Synthesis of new aldehyde derivatives from β -lapachone and nor-β -lapachone

Derivatives 6a and 6b , have been obtained in a chemoselective manner by a two step synthesis from β -lapachone ( 3a ) and nor-β -lapachone ( 3b ), respectively, using CH 2 N 2 in ether, which provided the lapachone-epoxide derivatives, 5a and 5b , followed by treatment with BF 3 .Et 2 O/CH 2 Cl 2 . Basic work-up of the reaction mixture of 5a or 5b with BF 3 .Et 2 O/CH 2 Cl 2 led instead to isolation of the rearranged derivatives 7a and 7b , respectively. An ab initio theoretical study [B3LYP/6-311+G(d)] on 3a indicated that the carbon atom, C6, carries a higher positive charge than that at C5 and hence predicts a greater reactivity towards nucleophiles at C6. Another important factor controlling the chemoselectivity in the reaction of 3a with CH 2 N 2 is the steric hindrance of the CH 2 groups of the pyran ring.


Introduction
Several old and new medical problems for mankind are driving research to find improved treatments.Paramount in this area is the research for more potent anticancer, antibacterial and antibiotic compounds.Compounds with quinonoid derived systems have shown particularly promise.A central feature of ortho and para-quinonoid based cytotoxins is their ability to generate semi-quinone radicals by bioreduction, which then accelerates intracellular hypoxic conditions. 1,2he drug arsenal for fighting against tropical diseases includes many natural and synthetic naphthoquinone derivatives, which have been studied extensively due to their abilities to interfere with the bio-activities of enzymes known as topoisomerases, a group of enzymes that are critical for DNA replication in cells.In addition, naphthoquinones have been shown to induce what are known as 'reactive oxygen species' that can cause damage to cells.Since the pioneer work of Wendel 1 in 1946, showing that certain 2-hydroxy-3-alkyl-naphthoquinones inhibited the growth of Plasmodium vivae, quinones have been the subject of much interest in chemotherapeutic studies, which have proved that the toxicity of naphthoquinones to Plasmodium sp. is due to their interaction with the mitochondrial respiratory chain of the parasites. 2This observation led Fieser and collaborators in the 1940´s to start an extensive search for new quinones for use in malaria chemotherapy. 2Subsequently, quinones have been studied for antitumor, 2 trypanocidal, 2 molluscicidal, 1 leiscmanicidal, 2 anti-inflammatory 3 and antifungal 4 activities.
Among significant biological active naphthoquinones are atovaquone 1, 5,6 a coenzyme Q analogue, which inhibits selectively P. Vivae by affecting its mitocondrial electron transport and lapachol 2, a versatile biologically active compound, present in the heartwood of several Tabebuia species from Central and South America, especially Tabebuia avellanedae Lorentz ex Grised (Bignoniaceae) 7, , 8 9 , see Figure 1. 12 Another very important quinone is β-lapachone 3a, a 1,2-naphthoquinone, Figure 1, also found in the heartwood of Tabebuia sp, whose extract has been used as a popular medicine for centuries.Recent investigations suggest 3a has potential application against numerous diseases.Its effectiveness at micromolar concentrations against a variety of cancer cells in cultures indicates its potential against tumor growth.4 15 The detailed mechanism of cell death induced by βlapachone has still to be revealed.It has been discovered that topoisomerases I 16 and II 17 , which act directing on DNA, were the biochemical targets involved in the apoptosis, but its role in cell death is not yet clear.Particularly promising is the remarkably powerful synergistic lethality between β-lapachone and taxol against several tumor cell lines implanted into mice. 18Enhanced lethality of X-rays and alkylating agents to tumor cells in culture was reported when β-lapachone was applied during the recovery period, because of inhibition of DNA lesion repair. 19,20In addition, β-lapachone also possesses a variety of pharmacological effects, including antibacterial, antifungal, and trypanocidal activities 21

Figure 1
Despite the vast pharmacological studies on β-lapachone 3a, only a few studies have been addressed to chemical modifications at the redox center.Recently, Burton and coworkers 22 reported the synthesis of monoarylimine o-quinones derived from β-lapachone 3a in an attempt to alter the redox cycling characteristics of the parent molecule.The phenylimine derivatives 3c or 3d were found to retain most of the cytotoxicity and selectivity of the β-lapachone 3a when tested in vitro in screen of 55 cancer cell lines.Pinto et al also have demonstrated changes in the trypanocidal activities by a select transformation of the o-quinone moieties of lapachones.For instance, the introduction of an oxazole nucleus (e.g. 4) in the β-lapachone (3a) structure showed a marked influence upon the trypanocidal activity. 23In recent studies, we have demonstrated that chemical modification at the redox moiety leads to alterations on some physical chemical properties. 24Therefore, the search for new compounds by modifying the quinonoid center is a subject of great interest, not only from the chemical but also from the pharmacological points of view.
We wish to report a novel synthetic pathway to prepare 6a-b and 7a-b structurally related to β-lapachone 3a and nor-β-lapachone 3b (its semi-synthetic lower homolog), respectively, and an investigation of the chemoselective nucleophilic addition at carbonyl C6 of 3a.

Results and Discussion
The synthetic route for preparing 6a-b and 7a-b from β-lapachone 3a and nor-β-lapachone 3b is outlined in Scheme 1.
As shown in Scheme 1 the synthesis of 6a-b and 7a-b were carried out in two steps.The first involved the preparation of lapachone-epoxide derivatives 5a-b, which were obtained in high yields from the reaction of lapachones 3a-b with diazomethane 25 in ether.The reaction of αlapachone 26,27 and β-lapachone (3a) with diazomethane leading to epoxide adducts had been previously described in the literature.Pinto et al tested 5a for blockage of cercaial skin penetration by Schistosoma mansoni.The activity of 5a was found to be 50% lower than those of lapachol (1) and β-lapachone (3a).

Scheme 1
NMR spectroscopic data for 5a have not been previously reported and hence have been obtained in this study.NMR spectroscopic techniques, such as 1 H X 1 H-2D NMR NOESY, clearly confirm that addition of diazomethane to 3a occurs at the carbonyl C6, e.g., coupling was observed between the hydrogen at C7 (δ = 7.22 ppm) and the hydrogens of the methylene group C11 (δ = 3.11 and 3.44 ppm).A similar observation was found for the addition of diazomethane to 3b, which produced the new epoxide 5b in high yield.The chemoselectivities found in the reactions of 3a and 3b with diazomethane is not surprising, since there are several reports in the literature indicating that the carbonyl nearest to the aromatic ring is the more reactive. 22,24n order to investigate the chemoselectivities of the carbonyls C6 (for 3a) and C5 (for 3b) we undertook a full geometry optimization of 3a and calculation of the geometry parameters for the optimized minima.The ab initio calculation was performed using the Gaussian 98W 28 package, with the Lee-Yang-Par 29 correlation functional (B3LYP) and the basis set, 6-311+G(d).Some selected calculated geometry parameters for the optimized minima can be compared, in Table 1, with values obtained from a report of an earlier X-ray structure determination. 30Both calculated and observed dihedral angles for the carbonyl indicated that it is almost in the plane of the aromatic system.All calculated geometry parameters are also in good agreement with the values determined in the X-ray study.The difference between the atomic charges for C5 and C6 are small, but they do indicate that C6 is more electrophilic than C5.This difference must account, partially at least, for the greater reactivity of C6 towards nucleophiles.In order to obtain information about the role of the pyran ring in the chemoselectivity of the carbonyls C6(for 3a) and C5 (for 3b), the addition of diazomethane to 4-methoxy-1,2naphthoquinone (8) was studied (Scheme 2).The reaction was not chemoselective as shown in reaction 1, where both epoxides 9 (80% yield) and 10 (8% yield), as well as 11 were produced.The formation of 10 in the reaction of 8, suggests that the chemoselectivity observed in reactions of 3a and 3b is partially, at least, due to the steric hindrance of the CH 2 group of the pyran ring towards the incoming diazomethane.In the second step of our synthetic route (see Scheme 1), different modes of isomerization of the epoxides 5a-b to either 6a-b or 7a-b could be effected.The reaction of the epoxides with BF 3 .OEt 2 in dichloromethane proceeded smoothly to give the aldehydes 6a-b in high yield.
However, the same reaction, but with a basic work up with NaHCO 3 , led to the isomeric aldehydes 7a-b also in high yields.It seems that the basic work-up before isolation of the products results isomerization of the aldehydes 6a-b to the aldehydes 7a-b.A similar behavior had been described for α and β-lapachone (3a), which can be formed from lapachol (2) depending of the pH of the medium.
The structure of the aldehydes, 6a-b and 7a-b, were investigated by 1 H and 13 C NMR in such 1D and 2D experiments as 1 Hx 1 H-COSY and HETCOR ( n J CH , n=1,2,3).These two set of aldehydes had different chemical shift values for the aldehyde hydrogens but the same molecular weight in the high resolution mass spectra.The multiplicities of the aromatic hydrogens of 6a-b are similar to those for 5a-b and also 3a-b.However, it was not possible to observe NOE between the aldehyde groups and the vicinal aromatic hydrogen in 6a-b, and so we could not confirm these structures using these techniques.
All doubts about the structures were solved by carrying out an x-ray structure determination of 6a.

Conclusions
A new method for obtaining aldehyde derivatives of β-lapachone (3a) and nor-β-lapachone (3b) is reported.The addition of diazomethane to the 1,2-naphthoquinones, (3a) and (3b), forming the epoxides 5a-b, occurs chemoselectively at the 1-carbonyl groups.These epoxides can be transformed in high yields to the aldehydes 6a-b or 7a-b depending on the experimental conditions.To the best of our knowledge, the preparation of 6a-b or 7a-b represents the first example of the chemoselective formation of aldehyde derivatives in two steps from 3a-b.Ab initio calculations of β-lapachone (3a) using B3LYP with basis set 6-311+G* agreed with the carbonyl regiospecifity on forming epoxides, but experimental evidence, obtained from the addition of diazomethane to 1-methoxy-3,4-naphthoquinone (9), also suggested an influence from the steric hindrance of the CH 2 group of the pyran ring is also involved in the chemoselectivity.The applicability of the present procedure for the preparation of other naphthoquinone-aldehydes is currently under investigation.

Experimental Section
General Procedures.Melting points were observed on a Reichert micro hotstage and are uncorrected.Analytical grade solvents were used.The solvents were previously purified as described in the literature. 31Methanol was distilled before being used.Column chromatography was performed on silica gel 60 (Merck 70-230 mesh).Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrum One spectrophotometer calibrated relative to the 1601.8cm -1 absorbance of polystyrene.Ultraviolet (UV) spectra were obtained on a Schimadizu spectrophotometer: wavelengths in nm and extinction coefficients, ε, in mol.cm -1 .NMR spectra in CDCl 3 solutions were recorded on a Varian Unity Plus VXR (300 MHz) instrument.Low resolution electron-impact mass spectra (70 eV) were measured in a Hewlett Packard 5985 instrument and high-resolution electron-impact mass spectra (70 eV) were obtained using a VG Auto Spec instrument.β-lapachone (3a) and nor-β-lapachone (3b) were prepared from lapachol by standard procedures 32 .Crystal data of 6a, space group Pbca, were collected on a Nonius KappaCCD diffractometer by the EPSRC X-Ray crystallographic service, based at the University of Southampton, UK.The structure solution and refinement were achieved using SHELX97 and SHELXL97. 33Full matrix least squares on F 2 converged to R = 0.482 [I > 2σ(I)].Atomic coordinates, bond lengths, angles and thermal parameters have been deposited at the Cambridge Crystallographic Data Centre, deposition number 212908.6-311+G(d) Ab initio calculations were carried out through the Gaussian 98W package running on a PC Pentium III 1.1 GHz.

Reaction of 4-methoxy-1,2-naphthoquinone with diazomethane
To a solution of 8 (367 mg, 0.195 mmol) in ether (10 mL) was added a ethereal solution (10 mL) of freshly prepared diazomethane.The mixture was kept in the refrigerator (5 o C) for 48 hours.

General procedure for preparing 6a-b
To solution of the appropriate epoxide in dichloromethane (20mL), externally cooled by ice and under a nitrogen atmosphere, was slowly added an ethereal solution of BF 3 .OEt 2 The reaction was stirred for 24h at room temperatures and then diluted with dichloromethane (10 mL).To this solution was added anhydrous sodium sulfate (1 g) and activated charcoal (3 g).The mixture was filtered and concentrated under reduced pressure leaving a viscous oil, which was quickly passed through a column chromatography eluting with hexane /ethyl acetate.

General procedure for preparing 7a-b
To solution of the appropriate epoxide in dichloromethane (20mL), externally cooled by ice and under nitrogen atmosphere, was slowly added an ethereal solution of BF 3. OEt 2 The reaction was stirred for 24 at room temperatures an then diluted with dichloromethane (10 mL).To this solution was added a saturated solution of sodium bicarbonate (10 mL).After stirring for 1h, the organic phase was separated, extracted with water (2x10mL), dried over anhydrous sodium sulfate and, concentrated under reduced pressure, leaving a viscous oil, which was quickly passed through a column chromatography eluting with hexane /ethyl acetate.
Scheme 2 Figure 2 shows a view of the molecule with the crystallographic atom numbering scheme.Displacement ellipsoids are drawn at the 30% probability level.All bond lengths and angles are in the expected ranges.As anticipated, an intramolecular hydrogen bond is formed between the phenolic OH group at C5 and the aldehydic oxygen, O3.The conformation of the C10b-C4a-C4-C3-C2-O1 ring, based on the Pucker parameters [Q = 0.48Å, θ = 52.2o , ϕ = 92.28o ] is a half chair with C2 and C3 on opposite sides of the best plane through the near-planar atoms, O1-C10b-C4a-C4.