Lapachol : an overview

Lapachol is a naphthoquinone that was first isolated by E. Paterno from Tabebuia avellanedae (Bignoniaceae) in 1882. A wide spectrum of therapeutic activities have been attributed to lapachol or its derivatives viz., anti-abscess, anti-ulcer, antileishmanial, anticarcinomic, antiedemic, anti-inflammatory, antimalarial, antiseptic, antitumor, antiviral, bactericidal, fungicidal, insectifugal, pesticidal, protisticidal, respiradepressant, schistosomicidal, termiticidal, and viricidal. Originally isolated from species of the Bignoniaceae family, lapachol can also be found in other families such as Verbenaceae, Proteaceae, Leguminosae, Sapotaceae, Scrophulariaceae, and Malvaceae. The interesting and most usefull knowledge on lapachol, which is reviewed in this paper, can be used as a starting point in future research endeavors.


Introduction
Compounds having a quinone as the core system have promising biological activity.A central feature of ortho-and para-quinonoid based cytotoxins is their ability to generate semiquinone radicals by bioreduction, which then accelerates intracellular hypoxic conditions. 1,2,3The drug arsenal currently available for fighting against tropical diseases includes many natural and synthetic naphthoquinone derivatives which have been extensively studied due to their ability to interfere with the bio-activities of enzymes known as topoisomerases, 3 a group of enzymes that are critical for DNA replication in cells.In addition, naphthoquinones have been shown to induce the so-called "reactive oxygen species" that can cause damage to cells. 3Since the pioneering work of Wendel 4 in 1946, who demonstrated that certain naphthoquinones inhibit the growth of Plasmodium vivae, and the work of Fieser et al. in the 1940's, an extensive search for new quinones for use in malaria chemotherapy has started. 5Subsequently, quinones have been studied for antitumor, 6 trypanocidal, 7 molluscicidal, 8 leiscmanicidal, 9 anti-inflammatory 10 and antifungal 11 activities.Lapachol 1 (Figure 1) was first isolated from Tabebuia avellanedae (Bignoniaceae) and in the years since the first review on lapachol, published in Portuguese in 2003 in Revista Brasiliera de Farmacia, 12 there has been a rapid increase in the information available on lapachol.

Tabebuia avellanedae
Lapachol (1) was first isolated by E. Paterno 33 from Tabebuia avellanedae in 1882.Tabebuia avellanedae (Figure 2) is a tree from the Bignoniaceae family.Commonly know as "pau d'arco" (alternative name: Lapacho, Pau d'Arco; Taheebo) in Brazil, and its inner bark is used as an analgesic, anti-inflammatory, antineoplasic 34 and diuretic by the local people in the north eastern parts of Brazil.Its anti-inflammatory, antimicrobial, and antineoplasic activities are cited in the literature and is supported by saponines, flavonoides, cumarines, and other natural antibiotics, such as derivatives of lapachol often included among the constituents of the extracted plant material. 34The aqueous and methanol extracts of T. avellanedae for instance also showed antifungal, antinociceptive and antiedematogenic activities. 34Species that contain lapachol and several biogenetically related naphthoquinones (e.g., tahaebo, pau d'arco and lapacho roxo) are widely used in American folk medicine for the treatment of cancer, lupus, infections, wounds, and many other diseases. 35

Paulownia kawakamii
Lapachol (1), a yellow coloring matter which occurs in the grain of a number of wooden trees, was suggested by Paterno 33 to have the structure of an amylene hydroxynaphthoquinone with both substituent groups in the quinone ring (structure 2, Figure 3).After Hooker 36 had presented convincing reasons for regarding the substance as a derivative of α-naphthoquinone, Paterno's structure 2 (Figure 3) remained uncertain only in respect to the position of the double bond in the prenyl side chain.Hooker 37 then carried out a synthesis of the compound corresponding to 2 by the condensation of isovaleraldehyde with hydroxynaphthoquinone and obtained an isomer of lapachol which however, was regarded as the o-quinone isomer of 2 (iso-β-lapachol) on account of its red color.Since the difference between the synthetic and the natural products extended to their hydroquinones, structure 2 could no longer be ascribed to that of lapachol.Of the remaining possibilities, structure 1 was given preference by Hooker, after finding that both lapachol and iso-β-lapachol could be converted into the same hydroxy compound, having the side chain of -CH=C(OH)CH(CH 3 ) 2 .Hooker's structure for lapachol (1) was thus largely influenced by the structure assigned to iso-β-lapachol, and this cannot be regarded as being established with certainty.Indeed, as a direct consequence, Fieser 38 undertook a synthesis of the compound corresponding to structure 1 by reaction of the silver salt of hydroxynaphthoquinone with γ,γdimethylallyl bromide (isoprene hydrobromide).Although the greater part of the silver salt was indeed converted into hydroxynaphthoquinone, a small amount of the normal alkylation product, viz., the 2-alkoxy-l,4-naphthoquinone, was produced together with a small amount of an acidic isomer to which structure 1 was assigned.The latter substance, 2-( γ,γ-dimethylallyl)-3-hydroxyl,4-naphthoquinone, was found to be identical with lapachol by direct comparison with a sample of the natural substance which was kindly furnished by Hooker.

Pharmacological activities of lapachol and its derivatives 4.1 Antitumor activity
In a 1968 study, lapachol demonstrated highly significant activity against cancerous tumors in rats. 39Then in 1974, the NCI reported that Phase I clinical trials failed to produce a therapeutic effect with lapachol without side effects and discontinued further cancer research. 40In a small study in 1980 with nine patients with various cancers (liver, kidney, breast, prostate and cervix), pure lapachol demonstrated an ability to shrink tumors and reduce feeling of pain caused by these tumors and achieved complete remissions in three of the patients. 41It is believed that the antitumor activity of lapachol may be due to its interaction with nucleic acids.Additionally it has been proposed that interaction of the naphthoquinone moiety between base pairs of the DNA helix occurs with subsequent inhibition of DNA replication and RNA synthesis. 42lthough lapachol has some beneficial effects, it is by no means a perfect anticancer drug.Despite the lack of significant toxicity even in large oral dose levels, sufficiently high blood levels were not attained to show a therapeutic effect. 43This led to the termination of further clinical development of lapachol.Because of its antitumor activity, it is an ideal candidate for systematic modification to develop an understanding of its structure-activity relationships and thus eventually to develop analogs with improved activity.

Anti-metastatic activity
Metastasis is the major process responsible for the death in cancer patients.Balassiano et al. 46 analyzed the effects of lapachol on a human cancer cell line and evaluated the potential of this substance as an anti-metastatic drug using an in vivo assay.The results of this study indicated that lapachol, in the maximal non-toxic concentration for HeLa cells of 400 µg/ml (corresponding to 10 12 molecules of the drug/cell), induces alterations in the protein profile and inhibits cellular invasiveness, thus representing an important anti-metastatic activity.

Antimicrobial and antifungal activity
Lapachol, like many napthoquinones, interferes with the electron transport system and inhibits the cell respiratory mechanism.In a study that was done in the 1940's, it was found that lapachol at a concentration of 100 mg/ml, inhibits the uptake of oxygen in Plasmodium Knowles by 74 % and the succinate oxidase system by 26 %.These findings lead to the conclusion that lapachol exhibits antimalarial activity against Plasmodium lapohurae via respiratory inhibition as a likely mechanism.However, the exact mechanism of action remains controversial.It is hypothesized that lapachol either inhibits the interaction between the cytochromes b and c or directly inhibits an unknown enzyme between the two cytochromes. 42It was also found that lapachol has activity against H. pylori, Staphylococcus, Streptococcus, Enterococcus, Bacillus and Clostridium species with an MIC ranging from 1.56 to 25 mcg/ml.8][49] As part of our systematic search for new bioactive lead structures from African medicinal plants, lapachol has been isolated from Newbouldia laevis and demonstrated antiplasmodial activity as well as antimicrobial activities against some Gram positive and Gram negative microorganisms. 19,50,51

Antiviral activity
Lapachol was found to be active against certain viral strains including herpes virus types I and II.Naphthoquinones have been documented to show effectiveness against four strains of the flu, polio and vesicular stomatitis virus.Lapachol and its enamine showed larvicidal and insecticidal activity against Artemia salina and Aedes aegypti, respectively and for cytotoxicity against A549 human breast cancer cells. 53he mechanism of action of these quinones is supposed to be via DNA and RNA polymerase and retrovirus reveres transcriptase inhibition.Furthermore, β-lapachone is presumed to interfere with the replication of the HIV-1 virus via transcriptase inhibition. 54It is reported that lapachol decreases the replication of viruses in human subjects but as yet there is no available clinical data. 54

Anti-inflammatory activity
T. avellanedae is believed to have an inhibitory effect on the histamine releasing cells which consequently leads to anti-inflammatory effects.Lapachol, in the preliminary study produced a significant anti-inflammatory action in rats. 55According to another in vitro study, lapachol and its analogs were shown to have antipsoriatic effects by inhibiting the growth of human karatinocyte cell line HaCaT and reducing inflammations.The authors of the study 56 concluded that lapachol has similar antipsoriatic activity to that of anthralin in inducing damage to the cell membranes of the karatinocyte cells.However, the authors did not report the degree of similarity or difference between the two agents. 56

Antiparasitic activity
Lapachol has been used as a topical barrier to trematodes specifically Shcistosoma mansion, which causes schistosomiasis.This parasite lives in water and enters the host by penetration through the skin.This pathogen can cause a complicated disease, which can sometimes be fatal.It has also been stated that oral lapachol formulation is effective against skin penetration.In addition, lapachol is claimed to have some effect against Trypanosoma cruzi, which causes trypanosomiasis or Chaga's disease.This disease can be present in either acute or chronic form and has no known cure to date. 42However, there is no documented data available in either humans or animal models.Schmeda-Hirschmann 57 reported the antiparasitic activity of lapachol and related naphthoquinones. 9reported that lapachol exhibits marked leishmanicidal activity in vitro.Recently, Lima et al. also reported the antileishmanial activity of lapachol and its derivatives. 52,58he mechanism by which lapachol induces lysis of intracellular amastigotes in vitro is not yet clear.However, a characteristic of the naphthoquinones (especially lapachol and β-lapachone) is that they interfere with the oxygen metabolism of the tumour cell, blocking cell respiration and generating free oxygen radicals.Besides free oxygen radicals, there is evidence that nitric oxide (NO) production, which follows the induction of nitric oxide synthetase by IFN-γ, plays an important role for the death of intracellular amastigotes in murine macrophages.NO is considered the most important metabolite involved in Leishmania killing in mice. 9

Molluscicidal activity
Lapachol was assayed for molluscicidal activity against Biomphalaria glabrata and showed significant molluscicidal activity. 59The naphthoquinone is bioactivated by P 450 reductase into reactive species which in turn promote DNA scission through the generation of superoxide anion radicals by redox cycling.Silva et al. 60 and Santana et al. 61 synthesized derivatives of lapachol for a molluscicidal assay.The partially hydrogenated lapachol derivative 6 (Figure 5), obtained from the catalytic reduction of lapachol, showed molluscicidal activity significantly higher than that of lapachol itself.These findings confirm the importance of lapachol as a pivatol starting material for the production of biologically active compounds. 60

Synthesis
Fieser's synthesis of lapachol was the first one of this molecule which helped to confirm the structural assignment (see section 3).The early approach employed as terminal step (5 % yield) an alkylation of the silver salt of 2-hydroxy-1,4-naphthoquinone with 1-bromo-3-methyl-2butene (Scheme 1).

Scheme 2
Ten years later Petti and Houghton 68,69

Scheme 3
An interesting alkylation method of lawsone (2-hydroxy-1,4-naphthoquinone) towards a preparative synthesis of lapachol on a large scale was recently reported. 70The lithium salt of lawsone was prepared in situ by the addition of lithium hydride to a frozen solution of the quinone in dimethylsulfoxide.As the solution thawed, the lithium salt was slowly formed and it was then alkylated with 3,3-dimethylallyl bromide, to afford the desired lapachol in 40 % yield (Scheme 4).

Scheme 4
In an attempt at improving the yield in lapachol synthesis, Kazantzi et al. 71 recently reported a new synthesis with an overall 43 % yield.In this instance lapachol was prepared by reaction between lawsone and 3-methylbut-2-en-1-ol in the presence of a catalysts such as Pd(Ph 3 P) 4 and 1-admantanecarboxylic acid (Scheme 5).

Scheme 5
In conclusion, the synthesis of lapachol was important for the varification of the structure but is of little practical value when considering the abundance this natural product as a metabolite in several tropical trees.

Scheme 6
The mode of incorporation of [1-l3 C]OSB into the naphthoquinone, juglone (27) in Juglans regia seedlings, and into lawsone (25) in Impatiens balsamina plants has been investigated. 72uglone carried approximately 50% of the label in each carbonyl group, indicating the participation of a symmetrical intermediate, whilst lawsone was specifically labeled at position 1, demonstrating asymmetrical incorporation (Scheme 7).1,4-Naphthoquinone ( 26) is known to be a precursor of juglone, and both 26 and the glucoside of its quinol form are found in Juglans regia. 72Thus, although 1,4-naphthoquinone ( 26) is therefore a likely intermediate in the formation of juglone (27), the decarboxylation and hydroxylation processes in the biosynthesis of lawsone (25)

Scheme 7
Melvalonic acid ( 28) is well established as the source of prenyl side chains in quinones.It is thus more probable that the prenyl side chain of lapachol is assembeled as a prenyl pyrophosphate (29) which then couples with 2-hydroxynaphthoquinone (25) to form lapachol (1) (Scheme 8).

Derivatives
A limited number of natural and semisynthetic derivatives were obtained because the functional groups of lapachol are not sufficiently suitable for structural modification.The first two natural analogues of lapachol to be isolated were β-lapachone (30) and α-lapachone (31) (Figure 6) which were present in Tabebuia avellanedae along with lapachol. 47The α-and β-lapachone were synthesized from lapachol using the Hooker acid catalyzed procedure. 22RKAT USA, Inc.  30) is a naturally occurring quinone derived from the lapacho tree, (Tabeuia avellanedae) native to Central and South America.The synthesis and chemistry of β-lapachone and related compounds was initially investigated in the late 19 th and early 20 th centuries by the chemist Samuel Hooker. 36,37Although some clinical studies on the bioactivity of naphthoparaquinones were reported early in 1940, it was Docampo's report of β-lapachone's potential as an anti-trypanosomal agent in 1977 that revitalized the study and characterization of this unique natural compound. 75revious studies demonstrated that β-lapachone can directly target DNA topoisomerases and inhibit their activity, which results in cytotoxicity. 76However, its inhibitory mode is quite distinct from that of other typical topoisomerase inhibitors, viz., camptothecin and its related compounds. 76β-Lapachone also exhibits a number of pharmacological actions including antibacterial, anti-fungal, anti-trypanocidal, and cytotoxic activities, which are linked to the formation of reactive oxygen species. 76

Anti-angiogenic activity
Neovascularization is an essential process in tumor development and thus it is conceivable that anti-angiogenic treatment may block tumor growth. 77In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration.β-Lapachone has been demonstrated to possess anti-cancer and anti-viral effects.Whether β-lapachone can induce endothelial cell death or has an anti-angiogenic effect is still uncertain. 77Kung et al. 77 recently investigated the in vitro effect of β-lapachone on endothelial cells, including the human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC).Kung et al. demonstrated that NO can attenuate the apoptotic effect of β-lapachone on human endothelial cells and suggest that β-lapachone may thus have potential as an antiangiogenic drug. 77

Anti-inflammatory activity
β-Lapachone is a chemotherapeutic agent that inhibits the expression of nitric oxide (NO) and inducible NO synthase (iNOS) in alveolar macrophages.Moon et al. 78 investigated the molecular mechanism of β-lapachone on lipopolysaccharide (LPS)-induced responses in BV2 microglia.They found that treatment of β-lapachone significantly inhibited NO and PGE2 release in LPSstimulated BV2 microglia. 78The inhibition of iNOS and COX-2 was also observed suggesting the blockage of transcriptional levels.In addition, β-lapachone attenuated the expression of mRNA and proteins of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in a dose-dependent manner.These results showed that β-lapachone may be useful as a potential anti-inflammatory agent for attenuating inflammatory diseases. 78

Anti-tumor activity by activation of the Mre11-Tel1p G1/S checkpoint
β-Lapachone is an anticancer agent that selectively induces cell death in several human cancer cell lines. 79However the precise mechanism of β-lapachone cytotoxicity is not yet fully understood.Menacho-Marquez and Mauricio 79 reported that β-lapachone treatment delayed cell cycle progression at the G1/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast, Saccharomyces cerevisiae. 79urthermore, β-lapachone induced phosphorylation of histone H2A at serine 129.These checkpoint responses were regulated by Mec1p and Tel1p kinases. 79Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following β-lapachone treatment in asynchronous cultures, but not for the G1 delay.The major and vital conclusion of all those findings indicated that β-lapachone activates a Mre11p-Tel1p checkpoint pathway in budding yeast.Given the conservative nature of the Mre11p-Tel1p pathway, these results suggest that activation of the Mre11-Tel1p checkpoint could be of significance for β-lapachone anti-tumor activity.

Use of β-lapachone in pancreatic cancers
Erik Bey et al. 80 discovered that β-lapachone, currently in phase II clinical trials for use in pancreatic cancers, is also effective against NSCLC (Non-Small Cell Lung Cancer).The authors found that NSCLC cells over express endogenous NAD(P)H:quinone oxidoreductase 1 (NQO1), similar to pancreatic cancers.In NQO1-positive cells, β-lapachone induced PARP-1-mediated cell death. 80Typically, PARP-1 facilitates DNA repair by resealing single strand breaks.When NQO1 is confronted with massive DNA damage, such as after β-lapachone treatment, it triggers µ-calpain cell death mechanisms.β-Lapachone was most effective when delivered in short, 2-to 4-h pulses.Downstream, the chemotherapeutic agent killed NSCLC cells independent of cell cycle or p53 status and in the absence of proapoptotic factors, according to the authors. 80ugh et al. 81 also found that cytotoxic in vitro effects of β-lapachone were inhibited with coadministration of dicumarol, a specific inhibitor of NQO1.In pre-established human pancreatic tumor xeno grafts in nude mice, β-lapachone demonstrated greater tumor growth inhibition when given intratumorally compared to when complexed with cyclodextrin to increase its bioavailability. 81n another study on β-lapachone, Park et al. 82,83 strongly suggested that NQO1 activity in tumors may be further and selectively elevated using either local radiotherapy or hyperthermia, both established cancer treatment modalities, to improve the cytotoxicity of β-lapachone against human cancer cells. 82,83,842.5 Use of β-lapachone in treatment of neuroendocrine tumors Larsson et al. 85 studied in vitro drug sensitivity screening using the fluorometric microculture cytotoxicity assay in one human pancreatic carcinoid and two human bronchial carcinoid cell lines.The aim of this study was to investigate drug sensitivity in neuroendocrine tumor cell lines.In addition, a normal human retinal pigment epithelial cell line was used for comparison.A total of 18 drugs, including β-lapachone, with different mechanisms of action were tested.These studies indicated that some of the tested compounds viz., β-lapachone, could possibly be used in clinical trials and demonstrate a therapeutic effect in patients suffering with neuroendocrine tumors.85

Selective necrotic cell death by DNA damaging activity
Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment.However, apoptotic pathway deficiencies are a hallmark of cancer cells. 86Sun et al. 86 proposed that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death.They showed that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways.While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells.Sun et al. 86 also found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo.All these data suggested that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy to eliminate cancer cells. 86

Use of β-lapachone in apoptosis induction
Woo et al. 87 studied the effects of β-lapachone on the growth of the human hepatoma cell line HepG2.The results showed that β-lapachone inhibits the viability of HepG2 by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. 87Reverse transcription-polymerase chain reactions and immunoblotting results indicated that treatment of cells with β-lapachone resulted in down-regulation of anti-apoptotic Bcl-2 and Bcl-XL and upregulation of pro-apoptotic Bax expression.However, β-lapachone treatment did not affect the inhibitor of apoptosis proteins family and the Fas/FasL system.Taken together, this study indicated that β-lapachone may have potential as a chemopreventive agent for liver cancer. 87imilarly Woo et al. 88 also investigated what effect β-lapachone would have on the cell growth and apoptosis in the human lung carcinoma cell line A549.Exposure of A549 cells to βlapachone resulted in growth inhibition and induction of apoptosis in a time-and dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy and flow cytometry anal.These findings provided important new insights into the possible molecular mechanisms of the anti-cancer activity of β-lapachone. 88ee et al. found that in the micromolar concentration range, β-lapachone inhibited the viability of human bladder carcinoma T24 89 and human prostate carcinoma DU145 90 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation.This investigation demonstrated that β-lapachone may be further studied as a promising agent for treatment of bladder cancer 89 as well as providing important new insights into the possible molecular mechanisms of the anti-cancer activity of β-lapachone. 90

β-Lapachone in combination treatment
Ablation of tumor colonies has been observed in a wide spectrum of human carcinoma cells in culture after treatment with a combination of β-lapachone and taxol, two low molecular mass compounds. 91They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells.This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. 91umi-Diaka et al. 92 studied the genistein and β-lapachone combination treatment to examine the role of NQOI in the signaling of genistein-and β-lapachone-induced apoptosis in human prostate carcinoma cells PC3.

Reaction of lapachol in pyridine
Jassbi et al. 93 isolated lapachol from heartwood shavings of Heterophragma quadriloculare and found it to be the most abundant naphthoquinone.To investigate the pyridine catalyzed product of lapachol, the main pigments in the H. quadriloculare plant, Jassbi et al. 94 heated lapachol (1)  in pyridine under reflux (Scheme 9) and isolated quadrilone (32), dehydro-α-lapachone (34), and adenophyllone (33).Dehydro-α-lapachone (xiliodone, 34) has also previously been synthesized from lapachol by FeCl 3 oxidation in the presence of pyridine, acetic anhydride and xilidione and was reported to have an antibiotic effective against Gram negative bacteria of the genus Brucella. 93Adenophyllone (33) is a naphthoquinone previously isolated by Jassbi et al. 93 from Heterophragma adenophyllum.The detailed mechanism describing the formation of compounds 32-34 has been described by Jassbi et al. 94  Scheme 10.Suggested mechanism for the formation of qaudrilone (32). 94The nucleophile may be pyridine or a conjugated base of a hydroxynaphthoquinone.

Perspectives
An evaluation of the therapeutic effects of lapachol started in the late 1960s by Rao, McBride and Oleson.After this study, many others confirmed the efficacy of this naphthoquinone as an antineoplastic agent.However, in 1974, the National Cancer Institute concluded that, due to the high concentrations necessary for this drug to act as an effective chemotherapeutic agent in human cancer which unfortunately also resulted in very toxic side effects, suspension of further studies on the action of lapachol as an antineoplastic agent would be terminated.However, none of these studies considered the effects of lapachol at a molecular level.This information forms the basis to develop the synthesis of new and novel lapachol analogues which can be used as the future starting point in future developments of this potentially potent biological molecule.

Table 1 .
Occurrence of lapachol in families and species In 1948 Gates and Moesta 67 reported a synthesis of lapachol by the condensation of leucoisonaphthazarin with isoprene (Scheme 2).
reported the first practical total synthesis of lapachol with a 35 % overall yield illustrated in Scheme 3.