New enamine derivatives of lapachol and biological activity

MAILCAR F. OLIVEIRA1, TELMA L.G. LEMOS1, MARCOS C. DE MATTOS1, TACIANA A. SEGUNDO2, GILVANDETE M.P. SANTIAGO2 and RAIMUNDO BRAZ-FILHO3 1Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará Cx. Postal 12200, 60451-970 Fortaleza, Ceará, Brazil 2Departamento de Farmácia, Universidade Federal do Ceará 3Setor de Química de Produtos Naturais-LCQUI-CCT, Universidade Estadual do Norte Fluminense 28015-620 Campos, Rio de Janeiro, Brasil


INTRODUCTION
Lapachol ( 1) is a natural quinone (see Figure 1) which has been isolated in very good yield from several species of Bignoniaceae found in Brazil, including Ceará State, where the species Tabebuia serratifolia is popularly known as ''ipê-amarelo'' (Correia 1984).From an ethanolic bark extract of a specimen of Tabebuia serratifolia lapachol (1) was obtained in 2.9% yield (Fernandes-de-Oliveira 2000).
Nitrogen containing indoloquinone derivatives have been recently evaluated as novel anticancer agents, and the amine moiety was identified as an important feature for oxic and hipoxic potency and for the ability to act as a substract for reductase enzymes (Naylor et al. 1997).
Aedes aegypti is the vector for the transmission of ''yellow fever'' or ''dengue'' and it is responsible for the contamination of the urban population in developing countries, specifically in Brazil, causing serious public health hazards (Pan American Health Organization 1994).
This prompted us to synthesize new nitrogen lapachol derivatives in order to examine their larvicidal activities against Aedes aegypti and Artemia salina as well as cytotoxicity against A549 human breast tumor cells.

General Experimental Procedure
Melting points (mp) were determined on a Mettler FP5 apparatus and are uncorrected. 1H-and 13 C-NMR spectra were obtained on a Bruker Avance DRX 500 MHz (500 MHz for 1 H and 125 MHz for 13 C) spectrometer.IR spectra were run on a Perkin-Elmer 1000 FT-IR spectrometer using KBr pellets.Lapachol (1) was obtained from an ethanolic extract of the bark of a specimen of Tabebuia serratifolia Bertol, Bignoniaceae, collected in Mulungu, Ceará, Brazil.

Larvicidal Activity
Larvicidal activity against Artemia salina was determined using a procedure described in the literature (McLaughlin 1991).Compounds 1-4 were tested in a concentration ranging from 1 to 500 ppm and LD 50 values were obtained using the Probit Program.Umbelliferone was included as a control (Table III).
Compounds 1-4 were tested against Aedes aegypti being placed in a beaker and dissolved *The number of bound hydrogens for each carbon signal was deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Homonuclear 2D 1 H-1 H-COSY spectra of 1-4 and heteronuclerar 2D 1 H-13 C-COSY-n J CH (n=1, HMQC) of 1 were also used in these assignments.
in DMSO (0.3 mL) and water (19.7 mL) at concentrations ranging from 1 to 500 ppm followed by addition of 50 larvae at the third stage.Mortality counts were made after 24 hours of treatment.A control solution was prepared using DMSO and water.Tests were done in tripiclate and the results are presented in Table III.

Citotoxicity Assay Against A549 Human Tumor Cells
Selective toxicity to hypoxic A549 human breast tumor cells was determined for all compounds using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay.Cells were  treated with drug for 3 hours at 37 o C under aerobic or hypoxic (N 2 ) conditions.The drug was then removed and the cells allowed to proliferate for 3 days prior to MTT assay.The IC 50 (air) and IC 50 (N 2 ) values are the concentrations required to kill 50% of the cells under aerobic and under hypoxic conditions, respectively.The results are derived from at least three independent experiments and are presented in Table IV.

RESULTS AND DISCUSSION
Reaction of lapachol (1) with the amines morpholine, piperidine and pyrrolidine gave the 2-aminenaphthalenedione derivatives 2-(4-morpholinyl)-3- 4) in relatively good yields.A solution of lapachol ( 1) in freshly re-distilled amine (morpholine or piperidine or pyrrolidine) was stirred for 6h at room temperature.The excess amine was evaporated under vacuum and the residue recrystallized from a mixture of EtOAc/ hexane to yield the corresponding enamine derivatives 2 (94%), 3 (79%) and 4 (77%).The reaction, is initiated by intermolecular attack of the nucleophile nitrogen on the 2-enol carbon atom (path a) or 2keto-carbonyl group (path b, much probable by the presence of a basic amine reagent) to produce the intermediates I and II, respectively, which dehydrate to give the corresponding enamine derivatives (2-4), as postulated and summarized in Figure 2.
The 13 C chemical shifts of the enamine derivatives 2-4 were compared with data reported in the literature (Giasuddin et al. 1978, Breitmaier and  Voelter 1987) for compounds 5-10 (Figure 4).As shown in Figure 4, the electronic effect of a nitrogen atom [reduced electron density at the α carbon (C-3) by inductive withdrawal effect and increased electron density at the β carbon by mesomeric effect] in 5 and 6 is practically identical, revealing δ C =1.1 ppm.Major differences ( δ C =2.4 to 10.4 ppm) were observed in the comparison of the 13 C chemical shifts of (Z)-1-pyrrolidinyl-1-( 7), (Z)-morpholinyl-1-( 8), (E)-1-pyrrolidinyl-1-( 9) and (E)-morpholinyl-1-propene (10), showing that, in fact, the electronic effects of the nitrogen atom in morpholinyl and pyrrolidinyl groups are significantly different, along with the anticipated shielding by a γ -effect which clearly allows to define the geometric isomer (E or Z).Comparative analysis of the chemical shifts of the non-hydrogenated carbon atoms C-1 [δ C 182.60 (2), 186.88 (3) and 187.17 (4)] and C-2 [δ C 154.74 (2), 164.47 (3) and 165.85 (4)] of the enamine derivatives 2-4 revealed major electron density reduction at C-2 of 3 (δ C 164.47) and 4 (δ C 165.85), which are in accord with values δ C 163.4 and 164.6 reported in the literature for the carbon atom C-3 of the model compounds 5 and 6, respectively (Figure 4).The chemical shifts of C-1 at δ C 186.88 (3) and 187.17 (4) compared with those corresponding to C-4 [δ C 183.97 (3) and 183.79 (4)] show major electron density at C-4 and may be justified by the mesomeric effect involving the conjugated unpaired electrons of the nitrogen atom.However, the chemical shifts of these carbon atoms C-1 and C-4 in the compounds 1 and 2 cannot be justified on the basis of electronic effects (meanly mesomeric in this case), since they reveal a lower electron density at C-4 [δ C 184.54 (1) and 184.58 ( 2)] than at C-1 [δ C 181.71 (1) and 182.60 (2)].Thus, these data show significant differences when compared with the values observed for the compounds 3 and 4 above considered (Figure 4).Consequently, the electronic participation of the nitrogen atom of the morpholinyl group of 2 is different from that conferred by the pyrrolidinyl and piperidinyl groups.Comparative analysis of the 13 C NMR spectral data reported in the literature (Giasuddin et al. 1978, Breitmaier andVoelter 1987) for the model compounds 7-10 allowed again to observe different participation of the nitrogen atom of the morpholinyl group (8: δ C 140.0; 10: δ C 141.0), indicating a major inductive effect at the α carbon and a minor mesomeric effect at the β carbon (8: δ C 107.5; 10: δ C 95.8) than those revealed by compounds 7 [δ C 137.6 (C-α) and 97.1 (C-β)] and 9 [δ C 136.6 (C-α) and 92.3 (C-β)], as shown in Figures 4  and 5.
All these data may be used to postulate the influence of other effects in the chemical shifts of C-1 and C-4 of lapachol ( 1) and its derivative 2, the same occurring with α and β carbon atoms of the compounds 8 and 10 also containing the morpholinyl    (Giasuddin et al. 1978, Breitmaier andVoelter 1987).
group as 2.Among other possible contributing effects can be included the participation of intramolecular hydrogen bonding in 1 and intermolecular deuterium bonding involving the deuterium of the solvent CDCl 3 , the carbonyl at C-1 and the nitrogen atom of 3 and 4 in the NMR experiments.However, in the case of 2, a repulsion between the non bonding electrons of chlorine (solvent) and the oxygen atom of the morpholinyl group prevents the formation of the deuterium bond (Figure 5) thus accounting for the minor changes of the chemical shifts of C-1 and C-2 of 2 when compared with 3 and 4.  (Giasuddin et al. 1978, Breitmaier andVoelter 1987).
In the compounds (Z)-( 8) and (E)-1-morpholinyl-1-propene (10) the postulated speculative intermolecular deuterium bonding (Figure 5) may involve the nitrogen and oxygen atoms of the morpholinyl group in a bark conformation (8a and 10a).Thus, the mesomeric effect involving the conjugated unpaired electrons of the nitrogen is attenuated and the inductive withdrawal effect encouraged.In this structural situation, the influence of these effects induces an electron density reduction and, consequently, major chemical shifts for the α and β carbon atoms of 8 and 10 when compared with those of 7 and 9.This postulation requires additional investigations involving solvent, concentration and temper-ature variations, including methyl ether and acetyl derivatives of lapachol ( 1) and other analogous compounds, using dried samples to assure the absence of H 2 O.
The larvicidal activity of compounds 1-4 using Artemia salina was evaluated (Table III).All compounds were found to be quite active.However, no significant difference was observed with substitution of the hydroxyl group by an amine at C-2 position.A slight decrease in activity was observed for compounds 3 and 4 compared to lapachol (1).LD 50 data are summarized in Table III.
In a bioassay against Aedes aegypti, lapachol (1), with a LD 50 of 20.79 ppm, was found to be more active than the amine derivatives with LD 50 values of 242.6, 899.4 and 397.0 ppm, respectively, showing the importance of the hydroxyl group at the C-2 position.The values of LD 50 of these bioassays are also summarized in Table III.
The cytotoxicity against A549 human cells was evaluated using compounds 1-4 (Table IV).On the basis of our experimental observations, lapachol (1) and their analogues 2-4 showed mM activity against cells kill, under aerobic as well as hypoxic conditions.Compound 2 is 10 fold more potent than its analogues, indicating the importance of the morpholine moiety in the toxicity of these compounds.

CONCLUSION
We have shown that lapachol (1) can be converted easily into enamine derivatives (2-4) using morpholine, piperidine and pyrrolidine.In a bioassay against Aedes aegypti, lapachol (1) was found to be more active than the amine derivatives 2-4, showing the importance of the hydroxyl group at the C-2 position.The morpholine moiety in compound 2 may be important in the toxicity against A549 human tumor cells.

Fig. 2 -
Fig. 2 -Proposed mechanism for the reaction of the amines morphiline, piperidine and pyrrolidine with lapachol (1) leading to the formation of the corresponding enamine derivatives 2-4.