-4-amino isoxazole (43) in aqueous solution. In neutral and alkaline aqueous solutions, the reaction between compounds 42 and 43

Fig. (4).

Fig. (5).

(ii) Cyclization Reactions of Lawsone Since natural phytochemicals such as naphthofuranodiones exhibit broad spectrum biological activity, the cyclization reactions have been developed for their preparation us-

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Fig. (7).

Fig. (8).

Fig. (9).

Fig. (10).

Fig. (11).

Fig. (12).

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Fig. (13).

Fig. (14).

Fig. (15).

Fig. (16).

yielded 2-hydroxy-N-(3-methyl-5-ethylisoxazol-4-yl)-1,4naphthoquinone-4-imine (44) respectively, while in acidic aqueous solution gave bisisoxazolylnaphthoquinone imine (45). By hydrolysis with acidic aqueous solutions at 800C in EtOH, compound 45 was converted to the diketone deriva-

tive (46), which is the tautomer of compound 44 Fig. (18) [40]. Thube et al. have used HartreeFock and hybrid density functional methods to establish the structure and energetics

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Fig. (17).

Fig. (18).

of keto- and nitrosophenol tautomers of 2-hydroxy-3-methyl1,4-naphthoquinone-1-oxime (47). The single crystal Xstructure of the keto form of this compound has been studied. Both theory and experiment predict that compound 47 exists predominantly as amphi conformer (keto form), which is more stable relative to the anti- and syn-conformers. This has partly been attributed to the five-membered ring formation as a result of N HO(2.06 A ) intra-molecular hydrogen bond formation. The x-ray crystal structure showed extended three-dimensional network comprising of C=O H intermolecular hydrogen bonds (1.87 A ) as well [41]. Some oxime derivatives of lawsone and its C-3 substituted analogs (48-52) have been prepared by interacting lawsone with hydroxylamine hydrochloride Fig. (19) [42-44]. In our group we have synthesized methyl-phenylazo-lawsone

Fig. (19).

derivatives (53-56) by coupling the diazotized anilines with lawsone in ethanol in presence of excess sodium acetate at temperatures below 100C. The single crystals x-ray structure

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of one of these compounds has shown that lawsone is present as the keto tuatomer (crystallized from polar acetonitrile solvent), as 1,2-naphthoquinone rather than 1,4-naphthoquinone species where the elongated C2-O2 bond length is favorable for the intra-molecular hydrogen bonding with the N1 nitrogen of the phenylazo group. The compound was found to be inhibitory towards human breast cancer MCF-7 cells [45]. c. O-C Bond Formation Hage et al. have synthesized atovaquone (57) derivatives substituted at the 3-hydroxy group with ester (58-60) and ether (61-63) functions which are active against the growth of malaria causing parasite Plasmodium falciparum Fig. (20). The compounds were found to be more potent than parent atovaquone, with IC50 values in the range of 1.2550 nM [46]. d. Other Analogs Buckle et al. have synthesized a series of substituted 2hydroxy-l,4-naphthoquinone (66-70) including its 3-nitro derivative (64). The 3-nitrolawsone derivatives were shown to be potent inhibitors of rat passive cutaneous anaphylaxis

(PCA) and having highest potency with alkyl substitution at C-6 and C-7 positions. The most potent compound (65) was found to produce 50% inhibition in the rat PCA test at doses of 10 M/kg following subcutaneous administration and showing activity after oral administration [47]. Tran et al. have synthesized 3-bromo-lawsone (71), 3-chloro-lawsone, 2-methoxy-3-bromolawsone (72) following the reaction conditions shown in Fig. (21) and evaluated their antifungal activities [48]. Similarly 3-Iodolawsone (73) was prepared from lawsone Fig. (22) by the treatment of iodating mixture (KIO3 and KI) [49]. Phthiocol (62) is another important lawsone analog Fig. (23) which has been studied for its metal chelating ability. It is prepared from the epoxide intermediate of menadione (74) which, upon treatment with concentrated sulfuric acid, yields phthiocol (2) [50]. 2. Metal Complexes of Lawsone The hydroxyl group in lawsone being vicinal to the quinone carbonyl is capable of forming coordination compounds with variety of transition metal ions. However, the ortho-hydroxyl group is known to give rise to keto  enol tautomerism thereby capable of generating the reduced paramagnetic semiquinone and catechol forms as shown in Fig.

Fig. (20).

Fig. (21).

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(24). The intermediate paramagnetic semiquinone form when complexed to paramagnetic transition metal ions provides possibility of spin-spin magnetic exchange interactions in which several intermediate spin states can be generated. The population of these intermediate spin states in such metal complexes prepared under specific conditions has been a matter of intense studies in several laboratories including our own.

from metal to quinonoidal ligands converting them to paramagnetic semiquinone species promoting ferromagnetic as well as anti-ferromagnetic exchanges leading to sub-normal magnetic moments and characteristic EPR and electrochemical features [53]. The first proof of semiquinone coordination in case of lawsone was provided by our group in 1989 in case of ferrous complexes (77) mainly on the basis of variable temperature magnetic susceptibility and low temperature EPR measurements [51]. This was later confirmed on the basis of single crystal X-ray structure determination wherein the bond lengths of the carbonyl group participating in metal coordination were found to be within the range of bond lengths typical of semiquinone moiety [54]. The mechanism of toxicity of Fe (II) and Fe (III) complexes of lawsone and its structural isomer juglone to isolated rat hepatocytes has been studied. All complexes were found to show a dose-dependent toxicity, which precedes cell death. The iron complexes of juglone were more toxic than those of lawsone indicating that the mechanisms of toxicity may be different. Electrochemical studies on these complexes showed that juglone facilitates formation of stable semiquinone species while lawsone does not. The low redox potential of lawsone makes it a poor substrate for metabolism by reductases [55]. The modulation of radiation induced lipid peroxidation in synaptosomes by these complexes has also been studied. At lower concentrations the complexes enhance lipid peroxidation predominantly through redox cycling while at higher concentrations they tend to limit lipid peroxidation through fast re-combination reactions [56]. The X-ray crystal structure of phenylazolawsone has been determined. In this compound the bond lengths of C(2)-O(2) (1.36A0) and C(4)-O(4) (1.26 A0) confirm the existence of the keto form of the naphthoquinone while the observed bond length for the N(1)-N(2) linkage (0.37 A0)is characteristic of the azo tautomer at least in the solid state. The copper complexes (78) of these ligands were found to possess 1:2 metal to ligand stoichiometry and square planar geometries with intermolecular stacking interactions resulting in antiferromagnetic exchange interactions. The copper compounds exhibited enhanced antiproliferative activity against human breast cancer MCF-7 cell-line, than the parent ligands, the highest being for the copper compound of 3-(3'-methyl phenylazo) lawsone [45]. Spectroscopic and electrochemical studies have been carried out on the six copper (II) complexes of Lawsone-1-thiosemicarbazone, [CuLCl](79), having pseudo-square-planar geometry. Their EPR spectra simulated with an axial spin-Hamiltonian, exhibit a four-line pattern with nitrogen super-hyperfine couplings originating from the imine/hydrazinic nitrogen atoms. The evaluation of covalency parameters suggested that the unpaired electron was localized in the dx2-y2 orbital and spends about 4245% of its time on the nitrogen donor site of the coordinated thiosemicarbazone ligand, reflecting the -acceptor property of the sulfur center as well as the charge accumulating character of the quinone molecules. The presence of a strong interaction leads to extensive delocalization through chelate rings formed by the tridentate ligands. Moderate covalency is observed in
-bonding while in- plane -bonding possess appreciable covalent character. A quasi-reversible Cu(II)/ Cu(I) redox couple is observed at relatively higher potential as a consequence of structural reorganizations [57].

Fig. (22).

Fig. (23).

Fig. (24).

Normally, the stoichiometric interaction between lawsone and divalent transition metal ions in aqueous methanolic medium generally yields metal complexes having [M (L)2(H2O)2] compositions, where the ligands are in their fully oxidized monoanionic form. The ferrous complexes have been particularly interesting for mimicking the quinone complex participating in photosynthetic electron transfer reactions in Photosystem II [51]. Octa-coordinated Lanthanide complexes of 3-amino-lawsone of general formula [M(3A2HNQ)3(H2O)2] (75) have been synthesized and characterized by Chikate [52]. IR and far-IR spectral data for these compounds indicated that phenolic oxygen and amino nitrogen constitute the donor atom set for the aminolawsone ligand, while coordinated water molecules are involved in the intermolecular hydrogen bonding interactions with the free quinone carbonyls. When the water ligands are replaced by hard pyridine nitrogen donor ligands as shown in the structure (76) the delocalization of electrons is promoted

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Our group has also studied iron (II), iron (III), cobalt (II) and copper (II) complexes of 2-thiosemicarbazido-1,4naphthoquinone (80). Under acidic medium, metal coordination is favored in the thione form while under basic conditions thiolato complexes are isolated. IR spectral data indicates that the ligand can act as neutral or mono-anionic tridentate donor, bonding through thione/ thiol tautomeric forms or both. The magnetic and Mssbauer parameters for iron (II) complexes reveal the presence of an intermediate S = 1 spin state possessing small zero-field splitting (ZFS) while in case of iron (III) complexes stabilization of the spintriplet state (S = 3/2) is observed with negligible contribution from ZFS component. In case of cobalt complexes the compounds are either diamagnetic or low-spin compounds. Evaluation of ESR covalency parameters indicated considerable delocalization of the unpaired electron present in the dx2y2 ground state in these tetragonally distorted octahedral complexes [57]. An interesting series of iron (III) complexes has also been described in case of these two C1- and C2substituted lawsone-thiosemicarbazone compounds. In case of Lawsone-1-thiosemicarbazone ligand (80) the ferric complex is ionic consisting of a cationic octahedral [FeL2]+ (81) species and a tetrahedral [FeCl4]
anion which exhibits an unusual mixed spin-states ferric compound as revealed from magnetic behavior with significant amount of exchange interactions mediated by intermolecular associations. The magnetic susceptibility data was fitted with S1 = 5/2 and S2 = 1/2 Heisengbergs exchange coupled model, ^H =
2JS1S2 and the magnetic exchange interactions are found to be of the order of
13.6 cm
1 indicating a moderate coupling between two paramagnetic centers present in different chemical and structural environment. The presence of spin-paired iron (III) cation having d2xzd2xzd1xz ground state is revealed from the EPR spectra with three prominent peaks while the high spin tetrahedral iron (III) anion exhibits characteristics g = 4 signal whose intensity increases with lowering of the temperature suggesting its influence on the magnetic properties of the complex molecule [58]. On the other hand Fe(III) complexes of 2-thiosemicabazido-1,4-naphthoquinone ligand having [FeLCl2] (82) stoichiometry are neutral compounds having highly distorted trigonal bipyramidal geometries [58]. The bismuth complex of lapachol having general formula,[Bi(Lp)2]Cl (83) has been synthesized and evaluated in a murine model of inflammatory angiogenesis induced by subcutaneous implantation of polyether polyurethane sponge discs. Intra-peritoneal (i.p.) administration of lapachol or [Bi(Lp)2]Cl reduced the hemoglobin content in the implants suggesting that reduction of neo-vascularization was caused by lapachol. In the oral administration only the bismuth complex decreased the hemoglobin content in the implants. Histological analysis showed that the components of the fibro-vascular tissue were decreased in groups treated with lapachol and bismuth complex. The anti-angiogenic and antiinflammatory activities have been attributed to the presence of the lapachol ligand. However, coordination to bismuth (III) could be an interesting strategy for improvement of lapachol's therapeutic properties [59]. A novel versatile tridentate lawsone analog, Viz. 3-[N-(2-pyridylmethyl) aminobenzyl]- 2-hydroxy-1,4-naphthoquinone (84), has been prepared via Mannich reaction of lawsone with 2-amino methylpyridine and benzaldehyde. The ligand on interaction

with copper chloride yielded two novel dinuclear copper(II) complexes, [Cu(L)(H2O)(-Cl)Cu(L)Cl] (85) and [CuCl(L) (-Cl)Cu(amp)Cl], whose relative yields were sensitive to temperature, reagent concentration and presence of a base. The X-ray crystal structures of [Cu(L)(H2O)(-Cl)Cu(L)Cl] and [CuCl(L)(-Cl)Cu(amp)Cl] have been determined which revealed that two copper atoms in complex 85 are connected by a single chloro bridge with a CuCu separation of 4.1342(8) and Cu(1)Cl(1)Cu(2) angle of 109.31(4)o. In the dimeric complex [CuCl(L)(-Cl)Cu(amp)Cl], the two copper atoms are held together by chloro and naphtholate bridges with CuCu inter-nuclear distance of 3.3476(9) . The bridging angles, viz. [Cu(1)Cl(2)Cu(2) and Cu(1) O(1)Cu(2) were found to be 83.31(3) and 109.70(9)o, respectively. The variable-temperature magnetic susceptibility measurements on compound 85 exhibited weak antiferromagnetic intramolecular coupling between the copper (II) centers, with coupling constant (J) of -5.7 cm-1 , while for complex (B) it was found to be -120 cm-1 respectively [60]. These researchers have also reported on the Zinc (II) and copper (II) complexes (86) of a tridentate Mannich base (87) derived from 2-hydroxy-1,4-naphthoquinone, pyridinecarboxyaldehyde and 2-aminomethylpyridine [61]. BIOLOGICAL ACTIVITIES OF LAWSONE AND ITS ANALOGS a. Anticancer Activity Kamei and co-workers have studied growth inhibitory effects of naphthaquinone (88) and anthraquinone (89-92) against human colon HCT-15 cancer cells and found them to be more effective than the benzoquinones. The flow cytometric analysis showed that growth inhibitory effects of lawsone and juglone were mediated via blocking the S-phase of cell cycle [62]. Su et al. have shown that Furano-1,2naphthoquinone (93), prepared from 2-hydroxy-1,4-naph thoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibited a growth inhibitory activity mediated via G(2)/M cell cycle arrest and apoptosis in A549 lung cancer cells. The apoptosis induced by compound 93 was accompanied by up-regulation of Bax and down-regulation of Bcl-2. The compound also suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, and induced activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signals. These reports suggest that compound 93induced G(2)/M cell cycle arrest and apoptosis in A549 cells are mediated via inactivation of EGFR pathway [63]. Further studies showed that 93 suppressed the phosphorylation of EGF receptor and activation of PI3K/Akt in Ca9-22 cells. The levels of downstream targets of Akt, including phosphoglycogen synthase kinase-3 (p-GSK-3), GSK-3, forkhead transcription factor (FKHR), and cyclin D1, were also decreased after treatment of compound 93. In addition, such treatment disrupted mitochondrial membrane potential resulting in release of cytochrome-C, and activation of both caspases-3 and 9 respectively. Taken together, these results indicate that the compound 93 induces apoptosis in Ca9-22 cells via inactivation of the EGF receptor-mediated survival pathway [64]. Two series of - and -pyranonaphthoquinones (94-96), when evaluated against four cancer cell lines (MDA-MB-

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Fig. (25).

Fig. (26).

Fig. (27).

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435, HL-60, HCT-8 and SF295 cells), exhibited potent activity, possibly indicating that these compounds have increased pro-oxidant property [65]. Akman et al. have investigated the mechanism of antitumor activity of the water-soluble derivative of menadione, viz. menadione sodium bisulfite (vitamin K3) (97), against murine leukemia L1210. The compound caused time- and concentration-dependent depletion of the acid-soluble thiol (GSH) pool as well as increased the rate of superoxide anion generation by the L1210 cells. It was concluded that tumor cell growth inhibition by vitamin K3 may be caused by GSH pool and/or NADPH depletion [66]. Silva et al. have synthesized new 1, 4-naphthoquinones structurally related to lapachol which are regarded as pterocarpan derivatives where the A-ring is substituted by the 1,4naphthoquinone nucleus. The compounds exhibit significant biological activities against proliferation of the MCF-7 human breast cancer cell line, where compound 98 was found to be most potent with IC50 value of 5.3
M [67]. Various lapachol derivatives have been synthesized and evaluated for their inhibitory effects on Epstein-Barr virus as a test for potential cancer chemo-preventive agents. These compounds (99-101) exhibited very high to moderate activities [68]. The cytotoxic activity of amino-derivatives of lapachol and lawsone were examined against Ehrlich carcinoma and human K562 leukemia cells. Cell viability was determined by MTT assay using vincristine (for K562 leukemia) and quercetin (for Ehrlich carcinoma) as positive controls. The results showed dose-dependent growth-inhibiting activities. The allyl-amine derivatives of lapachol (102) and lawsone (103) were the most active compounds against Ehrlich carcinoma with IC50 values of 16.94 and 23.89
M respectively [69]. Polyamine-naphthoquinone conjugates (104-106) were synthesized by nucleophilic displacement reactions involving 2methoxylawsone/lapachol and norlapachol with the polyamine N1-Boc-N5-Bn-spermidine. The compounds were active against human pro-myelocytic leukemia (HL-60), lung cancer (GLC4), Burkitt lymphoma (Daudi) and mouse breast tumor (Ehrlich carcinoma). DNA fragmentation was measured by quantification of the subG1 peak of the cell cycle. The amount of DNA fragmentation observed is compatible with the decrease in viability induced by the drugs. Kinetics of HL-60 DNA fragmentation and ROS formation indicated that production of ROS precedes cell death [70]. Eyong et al. have synthesized lapachol analogs by ozonolysis Fig. (25) resulting in unusual formation of a potent antitumor agent 2-acetylfuranonaphthoquinone (23) along with the expected aldehyde (108). Other biologically active furanonaphthoquinones were prepared from the reaction of lapachol with Cerium Ammonium Nitrate in dry acetonitrile. These compounds when evaluated for anticancer activity against human DU-145 prostate carcinoma cells revealed that compound 109, -lapachone (31b) and dehydro-lapachone diacetate (110) showed 100% inhibition at a concentration of 25 g/mL [71]. The pentacyclic 1,4-naph thoquinones (111-114) have been found to be cytotoxic to human leukemic cell lines K562 , Lucena-1 (MDR phenotype) and Daudi (Burkitt lymphoma). Previous data suggested that these compounds can be bioactivated in situ through reduction followed by rearrangement leading to enone species, which are powerful alkylating agents. In contrast, lapachol and -lapachone, which cannot be bioactivated by reduction,

showed little activity against these cell lines [72]. Boothman et al. have shown that 4-h treatment with 4 M -lapachone was found to enhance the lethality of X-rays against human laryngeal epidermoid carcinoma (HEp-2) cells [73]. These workers have further shown that -lapachone activated the DNA-unwinding activity of topoisomerase-I and inhibited the fast component of potentially lethal damage repair (PLDR) carried out by HEp-2 cells when present during or immediately following X-ray-irradiation. The compound specifically and synergistically enhances the cytotoxic effects of DNA-damaging agents or X-rays which induce DNA strand incisions against a radio-resistant human malignant melanoma (U1-Mel) cell line. The compound did not enhance the lethal effects of X-rays following prolonged drug exposures, indicating that -lapachone modifies the initially created DNA lesions or inhibits lesion repair but does not create lethal lesions by itself. The compound did not intercalate into DNA, nor did it inhibit topoisomerase-II or ligation carried out by mammalian T4 DNA ligases. Structurally similar analogs, -lapachone, lapachol, and dichloroallyl lawsone (126), did not enhance X-ray-induced cytotoxicity nor did they activate topoisomerase-I [74]. Huang and Pardee have examined the effects of lapachone on human colon cancer cells lines, viz SW480, SW620, and DLD1, with mutant or defective p53. The antiproliferative effects were assessed by colony formation assays, cell cycle analysis, and apoptosis assay including annexin-V staining and DNA laddering analysis. All three cell lines, SW480, SW620, and DLD1, were sensitive to the compound. However, these cells were arrested in different stages of S-phase. At 24 hr post-treatment, -lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by -lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1.These findings seem to suggest lapachone may prove to be a promising anticancer agent for targeting cancer cells that have defective or mutant p53 gene [75]. Choi and co-workers have investigated the effect of lapachone on the cell growth and apoptosis in human colon carcinoma tumor cell line HCT-116. The apoptotic effects were found to be associated with decrease in Bcl-2 expression, increase in caspase-3 activity, decrease in intact poly(ADP-ribose) polymerase protein levels and degradation of -catenin. The compound is also found to inhibit NF- B activity. The inhibition of the transcriptional activity of NFB-luciferase reporter plasmid by treatment with the compound suggested that -lapachone-induced apoptosis may partly be regulated through the inactivation of NF-B [76]. Li et al. have studied antiproliferative effects of -lapachone on human multiple myeloma cells by colony formation assay. The cytotoxicity of -lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. No apoptosis was observed in either quiescent or proliferative states, and in peripheral blood mononuclear cells freshly isolated from healthy donors after treatment with lapachone. The apoptosis induced by -lapachone in multiple myeloma cells was not blocked by either interleukin-6 or Bcl-2. The results suggest potential therapeutic application of -lapachone against multiple myeloma, particularly in overcoming drug resistance in relapsed patients [77]. Effect

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of -lapachone alone or in combination with mild heating on the clonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro has been determined. Incubation of FSaII and A549 tumor cells with -lapachone at 37 0C reduced the clonogenic survival of the cells in dose-dependent manner which was dependent on incubation time. It was observed that NQO1 level in the cancer cells increased within 1 hour after heating at 42 oC for one hour and remained elevated for >72 hours. The clonogenic cell death caused by -lapachone increased in parallel with the increase in NQO1 levels in heated cells. It was noted for the first time that mild heat shock can up-regulate NQO1 in tumor cells. The heat-induced up-regulation of NQO1 enhanced the anticancer effects of -lapachone in vitro and in vivo [78]. To enhance radiotherapeutic efficacy, Jeong et al. have used biocompatible gold nanoparticles (AuNP) as a vehicle for systemic delivery of -lapachone since its poor solubility and non-specific distribution prove detrimental to its pre-clinical evaluation and clinical application. Murine monoclonal anti-EGFR antibody was conjugated to the AuNPs/lap as a ligand for the purpose. The active tumor-targeting property of these AuNPs/lap conjugating anti-EGFR antibody was validated in vitro experiments using cell lines expressing EGFR at different levels. In mice bearing xenograft human tumors, the intravenous injection of AuNPs/lap exhibited highly enhanced radiotherapeutic efficacy indicating feasibility of further clinical application for human cancer treatment [79]. Another effort in improving the poor aqueous solubility and low bioavailability of -lapachone was made by Wang et al. by using -lapachone inclusion complexes with cyclodextrins (CDs). Sustained drug release was achieved when lapachone was complexed with
-CD or -CD which paved a way to tailor -lapachone release kinetics via cyclodextrin complexation, providing exciting opportunities for the intratumoral drug delivery of -lapachone [80]. Recently Dong et al. have complexed -lapachone with hydroxypropyl-cyclodextrin and have used the conjugate against prostate cancer cells over-expressing NAD(P)H:quinone oxidoreductase-1 enzyme. These workers were able to incorporate this conjugate into poly(D,L-lactide-co-glycolide) millirods which showed significant tumor growth inhibition compared with controls [81]. Blanco et al. have used a film sonication method which yielded -lapachone micelles with relatively high loading density. Release studies in phosphate-buffered saline (pH 7.4) showed the t1/2 time for 50% of drug release at 18 h. In vitro cytotoxicity assays were performed in NQO1-overexpressing H596 lung, DU-145 prostate and MDA-MB-231 breast cancer cells which showed a marked increase in toxicity in NQO1 positive cells over NQO1 negative cells [82]. Zheng and Li have investigated the loading of -lapachone, on a magnetite nanoparticle decorated with reduced graphene oxide (Fe3O4/rGO) and subsequently examining its in vitro anticancer efficacy. Reduced graphene oxide (rGO) with magnetic functionality was prepared via electrostatic interaction between positively charged magnetite Fe3O4 nanoparticles and negatively charged GO, followed by hydrazine reduction of GO to rGO. The prepared Fe3O4/rGO shows that Fe3O4 makes the Fe3O4/rGO hybrid magnetically separable for easy handling during drug loading and release. The Fe3O4/rGO hybrid exhibits significantly

higher loading capacity than that of Fe3O4/GO, suggesting that restoration of the graphene basal plane upon reduction of GO enhances the interaction between -lapachone and rGO. Cellular uptake studies using fluorescent labeled Fe3O4/rGO verified successful internalization of Fe3O4/rGO into the cytoplasm while rGO without hybridized Fe3O4 has poor uptake performance. Furthermore, -lapachone loaded Fe3O4/ rGO showed remarkably high cytotoxicity against MCF-7 breast cancer cells while the blank Fe3O4/rGO produced no cytotoxic effects. The cytotoxicity results suggest that Fe3O4/rGO is an efficient drug carrier for anticancer treatments. Thus, fine-tuning of the chemical structures of graphene oxides by reduction chemistry may provide a universal route for controlled loading and release of drugs or biomolecules to construct advanced delivery vehicles [83]. Lien and co-workers have found that apoptosis induced by -lapachone in DU-145 human prostate cancer cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins. The apoptosis was dosedependent and accompanied by cleavage of pro-caspase-12 and phosphorylation of p38, ERK, and JNK. It was also followed by activation of the executioner caspases, viz. caspase-7 and calpain respectively. However, pre-treatment with the general caspase inhibitor, viz. z-VAD-FMK, or calpain inhibitors including ALLM or ALLN, failed to prevent -lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with a known NQO1 inhibitor like dicoumarol or preventing an increase in intracellular calcium levels using an intracellular calcium chelator like BAPTAAM, substantially inhibited MAPK phosphorylation, and provided significant protection in -lapachone-treated cells. These findings showed that -lapachone-induced ER-stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of its anticancer effect [84]. Choi and group have observed an inhibitory effect of -lapachone in cultured human prostate cancer cells due to induction of apoptosis, confirmed through morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. DNA flow cytometric analysis revealed that -lapachone arrested the cell cycle progression at the G1-phase. The effects were associated with the downregulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. The compound suppressed the cyclin-dependent kinases (Cdks) and cyclin E-associated kinase activity without changing their expressions. It induced the levels of the Cdk inhibitor p21 (WAF1/CIP1) expression in a p53-independent manner. The p21 proteins that were induced by -lapachone were associated with Cdk2 [85]. Lee and co-workers have investigated possible mechanisms by which -lapachone exerts its anti-proliferative action in human prostate cancer DU145 cells. The increase in apoptosis was associated with a dose-dependent up-regulation in proapoptotic Bax expression, down-regulation of anti-apoptotic Bcl-2protein, and proteolytic activation of caspase-3 protease. These researchers found that -lapachone decreased the levels of cyclooxygenase (COX)-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. The compound markedly inhibited the

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activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by lapachone treatment. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of -lapachone [86]. NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by lapachone. The study undertaken by Terai and coworkers probed whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to -lapachone treatment by upregulating NQO1. The cytotoxicity of cisplatin and lapachone alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of -H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to -lapachone and the expression and enzymatic activity of NQO1 progressively increased during 24 h after cisplatin treatment. An inhibitor of NQO1, viz. dicoumarol, was found to nullify the cisplatin-induced increase in -lapachone sensitivity. The role of NQO1 in the cell death caused by -lapachone alone or in combination with cisplatin was further examined using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1positive but not of the NQO1-negative MDA-MB-231 cells to -lapachone treatment. Combined treatment with cisplatin and -lapachone suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It was, therefore, concluded that cisplatin markedly increases the sensitivity of these cancers to -lapachone in vitro and in vivo by up-regulating NQO1 [87]. The combination of paclitaxel (PTX) and -lapachone (LPC) was also found to induce apoptotic effects synergistically in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and -lapachone (1.5 M) caused biochemical and morphological signs of apoptosis at 48 h treatment and upregulation of apoptosis proteins and activation of Bid and caspases 3 and 6 proteins. The combination treatment also promoted p53 stabilization through lowering of Akt levels. Such lowering was thought to be responsible for the apoptotic action exerted by PTX/-lapachone combination [88]. Dong et al. have investigated whether or not NQO1 is involved in the action of -lapachone and whether the compound suppresses the radiation-induced NF-B activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-B in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-B activation. Treatment with 10 M of -lapachone for 4 h completely abrogated the radiation-induced increase in NF-B activation and the transcription of NF- B target genes such as bcl-2, gadd45 and cyclinD1. Moreover, the compound markedly suppressed the activation of I-B kinase-  (IKK) and subsequent phosphorylation of IB-
, thereby inhibiting NF- B activation. It was therefore concluded that -lapachone suppresses the radiation-induced activation of NF-B by interrupting the involvement of NQO1 in the activation of NF-B, thereby inhibiting the

transcription of survival signals. Consequently radiosensitization caused by -lapachone may, in part, be attributed to lapachone-induced suppression of NF-B activation [89]. Blanco et al. have reported a nano-therapeutic strategy that targets non-small cell lung cancer (NSCLC) through use of a bio-activatable agent such as -lapachone, and biocompatible nanocarriers such as polymeric micelles, to achieve drug stability, bioavailability, and targeted delivery. The -lapachone micelles produced by film sonication technique were small (approximately 30 nm), had core-shell architecture, and possessed favorable release kinetics. Pharmacokinetic analyses in mice bearing sub-cutaneous A549 lung tumors showed prolonged blood circulation (t1/2, approximately 28 h) and an increased accumulation in tumors. Antitumor efficacy analyses in mice bearing sub-cutaneous A549 lung tumors and orthotopic Lewis lung carcinoma models showed significant delay in tumor growth and increased survival [90]. These workers have also investigated the effect of -lapachone, on the cell growth and apoptosis in human lung carcinoma cell line A549. Exposure of A549 cells to lapachone resulted in growth inhibition and induction of apoptosis in a time- and dose-dependent manner. This increase in apoptosis was associated with a decrease in Bcl-2 expression and increase of Bax, and activation of caspase-3 and 9 respectively. The -lapachone treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the levels of human telomerase RNA (hTR) and c-myc expression were progressively downregulated by -lapachone treatment. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of lapachone [91]. Lee et al. have found that -lapachone inhibited the viability of human bladder carcinoma T24 cells by inducing apoptosis, as judged from the formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with -lapachone resulted in down-regulation of Bcl-2 expression and up-regulation of Bax expression. The induced apoptosis was associated with activation of caspase-3 and 9, inhibition of IAP expression, and degradation of poly (ADPribose) polymerase, and -catenin proteins respectively. At the same time Fas and FasL levels were inhibited upon treatment with -lapachone in a concentration-dependent manner. It was concluded that -lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway [92]. Woo et al. have investigated the effects of -lapachone on the growth of the human hepatoma HepG2 cell line and found that it inhibits the viability of these cells by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. Reverse transcription-polymerase chain reaction and immunoblotting results indicated that treatment of cells with -lapachone resulted in downregulation of anti-apoptotic Bcl-2 and Bcl-X(L) and upregulation of pro-apoptotic Bax expression. However, lapachone treatment did not affect the inhibitor of apoptosis proteins family and the Fas/FasL system [93]. Kim and coworkers have investigated functions of -lapachone in terms of anti-metastasis and anti-invasion abilities using human hepato carcinoma cell lines, viz. HepG2 and Hep3B respectively. The compound inhibited cell viability and migration of both cells as determined by MTT assay as well as wound

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healing assay. RT-PCR and western blot data revealed that -lapachone significantly increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, there was a down-regulation of Snail and upregulation of E-cadherin expression in -lapachone-treated HepG2 and Hep3B cells. The results strongly suggested that -lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin respectively [94]. Moon et al. have shown that treatment with -lapachone, induces cytotoxicity in human leukemia cells (U937, K562, HL60, and THP-1) through activation of caspase-3 and subsequent PARP cleavage. The observed induction of cell death was associated with decreased telomerase activity, which was ascribed to down-regulation of telomerase reverse transcriptase [95]. Several 3-arylamino and 3-alkoxy-nor-lapachone derivatives have been synthesized by Silva and co-workers which were found to be highly potent against cancer cells SF295 (central nervous system), HCT8 (colon), MDA-MB-435 (melanoma), and HL-60 (leukemia) with IC50 values below 2 M. The arylamino para-nitro (115) and 2,4dimethoxy (116) substituted naphthoquinones showed the best cytoxicity profile, while ortho-nitro (117) and 2,4dimethoxy substituted ones were more selective than doxorubicin and constitute promising new lead compounds in anticancer drug development [96]. In view of this finding thirty two compounds were synthesized in moderate to high yields and which showed growth inhibitory activity against cancer cells HL-60 (leukemia), MDA-MB-435 (melanoma), HCT-8 and SF295 (central nervous system). The most potent compound (118) exhibits IC50 values in the range 1.01-2.08 M respectively. The -lapachone-based 1,2,3-triazoles were also synthesized by this research group which showed best cytoxicity profile. The mechanistic studies seem to indicate that depletion of reduced glutathione (GSH) content and generation of reactive oxygen species (ROS) may be responsible for their observed cytoxicities [97]. From the methanolic extracts of solid callus cultures from two species of the closely related palaeotropical plant families Dioncophyllaceae and Ancistrocladaceae seven new natural naphthoquinones were isolated, which included dioncoquinones-A (119) and -B (120) from Triphyophyllum peltatum, and ancistroquinones-B (121), -C (122), -D (123), -E (124) and -F (125) from Ancistrocladus abbreviatus. Among these Dioncoquinones-A (119) and -B (120) strongly-induced apoptosis in human tumor cells derived from two different B cell malignancies, B cell lymphoma and multiple myeloma, without any significant toxicity towards normal peripheral mononuclear blood cells [98]. Dichloroallyl lawsone (126), which is a synthetic analog of the lapachol, is potentially useful in cancer chemotherapy. Unlike most anticancer agents, dichloroallyl lawsone is not significantly myelo-suppressive in animals although it induces acute cardiac toxicity in the rhesus monkey. This cardiac toxicity seems to be correlated with the maximal plasma concentration in the monkey. McKelvey et al. have studied pharmacokinetics of 126 in patients in an attempt to define safe dose limits for the Phase I clinical trial. These studies

suggested that in clinical trials the dose of dichloroallyl lawsone given by rapid intravenous infusion should not exceed 450 mg/m2 so that the maximal plasma drug concentration remains below 130 mg/L [99]. Acivicin (127) and dichloroallyl lawsone are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, Kemp et al. have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 L of an extract of cells grown in the presence of [14C] bicarbonate. Addition of dichloroallyl lawsone (25 M) results in a rapid depletion of uridine and cytidine nucleotides, carbamyl aspartate while dihydroorotate accumulates to high levels. The concentrations of orotate, orotidine and UMP increase transiently before reaching their original steady states. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP-- UDP initially followed by potent inhibition of dihydro-orotate [100]. Chau et al. have reported a dramatic elevation of hydrogen peroxide (H2O2) in human leukemia HL-60 cells following treatment with 1 M lapachone which can be effectively inhibited by treatment with antioxidant N-acetyl-L-cysteine (NAC), ascorbic acid,
-tocopherol. NAC strongly prevented -lapachone-induced apoptotic characteristics such as DNA fragmentation and apoptotic morphology. However, treatment of HL-60 cells with another topoisomerase inhibitor camptothecin (CPT) did not induce H2O2 production as compared to untreated cells. NAC also failed to block CPT-induced apoptosis. In agreement with these findings, these workers found that cancer cell lines K562, MCF-7, and SW620, containing high level of intracellular glutathione (GSH), were not elevated in H2O2 and were resistant to apoptosis by -lapachone treatment. In contrast, cancer cell lines such as, HL-60, U937, and Molt-4 having lower levels of GSH, readily underwent increase in H2O2 and were sensitive to this drug. These results suggest that intracellular H2O2 generation plays a crucial role in -lapachone-induced cell death and differentiation [101]. -lapachone has been found to inhibit DNA topoisomerases (Topos) by a mechanism distinct from that of other commonly known Topo inhibitors. Shiah and coworkers have demonstrated a pronounced elevation of H2O2 and O2- in human leukemia HL-60 cells treated with lapachone. Treatment with other Topo poisons, such as camptothecin (CPT), V-16, and GL331, did not have the same effect. On the other hand, treatment with antioxidant vitamin C effectively antagonized -lapachone-induced apoptosis. This suggested that a reactive oxygen species (ROS)-related pathway was involved in its apoptotic action. It was further shown that ROS acts as a mediator for JNK activation during -lapachone-induced apoptosis. These results indicate that -lapachone but not other topoisomerase inhibitors trigger apoptosis signaling [102]. b. Anti-Oxidant Activity Jacob and co-workers have studied the antioxidant activity of henna seeds in their extracts in petroleum ether, dichloromethane, ethanol and water respectively. Among these, the ethanol extract showed the best activity to scavenge DPPH radicals and inhibit lipid peroxidation [103].

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Among various fractions of Lawsonia inermis (Lythraceae), the n-butanolic fraction showed the highest antioxidant activity and potent capacity in preventing linoleic acid oxidation [104]. Hossain et al. have found that the leaf extract of Lawsonia inermis exhibits inhibition of lipid peroxidation as well as scavenging of DPPH radicals [105]. Al-Omar has studied effect of lawsone on generation of superoxide anion and hydrogen peroxide in phenanthridine oxidation by guinea pig aldehyde oxidase. The compound was found to specifically inhibit aldehyde oxidase and inhibit production of superoxide anion as well as substrate oxidation more potently than hydrogen peroxide [106]. Mikhaeil et al. have isolated lawsone and its analog, viz. 2-methoxy-3-methyl1,4-naphthoquinone (128) by bioassay-guided fractionation of the methanolic extract of henna leaves (Lawsonia inermis L.) and have evaluated its immunomodulatory as well as free radical scavenging activity where the analog was found to be more potent [107]. Guha and coworkers have studied the protective effect of aqueous and methanolic extracts of Lawsonia inermis Linn against Cr(VI)-induced cellular damage at concentrations of 20-60 g/mL which showed significant potential in scavenging free radicals (DPPH and ABTS+) and Fe3+, as well as lipid peroxidation and DNA damage caused by exposure of pBR322 to Cr(VI)-UV. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in human breast cancer MDA-MB-435 cells [108]. Since the antioxidant effects of these plants have been implicated in hepatoprotection, Bhandarkar and Khan have studied hepatoprotective activity of the bark extract of Lawsonia alba against carbon tetrachloride-induced hepatic damages in rats after oral dosing for 10 days. The treatment was found to produce significant protection against CCl4-induced elevation in serum marker enzymes, serum bilirubin, liver lipid peroxidation and reduction in total serum protein, liver glutathione, glutathione peroxidase, glutathione-s-transferase, glycogen, superoxide dismutase and catalase activity [109]. Similarly the hepato-protective activity of the ethanolic extract of the dried leaves of L. inermis and its fractions has also been evaluated against CCl4 induced hepatotoxicity in mice [110, 111]. Ahmed et al. have studied the hepatoprotective activity of the 50% ethanol extract of the bark of Lawsonia alba syn. and L. inermis against the carbon tetrachloride-induced oxidative stress. Pre-treatment of rats with doses of 250 and 500 mg/kg of the plant extract significantly lowered the enhanced serum transaminases (GOT and GPT) and LDH levels in a dose-dependent manner. Pretreatment of rats with the extract also inhibited the peroxidation of microsomal lipids [112]. The immuo-modulatory profile of lawsone has been studied using in vitro immunoassay, and lymphocyte transformation assay. The ABTS [2,2'azino-bis (3-ethyl benzthiazoline-6-sulfonic acid)], free radical scavenging assay showed that lawsone exhibited antioxidant activity equivalent to that of ascorbic acid [107]. c. Anti-Inflammatory Activity Crude ethanolic extract of Lawsonia inermis L. after fractionation using liquid-liquid extraction procedure was tested for anti-inflammatory, analgesic, and antipyretic effects in rats. The n-butanol and chloroform fractions showed more potent anti-inflammatory, analgesic, and antipyretic effects than the crude extracts, while the aqueous extract showed significantly less effect. The chloroform extract

yielded lawsone. It was found to possess significant antiinflammatory, analgesic, and antipyretic activity and it significantly potentiated the pentobarbitone-induced sleeping time. The anti-inflammatory effect of lawsone (500 mg/kg) was not significantly different from that of the reference drug phenylbutazone (100 mg/kg) [113]. Gupta and coworkers have reported on the anti-inflammatory activity of ethanolic extract of leaves of Lawsonia inermis in inflammation induced by cotton pellet, croton oil and formalin. Lawsone at a dose of 20 mg/kg was found to reduce ankle, paw diameter and granuloma weight, indicating potent anti-inflammatory activity [114]. Wurm and Baumann have concluded that the ortho-hydroxyl group to the quinone carbonyl and planarity of the lawsone molecule are important requirements for the potent anti-inflammatory activity and compounds satisfying these such as 2-ethyl-5-hydroxy-1,4-naphthoquinone (129) and 2-methyl-3-hydroxy-1,4-naphthoquinone (Phthiocol, 2) indeed possess potent anti-inflammatory activity [115]. Furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives (131137) have been synthesized from compound 130 which in turn was prepared from lawsone in one pot reaction Fig. (26). In the CLP rat animal model, compound 137 was found to be more active than the standard hydrocortisone drug used as positive control in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased remarkably by the pre-treatment with compound 137 at a dosage of 10 mg/kg [116]. Buckle et al. have synthesized nitrolawsone (138) which was found to be potent inhibitor of rat passive cutaneous anaphylaxis (PCA) whose and potency can be elevated with alkyl substitution at both C-6 and C-7 positions. Compounds 139 and 140 produced 50% inhibition in the rat PCA test at doses of 10
M/kg administered subcutaneously. The compounds showed activity even after oral administration [117]. Lira et al. have evaluated the topical formulation of lapachol by invitro release studies. The formulation showing the highest release rates were selected and assessed through skin permeation and retention experiments. It was observed that the gel formulation retains higher amount of lapachol while gelcream formulation provides significantly higher permeation. Anti-nociceptive and anti-edematogenic activities of the most promising formulations were evaluated. It was observed that lapachol gel reduced increase in the hind-paw volume induced by carrageenan injection and reduced the nociception produced by acetic acid when used topically. These results suggest that topical delivery of lapachol gel formulation may be an effective medication for both dermal and sub-dermal injuries [118]. Moon et al. have investigated the molecular mechanism of -lapachone effect on lipopolysaccharide (LPS)-induced responses in BV2 microglia. The compound significantly inhibited NO and PGE2 release in LPS-stimulated BV2 microglia. The inhibition of iNOS and COX-2 was also observed, suggesting blockage at transcriptional levels. In addition, the compound also attenuated the expression of mRNA and proteins of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)- in a dose-dependent manner. Moreover, the compound suppressed NF-B activation by blocking IB degradation, indicating that it may be useful as a potential anti-inflammatory agent for treating inflammatory disorders [119].

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d. Miscellaneous Activities Andrade-Neto et al. have evaluated antimalarial activity of benzo[a]phenazines synthesized from 1,2-naphthoqui none, lapachol, and -lapachone against Plasmodium falciparum in vitro using isolates of parasites with various susceptibilities to chloroquine and/or mefloquine. The growth of the parasite in the presence of these test drugs was measured by incorporation of [(3)H]-hipoxanthine in comparison to chloroquine as standard antimalarial compound. The 3sulfonic acid--lapachone-derived phenazine (141) was the most active compound exhibiting 98% inhibition of parasi-

taemia employing long term treatment subcutaneously, whereas the phenazine from 3-bromo--lapachone (142) was inactive [120]. The anti-mycobacterial activity of lapachol as well as its influence on macrophage functions has been investigated by Oliveira and group. Lapachol did not induce apoptosis/necrosis of THP-1 macrophages at
32 g/m concentration while Mycobacterium avium liquid growth was arrested at 32 g/mL and intra-macrophage proliferation at 16 g/mL concentrations. The main immunomodulatory effects of the compound included an up-regulation of interferon--receptor 1 (IFN-R1), histocompatibility complex class II (MHCII) surface expression, and marked inhibition

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of IL-10 secretion. The compound did not affect resting IFN or toll-like receptor 2 (TLR2)-induced levels of oxygen and key proteins of nitrogen metabolism nor the TLR2-mediated secretion of TNF-
. It did not induce either oxidative or endoplasmic reticulum (ER) stress. It inhibited the surface expression of the co-stimulatory molecule CD86 but not those of CD80 and CD83 respectively. The results indicated that the substituted lapachol compounds exhibit pronounced anti-mycobacterial activity that is more efficient intra- than extra-cellularly. These compounds exerted immuno-modu latory effects some of which may enhance the capacity of the host cell to control mycobacterial growth [121]. Kung et al. have investigated the effects of -lapachone on wound healing and its underlying mechanism. Their work demonstrated that a low dose of -lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without -lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in -lapachone-treated animals than in un-treated ones. In addition, -lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. These results suggest that -lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing [122]. Oliveira et al. have synthesized several 1,4-naphthoquinone derivatives having a hydrazino side chain from 3-diazo-naphthalene1,2,4-trione (42) and tested them as potential antimicrobial agents. These naphthoquinone derivatives showed greater antibacterial activity by disk method. Studies on the minimal inhibitory concentration (MIC) for Staphylococcus aureus indicated that compound 143 has an activity twofold greater than lapachol (30) while a comparable activity with vancomycin [39]. Fourteen ferrocenyl amino-hydroxy-naphthoqui nones, which are analogs of atovaquone were synthesized and tested against two apicomplexan parasites of medical importance, viz. Toxoplasma gondii and Plasmodium falciparum. Three of these derivatives 14-16 were found to be significantly active against T. gondii. Moreover, they were also effective against the atovaquone-resistant strain of T. gondii [31]. Antifungal activity of lawsone methyl ether (144) as mouthwash have been studied in vitro and in vivo and the compound was found to be potent enough to go into clinical trials by little adjustment in concentration of compound 144 in mouthwash [123]. A series of C-3 halo substituted 1,4-naphthoquinone derivatives (3, 145-146, 147a-f) have been synthesized and studied for their antifungal activities against C. albicans ATCC10231, C. albicans 955, T. mentagrophytes and M. gypseum Fig. (27). The results indicate that compound 2-hydroxy-3-chloro-1,4-naphthoquinone (3), 2-(N-acetyl)- acetamido-3-chloro- 1,4-napthoquinone (145) and 2-(N-acetyl)-acetamido-3-chloro- 1,4- napthoquinone (146) have potent antifungal activity. Among these compounds, 3 and 146 showed better activity than even standard clinically used antifungal drug, viz.clotrimazole against C. albicans ATCC10231. The C-3 halo substitution was found to be crucial for the antifungal activity [48]. Gardner has investigated the quinoidal pigment associated

with long grown cultures of Mycobacterium tuberculosis, viz. phthiocol and pyocyanine produced by Pseudomonas aeruginosa, for their effects on superoxide radical production in cultured human lung epithelial-like A549 cells. Elevated superoxide radical levels were detected in cells exposed to < 25 M phthiocol and < 2 M pyocyanine concentrations in neutral pH medium, wherein both agents impaired cell growth. DT-diaphorase, was a significant source of phthiocol- and pyocyanine-mediated O2- generation in cells. These two compounds operating through O2-/H2O2 cycle lead to inhibition of host cell aconitase activity, and other host cell functions, which may contribute to the pathogenicity of M. tuberculosis and P. aeruginosa [124]. SUMMARY Henna happens to be an ornamental plant usually employed for decorating skin, hairs and fingernails especially at the times of festivals. In recent times henna paste has been used for body art paintings and designs in western countries. However, in indigenous systems of Ayurved and Unani the plant has been regarded as providing non-toxic therapeutic agent for wounds, inflammation, persistent tuberculosis and tumors when used in combination with other ingredients. When translated into modern scientific terminology it amounts to transforming its active constituents into various analogs for specific therapeutic purposes. The account of such lawsone analogs as described in the present review provides adequate evidence for the efficacy of some of these compounds against certain cancers. It is of interest to note that lawsone itself along with its analogs exhibits potent antioxidant and anti-inflammatory properties which are the hallmarks of many anticancer phytochemicals. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS RP and PD acknowledge CSIR, New Delhi, INDIA for providing Junior and Senior Research Fellowships. We are thankful to Miss Pradnya Kedari, Department of Botany and Mrs. Nilima Vyas, Department of Chemistry, University of Pune for providing plant material and pictures of Henna powder and hand tattoos. REFERENCES
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Received: ????????????

Revised: ????????????

Accepted: ???????????????