Synthesis of novel naphthoquinone-spermidine conjugates and their effects on DNA-topoisomerases I and II-alpha

Abstracts

Novel derivatives of lapachol 2, nor-lapachol 3 and lawsone 4 have been synthesized by nucleophilic displacement of the methoxynaphthoquinones 2a, 3a and 4a with the polyamine (PA) N¹-Boc-N5-Bn-spermidine 1a. The respective products 2b-4b were obtained in good yields and characterized by spectroscopic and analytical methods. The inhibitory action of these naphthoquinone-PA conjugates on DNA-topoisomerases (topo) I and II-alpha was evaluated by relaxation assay of supercoiled DNA plasmid. All compounds (1a 2b, 3b and 4b) presented significant inhibition of topo II-alpha catalytic activity at the 2 µM dose. Considering that only PA 1a did not inhibit the enzyme catalytic activity at the 0.2 µM dose, the appended naphthoquinone moiety acts as a "value added" fragment. Compounds 1a 2b, 3b and 4b did not inhibit the enzyme DNA-topo I at the 200 µM dose.

spermidine; lapachol; lawsone; nor-lapachol; DNA-topoisomerase II-alpha

Novos derivados do lapachol 2, nor-lapachol 3 e da lausona 4 foram sintetizados através do deslocamento nucleofílico das metoxinaftoquinonas 2a, 3a e 4a pela poliamina (PA) N¹-Boc-N5-Bn-espermidina 1a. Os produtos, 2b, 3b e 4b, respectivamente, foram obtidos em bons rendimentos e caracterizados por métodos espectroscópicos e analíticos. Os ensaios preliminares de inibição das enzimas topoisomerases (topo) I e II-alfa mostraram-se promissores: todos os compostos (1a 2b, 3b e 4b) inibiram a atividade catalítica da enzima topo II-alfa na dose de 2 µM. Considerando que somente a PA 1a não inibiu a atividade da enzima na dose de 0,2 µM, as naftoquinonas apresentam-se como fragmentos em potencial para melhorar a atividade de PAs. Nenhum dos compostos inibiu a topo I na dose de 200 µM.

COMMUNICATION

Synthesis of novel naphthoquinone-spermidine conjugates and their effects on DNA-topoisomerases I and II-a

Andréa S. Cunha^I; Edson L. S. Lima^{*, I}; Angelo C. Pinto^{*, I}; Andressa Esteves-Souza^I; Aurea Echevarria^II; Celso A. Camara^III; Maria D. Vargas^{*, IV}; José C. Torres^V

^IInstituto de Química, Universidade Federal do Rio de Janeiro, 21945-970 Rio de Janeiro - RJ, Brazil

^IIDepartamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro, Seropédica - RJ, Brazil

^IIILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa - PB, Brazil

^IVInstituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, 24020-150 Niterói - RJ, Brazil

^VCentro Federal de Educação Tecnológica de Química de Nilópolis, Unidade Nilópolis, 26530-060 Nilópolis - RJ, Brazil

ABSTRACT

Novel derivatives of lapachol 2, nor-lapachol 3 and lawsone 4 have been synthesized by nucleophilic displacement of the methoxynaphthoquinones 2a, 3a and 4a with the polyamine (PA) N¹-Boc-N⁵-Bn-spermidine 1a. The respective products 2b-4b were obtained in good yields and characterized by spectroscopic and analytical methods. The inhibitory action of these naphthoquinone-PA conjugates on DNA-topoisomerases (topo) I and II-a was evaluated by relaxation assay of supercoiled DNA plasmid. All compounds (1a 2b, 3b and 4b) presented significant inhibition of topo II-a catalytic activity at the 2 µM dose. Considering that only PA 1a did not inhibit the enzyme catalytic activity at the 0.2 µM dose, the appended naphthoquinone moiety acts as a "value added" fragment. Compounds 1a 2b, 3b and 4b did not inhibit the enzyme DNA-topo I at the 200 µM dose.

Keywords: spermidine, lapachol, lawsone, nor-lapachol, DNA-topoisomerase II-a

RESUMO

Novos derivados do lapachol 2, nor-lapachol 3 e da lausona 4 foram sintetizados através do deslocamento nucleofílico das metoxinaftoquinonas 2a, 3a e 4a pela poliamina (PA) N¹-Boc-N⁵-Bn-espermidina 1a. Os produtos, 2b, 3b e 4b, respectivamente, foram obtidos em bons rendimentos e caracterizados por métodos espectroscópicos e analíticos. Os ensaios preliminares de inibição das enzimas topoisomerases (topo) I e II-a mostraram-se promissores: todos os compostos (1a 2b, 3b e 4b) inibiram a atividade catalítica da enzima topo II-a na dose de 2 µM. Considerando que somente a PA 1a não inibiu a atividade da enzima na dose de 0,2 µM, as naftoquinonas apresentam-se como fragmentos em potencial para melhorar a atividade de PAs. Nenhum dos compostos inibiu a topo I na dose de 200 µM.

Introduction

Polyamines (PAs) spermidine, spermine and putrescine occur in the cells of living organisms where they fulfill an array of physiological roles.¹ Because they are involved in optimum growth and replication of various cell types, and are present in higher concentrations in rapidly proliferating cells² they were identified as potential targets for the development of anticancer drugs. Initial efforts were focused on the design and synthesis of selective inhibitors of the PA biosynthetic enzymes however their action was not sufficient to inhibit tumor growth.³ The discovery that tissues with a high demand of PAs contain active polyamine transporter (PAT) for importing exogenous PAs⁴ redirected the focus of interest to the PA uptake system from the cellular environment, and several potent inhibitors became known.⁵ Anticancer therapies have tried to use the PAT to convey citotoxic and genotoxic agents to rapidly proliferating cells.⁶ Currently, the most actively pursued approach is based on tumor growth inhibition by cytotoxic structural analogues of the natural PAs, however the PA transport system also offers the possibility to improve transport and accumulation by tumors of compounds which are tethered to PA structures. One of the recent strategies involved the grafting of PAs onto DNA intercalators⁷ (e.g. linear tricyclic systems) whose primary target is a DNA topoisomerase enzyme.^6,8

As part of our interest in the synthesis of PAs⁹ and in the preparation of aminonaphthoquinone derivatives with pharmacological activities,^10,11 we became interested in linking spermidine (1) to lapachol (2), nor-lapachol (3) and lawsone (4). The prenyl naphthoquinone 2 is readily isolated from several species of Tabebuia sp (Bignoniaceae) abundant in South America, and derivative 3 is obtained from 2 by the Hooker oxidative degradation.¹² A number of synthetic derivatives of lapachol, such as mono-(arylimines)-o-quinones derived from b-lapachone showed cytotoxicity against human cancer cells.^13,14 Naphthoquinones related to lapachol 2 have been shown to exhibit notable cancer preventive potential.¹⁵ Furthermore, various amine derivatives of 2¹¹ have been shown to interact with DNA.¹⁶

In an earlier communication⁹ we described a protocol for selective handling of spermidine amino groups. We report herein the coupling of a protected derivative of spermidine with the methoxylated derivatives of compounds 2-4 to yield the first naphthoquinones tethered to a PA.

Results and Discussion

Synthesis of the spermidine-quinone conjugates

The novel spermidine-quinone conjugates were synthesized as follows: i) methylation of lapachol 2 and nor-lapachol 3 with dimethylsulphate in acetone and potassium carbonate as described previously, to yield 2a (77%)¹⁰ and 3a (71%),¹¹ respectively, and synthesis of methoxylawsone 4a from the sodium salt of 1,2-naphthoquinone-4-sulfonic acid;¹⁷ii) preparation of the protected derivative of spermidine 1a in a four-step synthesis;⁹iii) nucleophilic displacement of the methoxyquinones 2a, 3a and 4a with compound 1a (Scheme 1).

The reaction of 1a with methoxylapachol 2a under the conditions used for the synthesis of amine derivatives of lapachol, i.e. in methanol, under reflux, did not occur after 24 h, according to TLC, however, when it was carried out in the same solvent in a sealed tube, at 90 ºC for 20 h, the desired product 2b was obtained 75% yield (Scheme 1). The reactions of 1a with the other methoxynaphthoquinones were then carried out under the same conditions and the respective substitution products 3b and 4b were also obtained in 61 and 98% yields, respectively. These compounds were characterized by analytical and spectroscopic methods.¹⁸ Their ¹H and ¹³C NMR spectra showed clearly the naphthoquinone hydrogen and carbon signals, as well as those of the protected spermidine moiety.

Relaxation assays of DNA topoisomerases (topo) I and II-a

The conversion of pBR322 supercoiled plasmid DNA to the relaxed form by the enzymes topo I and II-a was examined in the presence of PA 1a and adducts 2b-4b.¹⁹ DNA topoisomerases are enzymes that modulate the topological state of DNA. They are targets for many effective drugs in cancer treatment.²⁰

The effects of the compounds on the catalytic activity of DNA topo II-a enzyme were observed in the relaxation assays using pBR322 in the presence of ATP.²¹ PA 1a and conjugates 2b, 4b and 3b were evaluated at 2 µM (lanes 4-7, respectively) and 0.2 µM (lanes 8-11, respectively) (Figure 1). All compounds presented significant inhibition of topo II-a catalytic activity at the 2 µM dose. Etoposide was used as the positive control. Interestingly, at the 0.2 µM dose, only PA 1a did not inhibit the enzyme catalytic activity. Since only partial inhibition of the enzyme activity by lapachol 2 has been observed at the 200 µM dose,¹⁷ it is clear that the PA appendage has led to a dramatic decrease in the concentration of the adduct 2b necessary for inhibition of the enzyme activity. In contrast, none of the compounds inhibited the enzyme DNA-topo I at the 200 µM dose.

Conclusions

Although a number of PA conjugates have been described in the literature,^6,8,22-23 to our knowledge, these are the first naphthoquinone spermidine adducts known to date. Preliminary results of the relaxation assays of DNA topo I and II-a indicate that the enzyme topo II-a is a target of PA-naphthoquinone conjugates 2b-4b at relatively low concentrations, and also point to the importance of the naphthoquinone moiety in improving the inhibitory activity of PA 1a. Further biological evaluation is in progress to determine the potency of these compounds. Considering the large spectra of pharmacological activities shown by lapachol and analogous naphthoquinones, studies are in progress to assess other pharmacological activities of compounds 2b-4b.

Acknowledgments

The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenadoria de Apoio à Pesquisa do Ensino Superior) PADCT-FAPERJ and FAPERJ-PRONEX for research grants and fellowships.

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18. Experimental. To a solution of 1a (110.0 mg; 0.40 mmol) in MeOH (2 mL) in a sealed tube at rt, was added a solution of 2a (100.0 mg; 0.30 mmol) in MeOH (5 mL). The reaction was stirred at 90 ºC under argon, for 20 h, after which time the solvent was removed under reduced pressure. The product was purified by flash chromatography (hexane/EtOAc, 9:1) and obtained as the reddish brown oil 2b (174.0 mg, 75%). R_f = 0.7 (hexane/EtOAc, 1:1). IR (film) n_max/cm^-1: 3347, 3063, 3027, 2930, 2863, 1712, 1669, 1602, 1570, 1515, 1271, 721, 698. ¹H NMR (200 MHz, CDCl₃): d 1.43 (s, 9H), 1.60 (s, 4H), 1.68 (brs, 3H), 1.73 (brs, 3H), 2.42 (t, J 6.4 Hz, 2H), 2.48 (t, J 6.4 Hz, 2H), 3.15 (m, 2H), 3.36-3.45 (m, 4H), 3.52 (s, 2H), 5.06 (m, 1H), 5.25 (m, 1H), 5.67 (m, 1H), 7.56 (dt, J 7.5 and 2.0 Hz, 1H), 7.67 (dt, J 7.5 and 2.0 Hz, 1H), 7.99 (dd, J 7.5 and 2.0, 1H) and 8.09 (dd, J 7.5 and 2.0 Hz, 1H).¹³C NMR (50 MHz, CDCl₃): d 18.2, 23.8, 24.3, 25.8, 26.6, 28.6, 29.2, 39.3, 45.0, 52.7, 53.4, 58.9, 77.9, 115.4, 123.2, 126.2, 127.2, 128.7, 130.5, 132.0, 133.6, 134.4, 139.4, 145.7, 156.1, 183.1. MS (QTof micromass spectrometer) Found: 560.3488 [2b+1]⁺. Calc. for [C₃₄H₄₆N₃O₄ ]⁺ 560.34883. Similar procedure was followed for the syntheses of compounds 3b and 4b. Product 3b was purified as above using hexane/EtOAc 8:2 as eluent, and obtained as a brown-reddish oil. 3b (132 mg, 61%). R_f = 0.50 (hexane/EtOAc, 1:1). ¹H NMR (200 MHz, CDCl₃): d 1.43 (s, 9H), 1.48 (brs, 3H), 1.56-1.68 (m, 4H), 1.91 (brs, 3H), 2.38 (t, J 6.5 Hz, 4H), 2.46 (t, J 6.5 Hz, 2H), 3.14 (t, J 6.2 Hz, 4H), 3.52 (s, 2H), 5.38 (m, 1H), 5.92 m, 1H), 6.10 (m, 1H), 7.26-7.35 (m, 5H), 7.55 (dt, J 7.5 and 1.6 Hz, 1H), 7.63 (dt, J 7.5 and 1.6 Hz, 1H), 8.03 (dd, J 7.5 and 1.6 Hz, 1H) and 8.06 (dd, J 7.5 and 1.6 Hz, 1H). ¹³C NMR (50 MHz, CDCl₃): d 20.3, 24.4, 25.6, 27.0, 28.1, 28.6, 28.7, 44.4, 53.0, 53.5, 59.0, 79.0, 113.5, 118.1, 126.2, 126.5, 127.2, 128.5, 129.0, 130.1, 132.1, 133.8, 134.7, 138.7, 139.5, 144.8, 156.2, 183.1, 183.5. MS Found: 546.3332 [3b+1]⁺. Calc. for [C₃₃H₄₄N₃O₄ ]⁺ 546.33318. Product 4b was purified as above using hexane/EtOAc 8:2 as eluent and obtained as a brown-reddish oil (307.8.0 mg, 98%). R_f = 0.43 (hexane/EtOAc, 1:1). IR (film) n_max/cm^-1: 3353, 3063, 3003, 2973, 2805, 1708, 1680, 1606, 1509, 1254, 1171, 729. ¹H NMR (200 MHz, CDCl₃): d 1.44 (s, 9H), 1,59 (m, 6H), 2.43 (t, J 6.2 Hz, 2H), 2.50 (t, J 6.2 Hz, 2H), 3.11 (m, 4H), 3.53 (s, 2H), 5.28 (brs, 1H), 5.68 (s, 1H), 5.99 (s, 1H), 7.30 (m, 5H), 7.61 (brt, J 7.5 Hz, 1H), 7.73 (brt, J 7.5 Hz, 1H), 8.05 (d, J 7.5 Hz, 1H), 8.11 (d, J 7.5 Hz, 1H). ¹³C NMR (50 MHz, CDCl₃): d 24.6, 25.9, 26.1, 28.5, 34.0, 42.4, 52.4, 53.1, 58.9, 78.9, 126.2-129.0, 131.9, 134.7, 139.2, 149.2, 157.3, 183.1, 184.3. MS Found: 492.2862 [4b+1]⁺. Calcd for [C₂₉H₃₈N₃O₄ ]⁺ 492.28623.

19. Relaxation assay of DNA topoisomerases I (topo I) and II-a (topo II-a). Tðhe enzymatic activity was analyzed¹⁴ by the DNA relaxation assay according to the protocol described by topoGEN (topogGEN, Columbus, OH, USA). One unit of DNA topo I (from wheat germ, Sigma Co) and topo II-a (human recombinant in E. coli, Invitrogen) enzymes was incubated with 0.25 µg of pBR322 DNA (Sigma Co), in the presence or absence of test compounds, in 10 µL of a mixture containing 100 mmol L^-1 Tris, pH 7.9, 500 mmol L^-1 NaCl, 500 mmol L^-1 KCl, 50 mmol L^-1 MgCl₂, 1 mmol L^-1 EDTA, 0.15 mg mL^-1 BSA and 10 mmol L^-1 ATP for 30 min at 37 °C. The reaction was terminated by the addition of 1µL of a stop solution consisting of 50% glycerol, 10% sodium dodecyl sulfate (SDS) and 25% bromophenol blue. Electrophoresis was carried out over 1% agarose gel plates, equilibrated with TAE buffer (50 x stock: 242g Tris-base, 57.1 mL glacial acetic acid and 100 mL of 0.5 mol L^-1 EDTA) for 2.5 h at 60 V. The gels were stained with ethidium bromide solution (0.5 µg mL^-1) after electrophoresis for 30 min, washed with water and photographed under UV light with a digital camera.

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Received: October 13, 2005

Published on the web: April 24, 2006

Publication Dates

• Publication in this collection
  25 May 2006
• Date of issue
  June 2006

History

• Accepted
  24 Apr 2006
• Received
  13 Oct 2005