Molluscicidal activity of 2-hydroxy-[1,4]naphthoquinone and derivatives

BIOLOGICAL SCIENCES

Molluscicidal activity of 2-hydroxy-[1,4]naphthoquinone and derivatives

Celso A. Camara^I; Tania M.S. Silva^II; Thiago G. da-Silva^II; Rodrigo M. Martins^II; Ticiano P. Barbosa^II; Angelo C. Pinto^III; Maria D. Vargas^IV

^IDepartamento de Química, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-030 Recife, PE, Brasil

^IILaboratório de Tecnologia Farmacêutica, LTF-UFPB, Campus I, Cidade Universitária, Caixa Postal 5009, 58051-970 João Pessoa, PB, Brasil

^IIIDepartamento de Química Orgânica, Instituto de Química-CT, Bloco A, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590, Rio de Janeiro, RJ, Brasil

^IVInstituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista, Campus do Valonguinho, Centro, 24020-150, Niterói, RJ, Brasil

^{Correspondence to}

ABSTRACT

The toxic profile of lawsone (2-hydroxy-[1,4]naphthoquinone) and a series of [1,4]naphthoquinone derivatives was evaluated against the brine shrimp Artemia salina and against the mollusk Biomphalaria glabrata, the main transmitting vector of schistosomiasis in Brazil. Of the seventeen compounds tested nine fell below the threshold of 100 µg/mL set for potential molluscicidal activity by the World Health Organization. As a general rule derivatives with non-polar substituents presented the highest molluscicidal activities. These substances showed significant toxicity in A. salina lethality bioassay.

Key words:Biomphalaria glabrata, Artemia salina, toxicity, quinones, schistosomiasis, molluscicidal activity, lawsone, 1,4-naphthoquinone.

RESUMO

A toxicidade da lausona (2-hidroxi-1,4)-naftoquinona e de diversos derivados foi avaliada frente à Artemia salina e ao molusco Biomphalaria glabrata, o principal vetor de transmissão da esquistossomose no Brasil. Entre os dezessete compostos testados, nove apresentaram um perfil de toxicidade menor que 100 µg/mL, sendo potenciais agentes moluscicidas de acordo com as designações da Organização Mundial da Saúde. No presente estudo, os compostos contendo substituintes apolares exibiram as maiores atividades. Estes compostos também se mostraram significantemente tóxicos frente à A. salina.

Palavras-chave:Biomphalaria glabrata, Artemia salina,toxicidade, quinonas, esquistossomose, atividade moluscicida, lausona, 1,4-naftoquinona.

INTRODUCTION

Among human parasitic diseases, schistosomiasis (also called bilharziasis) remains one of the most prevalent parasitic infections, with significant economic and public health consequences (Chitsulo et al. 2000). Mortality due to schistosomiasis was estimated at 11,000 deaths per year (WHO 2005). The aquatic gastropod mollusk Biomphalaria glabrata (Perrett and Whitfield1996, Verjovski-Almeida and DeMarco 2004) is themain intermediate host of schistosomiasis in SouthAmerica. Infection usually occurs by contact with water containing infected snails. Permanent solutions to this problem include restriction of human contact with polluted water and prevention of further contamination of the environment (Chitsulo et al. 2000). The use of molluscicides as a prophylactic treatment involves breaking the life cycle of Schistosoma mansoni (the etiological agent of schistosomiasis) through the destruction of its intermediate host, the snail B. glabrata (Perrett and Whitfield 1996, Bezerra et al. 2002). This constitutes the weakest link in the transmission cycle and is the logical point of attack to control the disease. The search for molluscicides is of great interest for potential focal control of parasitary diseases, especially schistosomiasis, in endemic countries (Pointier and Giboda 1999, Capron 1998). New, safe, and effective molluscicides are urgently needed, but in order to be useful the products must be stable, inexpensive and easy to apply, and must show high selective toxicity to the target pest (Singh et al. 1996).

In our continuing search for bioactive substances, either natural (Silva et al. 2007a, b, 2006, 2005a) or synthetic (Silva et al. 2005b, Barbosa et al. 2005, Vasconcellos et al. 2006) we have recently described some effective molluscicide compounds prepared from natural

2-hydroxy-[3-methyl-(2-buten)]-1,4-naphthoquinone

(lapachol). Studies on the amine and aza-antraquinones derivatives of lapachol and nor-lapachol (2-hydroxy-[3-methyl-(2-propen)]-1,4-naphthoquinone) showed that in general the compounds containing the most polar groups bonded to the nitrogen generally exhibited the lowest molluscicidal activities and that the lapachol derivatives were slightly more active than the nor-lapachol ones(Silva et al. 2005b, Barbosa et al. 2005). In order to find out whether a side chain on position 3 of the aminonaphthoquinone is necessary for activity, we have evaluated, and describe herein, both molluscicidal activity and brine shrimp toxicity of readily available 2-amino-derivatives of lawsone 1 (2-hydroxy-1,4-naphthoquinone) (Fieser and Martin 1955), whose structures are shown in Figure 1.

EXPERIMENTAL

SYNTHESIS OF THE COMPOUNDS

Seventeen derivatives of [1,4]naphthoquinone were prepared by know methods and their structures were confirmed by infrared spectroscopy, NMR, and mass spectrometry. Some of the title compounds are analogues of 2-amino-derivatives of lapachol and nor-lapachol reported elsewhere (Silva et al. 2005, Barbosa et al. 2005). All the nitrogen compounds were readily obtainedby nucleophilic displacement of the methoxyl groupof 2-methoxy-1,4-naphthoquinone 2 (Fieser and Martin 1955) with the appropriate amines. Except for compound 13, all compounds were described previously in the literature: 2-bromo-3-methoxy-1,4-naphthoquinone 3 (Fieser and Brown 1949), 2,3-dibromo-1,4-naphthoquinone 4 (Kohn and Schwarz 1926), 2-azido-1,4-naphthoquinone 5 (Molina et al. 1995), 2-nitrogen derivatives 6-16 (Aristoff and Johnson 1992, Bowman et al. 1969,Couladouros et al. 1997, Fieser et al. 1948, Johnson et al. 1997) and cyclic derivative 17 (Kallmayer and Seyfang 1980). All compounds were purified by chromatographic techniques, especially flash chromatography, using silica-gel-60 (230-400 Mesh, Fluka), and fully characterized by analytical and spectroscopic methods. Melting points were obtained from an electrically heatedmetal block apparatus (Quimis) and compared with the literature data. FTIR spectra were obtained in a Bomen-Michelson spectrophotometer using KBr film, NMRspectra, in a Varian-Mercury 200 MHz for ¹H and 50.3 MHz for ¹³C, with CDCl₃ or DMSO-d6 as solvents, and HR mass spectra, on a VG Autospec spectrometer (electron-impact at 70 eV).

MOLLUSCICIDAL ASSAYS

The bioassays were carried out as described previously (Silva et al. 2005b, 2007a, b) by dissolving the sample first in dimethyl sulfoxide (DMSO) and then adding dechlorinated water, to give a solution 0.1% in DMSO. Ten adult snails (9-16 mm in diameter) were placed in a beaker, containing 250 mL of the molluscicide suspension at four appropriate concentrations (1000, 100, 10 and 1 µg/mL). Each test concentration was set in duplicate. Snails were exposed to the potential molluscicide for 24 h, at room temperature, and were kept under normal diurnal lighting. After 24 h, the suspension was decanted; the snails were washed with water and offered lettuce leaves as food. The tested snails were then left in water for another 24 h, and at the end of this period were examined to assess mortality. Snails were considered dead if they either remained motionless or did not respond to the presence of food, or if the shell looked discolored. In order to verify the snails' susceptibility, two control sets were used: one with cupric carbonate at 50 ppm and the other containing 0.1% DMSO dechlorinated water. The concentrations that kill 90% (LC₉₀), 50% (LC₅₀) and 10% (LC₁₀) of the exposed snails (that would have survived in the negative-control cultures) was estimated by probit analysis, using the Origin 6.0 software package (Microcal Software, Northampton, MA). In this study, B. glabrata snails were grown in the laboratory (Laboratório de Tecnologia Farmacêutica, UFPB) from stock which originated from Universidade Federal de Pernambuco (Professor F.F. Amancio, CCB-UFPE). They were not infected by trematodes and were grown in transparent containers with appropriate sourcesof food, light and temperature.

TOXICITY AGAINST Artemia salina

The brine shrimp lethality bioassay was performedfollowing the reported procedure (Barbosa et al. 2005). The growth medium was prepared with sea water of in a small tank divided into two compartments. The shrimp eggs were added to the covered compartment. A lamp was placed above the open side of the tank to attract hatched shrimps through perforations in the partition wall. After 48 h the shrimps are mature as nauplii and ready for the assay. Test compounds were dissolved in three drops of Cremophor^®, 2 mL of DMSO and sea water to complete 5 mL of total volume. Appropriate volumes were then added to tubes with 5 mL ofsea water containing 10 nauplii to afford the five desired concentrations, in quadruplicate for each concentration. The control samples containing Cremophor^® and DMSO, under the same conditions, do not cause significant brine shrimp mortality. After 24 h incubation under light, the number of dead and survivor brine shrimps in each tube was counted. The LC₅₀ values were calculated by graphics from drug concentration vs. lethality percentage using a Probit scale adjust. Data analysis was performed with Origin 6.0 software.

RESULTS AND DISCUSSION

All compounds, except for the glycine derivative 15, showed toxicity in the brine shrimp lethality bioassay, with toxicities ranging from LC₅₀ = 3.1 (4) to LC₅₀ = 163.5 µg/mL (12), as summarized in Table I. Substitution at positions 2 and 3 of the naphthoquinone nucleus resulted in a series of compounds with increased toxicity compared with lawsone 1. For example, substitution of the hydroxyl group in 1 (LC₅₀ = 97.3 µg/mL) for a methoxyl group in 2 (LC₅₀ = 14.6 µg/mL) led to a remarkable six fold enhancement of the toxicity. Introduction of a bromine atom at position 3 in compound 3 (LC₅₀ = 10.2 µg/mL) and of two bromine atoms in compound 4 (LC₅₀ = 3.1 µg/mL) resulted, respectively, in ten and thirty fold toxicity enhancement compared to compound 1. The presence of an azido group in 5 (LC₅₀ = 26.1 µg/mL) in place of the hydroxyl group in 1 also resulted in toxicity enhancement. Although the amino group in compound 6 (LC₅₀ = 14.4 µg/mL showed similar profile to the methoxyl group of 2, further substitution at the nitrogen atom led to an overall loss of activity, the toxicity in the 2-aminonaphthoquinone series ranging from LC₅₀ = 10.1 µg/mL (amine 7) to LC₅₀ = 163.5 µg/mL (compound 12). This series also includes a non-toxic derivative 15 with a glycine moiety. Decrease in polarity of the group attached to the nitrogen atom did not show any clear correlation with toxicity (e.g. compounds 10 and 9 presented similar activity profiles), and neither did the introduction of oxygen functionalities attached to the 2-amino groups [e.g. compounds 13 (LC₅₀ = 21.7 µg/mL), 2 (LC₅₀ = 14.6 µg/mL) and 6 (LC₅₀ = 14.4 µg/mL)].

As shown in Table I of the seventeen compounds tested nine fell below the threshold of 100 µg/mL set for potential molluscicidal activity by the World Health Organization (WHO 1965). The results of the assays of compounds 1-17 indicated that the dibromonaphthoquinone 3 (LC₅₀ = 2.1 µg/mL), which contains two non-polar groups, is the most active of the series, followed by the azido derivative 5 (LC₅₀ = 7.4 µg/mL). These two compounds also figure as the most toxic in the brine shrimp assay for overall toxicity profile. As a general rule, derivatives with non-polar substituents (e.g., 1-6 in Table I) present the highest molluscicidal activities. The presence of a glycine moiety results in inactive compounds, as found previously for analogous derivatives of lapachol (Silva et al. 2005b) and nor-lapachol (Barbosa et al. 2005). The appended moieties of derivatives 13 and 14 led to inactivity in the present study and in the nor-lapachol series (Barbosa et al. 2005). In the lapachol series, however, the corresponding compounds exhibit median to low activities (LC₅₀ = 54.9 and 72.6 µg/mL, respectively) (Silva et al. 2005b).

In general, the amine derivatives of lapachol and nor-lapachol showed higher moslluscicidal activity than those of lawsone 1 (Silva et al. 2005b, Barbosa et al.2005), probably for being less polar than 1, as a result of the presence of a lipophilic side chain; this result is in agreement with the general trend discussed above. Furthermore, the desired lowest toxicity in thebrine shrimp assay/highest molluscicidal activity, although not verified for the most active compounds (3 and 5), was achieved in the nor-lapachol series (Barbosa et al. 2005) which suggests that the side chain is an important tool in tuning this selectivity.

Thus although several compounds containing polar substituents exhibited high toxicity in the brine shrimp assay, as a general rule these compounds did not show molluscicidal activity (e.g., compounds 13 and 16).

The bioactivity of lawsone 1 and derivatives 2-17 shows a clear correlation with the presence of non-polar groups appended to the 1,4-naphthoquinone nucleus of the tested compounds. These results are in agreement with our previous findings with a similar series of 2-amino-naphthoquinones synthesized from lapachol and nor-lapachol.

ACKNOWLEDGMENTS

This work was supported by grants from ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Apoio a Núcleos de Excelência (PRONEX-CNPq), Instituto do Milênio do Semi-Árido (IMSEAR-CNPq), Rede Nordestina de Biotecnologia (RENORBIO) and Programa Institucional deBolsas de Iniciação Científica (PIBIC-UFPb).

Manuscript received on October 10, 2007; accepted for publication on January 21, 2008; contributed by ANGELO C. PINTO^* AND MARIA D. VARGAS^*

• ARISTOFF PA AND JOHNSON PD. 1992. Synthesis of CBI-PDE-I-dimer, the benzannelated analog of CC-1065. J Org Chem 57: 62346239.
• BARBOSA TP, CAMARA CA, SILVA TMS, MARTINS RM, PINTO AC AND VARGAS MD. 2005. New 1,2,3,4-tetrahydro-1-aza-anthraquinones and 2-aminoalkyl compounds from nor-lapachol with molluscicidal activity. Bioorg Med Chem 13: 64646469.
• BEZERRA JCB, SILVA IA, FERREIRA HD, FERRI PH AND SANTOS SC. 2002. Molluscicidal activity against Biomphalaria glabrata of Brazilian Cerrado medicinal plants. Fitoterapia 73: 428430.
• BOWMAN DF, MIDDLETON BS AND INGOLD KU. 1969. The oxidation of amines with peroxy radicals. N-phenyl-2-naphthylamine. J Org Chem 34: 34563461.
• CAPRON A. 1998. Schistosomiasis: Forty years' war on the worm. Parasitol Today 14: 379384.
• CHITSULO L, ENGELS D, MONTRESOR A AND SAVIOLI L. 2000. The global status of schistosomiasis and its control. Acta Tropica 77: 4151.
• COULADOUROS EA, PLYTA ZF, HAROUTOUNIAN SA AND PAPAGEORGIOU VP. 1997. Efficient synthesis of amino-naphthoquinones and azidobenzohydroquinones: Mechanistic consideration soft here action of hydrazoi cacid with quinones. An overview. J Org Chem 62: 0610.
• FIESER LF AND BROWN RH. 1949. Synthesis of naphthoquinones for studies of the inhibition of enzyme systems. J Am Chem Soc 71: 36093614.
• FIESER LF AND MARTIN EL. 1955. 2-Hydroxy-1,4-naphthoquinone. Org Synth Coll 3: 465468.
• FIESER LF ET AL. 1948. Naphthoquinone antimalarials. X. Miscellaneous compounds with oxygen, halogen, or nitrogen in the side chain. J Am Chem Soc 70: 32063211.
• JOHNSON MG, KIYOKAWA H, TANI S, KOYAMA J, MORRIS-NATSCHKE SL, MAUGER A, BOWERS-DAINES MM, LANGE BC AND LEE K. 1997. Antitumor agents. CLXVII. Synthesis and structure-activity correlations of the cytotoxic anthraquinone 1,4-bis-(2,3-epoxypropylamino)-9,10-anthracenedione, and of related compounds. Bioorg Med Chem 5: 14691479.
• KALLMAYER HJ AND SEYFANG KH. 1980. Quinone amine reactions. 4. 2,3,4-trihydrobenzo[f]quinoxalin-6-ones. Arch Pharmazie 313: 603611.
• KOHN M AND SCHWARZ L. 1926. Bromophenols. XVII. Brominated á-naphthoquinones. Monatsh Chem 46: 347353.
• MOLINA P, PASTOR A, VILAPLANA MJ AND FOCESFOCES C. 1995. Vinyliminophosphorane-mediated preparation of 2-arylquinoline and 4-aryl-1-azaanthraquinone derivatives X-Ray crystal-structure of 1,2-dihydro-3Hindazolo[2,3-a]quinolin-4-one. Tetrahedron 51: 12651276.
• PERRETT S AND WHITFIELD PJ. 1996. Currently available molluscicides. Parasitol Today 12: 156159.
• POINTIER J-P AND GIBODA M. 1999. The case for biological control of snail intermediate hosts of Schistosoma mansoni Parasitol Today 15: 395397.
• SILVA TMS, BATISTA MM, CAMARA CA AND AGRA MF. 2005a. Molluscicidal activity of some Brazilian Solanum spp. (Solanaceae) against Biomphalaria glabrata Ann Trop Med Parasitol 99: 419425.
• SILVA TMS, CAMARA CA, BARBOSA TP, SOARES AZ, CUNHA LC, PINTO AC AND VARGAS MD. 2005b. Molluscicidal activity of synthetic lapachol amino and hydrogenated derivatives. Bioorg Med Chem 13: 193196.
• SILVA TMS, CAMARA CA, AGRA MF, CARVALHO MG, FRANA MT, BRANDOLINI SVPB, PASCHOAL LS AND BRAZ-FILHO R. 2006. Molluscicidal activity of Solanum species of the Northeast of Brazil on Biomphalaria glabrata Fitoterapia 77: 449452.
• SILVA TMS, DA SILVA TG, MARTINS RM, MAIA GLA, CABRAL AGS, CAMARA CA, AGRA MF AND BARBOSA-FILHO JM. 2007a. Molluscicidal activities of six species of Bignoniaceae from north-eastern Brazil, as measured against Biomphalaria glabrata under laboratory conditions. Ann Trop Med Parasitol 101: 359365.
• SILVA TMS, NASCIMENTO RJB, BATISTA MM, AGRA MF, BARBOSA-FILHO JM AND CAMARA CA. 2007b. Brine shrimp bioassay of some species of Solanum from Northeastern Brazil. Rev Bras Farmacogn 17: 3538.
• SINGH OK, MISRA TM AND AGRAWAL RA. 1996. Molluscicides of plant origin. Biol Agric Hort 13: 205252.
• VASCONCELLOS MLAA, SILVA TMS, CAMARA CA, MARTINS RM, LACERDA KM, LOPES HM, PEREIRA VL, DE SOUZA ROMA AND CRESPO LT. 2006. Baylis-Hillman adducts with molluscicidal activity against Biomphalaria glabrata Pest Manag Sci 62: 288292.
• VERJOVSKI-ALMEIDA S AND DEMARCO R. 2004. Genoma contra a Esquistossomose. Scient Am Brasil 28: 5461.
• WHO WORLD HEALTH ORGANIZATION. 1965. Molluscicide screening and evaluation. Bull WHO33: 567581. Available at:<http://whqlibdoc.who.int/bulletin/1965/Vol33/Vol33-No4/bulletin_1965_33(4)_memoranda.pdf> (accessed 8th January 2008).
• WHO WORLD HEALTH ORGANIZATION. 2005. Schistosomiasis: Western Pacific, Available at: <http://www.wpro.who.int/health_topics/schistosomiasis/> (accessed 25th February 2008).

*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  14 May 2008
• Date of issue
  June 2008

History

• Accepted
  21 Jan 2008
• Received
  10 Oct 2007