Novel 2-( R-phenyl ) amino-3-( 2-methylpropenyl )-[ 1 , 4 ]-naphthoquinones : Synthesis , Characterization , Electrochemical Behavior and Antitumor Activity

Novas 2-(R-fenil)amino-3-(2-metilpropenil)-[1,4]-naftoquinonas (R = H, 4-OMe, 4-Ferrocenil, 4-Me, 3-Me, 4-I, 3-I, 4-CN, 3-CN, 4-NO 2 e 3-NO 2 ) derivadas do nor-lapachol [2-hidroxi-3-(2metilpropenil)-1,4-naftoquinona] foram obtidas em bons rendimentos. A estrutura dos compostos foi proposta com base em estudos de difração de raios-X (R = OMe, 2b), dados de RMN de H e C e cálculos teóricos utilizando o funcional B3LYP e a base 6-311+G(2d,p). Os potenciais de meia-onda das aminonaftoquinonas e o deslocamento químico do hidrogênio da cadeia 3-propenil dos compostos 2a-k mostraram boa correlação com as constantes de Hammett dos substituintes presentes no anel fenileno. A avaliação da citotoxicidade evidenciou atividade antitumoral promissora para o substrato metóxi-nor-lapachol 1 e o derivado 4-ferrocenil 2c.


Introduction
Naphthoquinones are widely distributed in nature and some of these molecules have an important role in the biochemistry of microbial energy production, by means of photosynthesis and respiratory chain. 1 Compounds containing the quinone group are known for exhibiting antitumor, 2 trypanocide, 3 moluscicide, 4 fungicide 5 and antimalarial 6 activities.[9][10][11][12] Biological activity of quinones is often related to their electrochemical behavior. 13The ability to accept one or two electrons to form the corresponding radical anion (Q •-) or dianion (Q 2-) species is believed to induce formation of reactive oxygen species, responsible for the oxidative stress in cells. 14The electron-accepting capacity of naphthoquinones may be tuned by carbonyl position changes (1,2-x 1,4-naphthoquinones) 15 or different substituents or functions attached to the naphthoquinone moiety, and the use of electrochemical methods to study this type of molecules has proven to be useful. 16,17We reported recently 18 the synthesis of a series of 2-arylamino-1,4-naphthoquinones from 2-methoxy-1,4-naphthoquinone and arylamines in the presence of MgCl 2 • 6H 2 O and p-toluenesulfonic acid as catalysts.The reactions of both electron-donor and electron-attracting substituted anilines having given good yields of the respective products, we decided to investigate the analogous reactions of the methoxy-derivatives of nor-lapachol [2-hydroxy-3-(2methyl-propenyl)-1,4-naphthoquinone] 4 and lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone]. 190][11][12] The incorporation of polyamines to this quinone, for example, has led to significant increase in the DNA topoisomerase II-a inhibition, compared to the original naphthoquinone. 13,14urthermore, arylamino derivatives of nor-a and nor-b lapachones present potent antitumor 11 and trypanocide activities. 9erein is the first report on the synthesis and characterization of the novel 2-arylamine derivatives of norlapachol 2a-k, including 2-(4-ferrocenyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone 2c (Figure 1) and cytotoxic screening against the several cancer cell lines (SF-295, HCT-8, MDAMB-435 and HL-60).Because correlations between electrochemical potentials and the inhibitory activity of naphthoquinones on Epstein-Barr virus early antigen activation 21 and their cytotoxicity 22 has been reported, we also investigated the redox properties of these compounds by cyclic voltammetry.

Syntheses
The compounds 2a-k (Figure 2) were synthesized from 2-methoxy-3-(2-methylpropenyl)- [1,4]-naphthoquinone with various aromatic amines, in the presence of the catalysts 4-toluenesulfonic acid and MgCl 2 • 6H 2 O in methanol under reflux. 18The products are stable in the solid state and in solution.Compounds 2a, 2b were obtained in a pure state, whereas 2c-k were purified by column chromatography using a mixture of ethyl acetate / hexane (1:5) as eluent.They were obtained in yields ranging from 84 to 73% and formulated on the basis of analytical and spectroscopic data (see Experimental).
The 1 H NMR and infrared spectra of compounds 2a-k are consistent with their composition and structure.The 1 H NMR spectra exhibit signals in the d 7.5-8.2ppm region as double dublets and triple dublets, attributed to the four naphthoquinone aromatic hydrogens H5-H8.Attributions were made on the basis of 1 H x 1 H-COSY experiments, J values and multiplicity.All expected resonances were observed in the 13 C NMR spectra of compounds 2a-k.The carbonyl peaks appear around d 183 and 180, and those attributed to C2 bound to the nitrogen at about d 145.The other chemical shifts are compatible with the structures proposed for these compounds.We observed that the chemical shift of H18 (Figure 2) in the 1 H NMR spectra of 2a-k is directly influenced by the nature of the R substituent group in the phenylamino ring [5.80 (4-OMe) < d H(18) < 6.08 (4-NO 2 )].

X-ray structure / Theoretical calculations
The structure of 2-(4-methoxy-phenyl)amino-3-(2methylpropenyl)-1,4-naphthoquinone 2b was determined by a single crystal X-ray diffraction study (Figure 3).The average C-C, C-O, C=O and C-N bond lengths are in good agreement with the literature. 36The naphthoquinone ring system of 2b is approximately planar, the dihedral angle between the naphthoquinone plane and the arylamine phenyl ring being 47.3° (1) In addition the other C=O group interacts via O2 ii with H5 to the C5 forming now a ten-membered ring.For more details of the crystal structure, see Supplementary material.
Starting from the experimental structure of 2b (Figure 3), the geometries of 2a-2e and 2h-2i were fully optimized with the B3LYP/6-31G(d) method. 37Energies and molecular properties were obtained from a singlepoint calculation on the optimized geometries using the 6-311+G(2d,p) basis set 38 and the B3LYP functional. 39To confirm that the most stable conformation in the gas-phase is similar to that found in the solid state the geometry of an alternative conformation for 2b, with the 2-methylpropenyl group bonded to position 3 of the naphthoquinone ring rotated by 180 o was also optimized.This alternative conformation is 0.4 kcal mol -1 less stable than the solid state conformation.The barrier for conversion between the two conformers calculated at the 6-31G(d) level is 5.5 kcal mol -1 .
Calculations of the absolute 1 H NMR chemical shifts using the GIAO approach 40 confirmed that electronattracting groups yield higher d H (18) values than electrondonor groups.Calculations including solvent (chloroform) show essentially the same behavior.Interestingly, the d H (18) value for the alternative conformation with the 2-methylpropenyl side chain rotated by 180 o is shifted highfield by 1.67 ppm, compared to the same hydrogen in the most stable conformation.The fact that the experimental values are intermediate between the calculated values for the two conformations suggests that these conformations are in equilibrium in solution, although the variable temperature 1 H NMR spectra of 2a do not show broadening of H18 down to -90 o C in CD 2 Cl 2 .

UV-Vis spectra
The UV-Vis spectra of the compounds obtained in CHCl 3 show two absorption bands.PBE1PBE/6-311+G(2d,p) calculations indicate that the band in the 275-290 nm region can be attributed to the aromatic and quinone p-p * transitions and the low-energy band in the visible region between 456 and 512 nm is attributed predominantly to p phenyl-p * naphthoquinone transitions.Electron-donor substituents blue shift the latter band, whereas electronattracting groups red shift it.

Cyclic voltammetry
The redox behavior of compounds 2a-k was evaluated by cyclic voltammetry (CV) at room temperature in acetonitrile/Bu 4 NPF 6 (0.1 mol L -1 ).The CVs were obtained in the potential range from +1.3 to -2.1V vs FcH/FcH + as internal standard (Table 1).Two quasi-reversible pairs of waves were observed for compounds 2a-i in the negative region of the CV, which are attributed to the one-electron transfer to the naphthoquinone moiety.The redox potentials of the naphthoquinone unit are directly influenced by the substituents in the phenylamino ring: electron-donor groups present lower E 1/2 when compared to electron-releasing groups.The complexity of the CV observed for 2j indicated that the nitro group is also electroactive in the cathodic region studied 41 and because the reduction potentials for the nitro and the quinone moieties are similar, we were unable to assess the voltammetric parameters for this derivative.The data indicate that derivative 2c, R= ferrocenyl (Fc, E 1/2 = -1.23 V) exhibit electronic properties similar to compound 2b.This was also observed for the Fc-arylamine derivative of lawsone. 18od correlation of the E 1/2 (1) potentials with the s p and s p -Hammett constants 42 was obtained (Figures 6 and 7, respectively) except for the Fc group (Figure 7) for which the low s p -value (-0.03) 43 has been correlated to low resonance contribution.The linear correlation coefficients for both plots suggest that all naphthoquinones of this series are reduced by the same mechanism.E 1/2 (1) potentials do not show linear correlation with s m values.

Antitumor assays
The antitumor screening of compounds 2a-k was carried out against three cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast) through an MTT assay 35 and the results, summarized in the Table 1.Voltammetric data (V) for 2a-k vs FcH/FcH + as internal standard Supplementary information, show that the Fc-derivative 2c and the methoxy-substrate 1 presented significant proliferation inhibition against MDA-MB435, higher than the positive control doxorubicin (DOX).In a second set of experiments, four cell lines were used for IC 50 determination of previously selected compounds (1 and 2c).Only methoxynor-lapachol 1 was highly active against MDA-MB435 and moderately active against HL-60 and HCT-8 cell lines (Table 2).The loss of activity of the ferrocenyl derivative 2c may be due to decomposition during dilution and defreezing of the solution, since this compound is slightly unstable in solution in the presence of oxygen.

Materials and methods
Reagents and solvents were used without further purification.Microanalyses were performed using a Perkin-Elmer CHN 2400 micro analyser at the Central Analítica, Instituto de Química, USP-São Paulo, Brazil.Melting points were obtained with a Mel-Temp II, Laboratory Devices-USA apparatus and are uncorrected.IR spectra (KBr pellets) were recorded on a FT-IR Spectrum One (Perkin Elmer) spectrophotometer. 1 H and 13 C NMR spectra were recorded with a Varian Unit Plus 300 MHz spectrometer in CDCl 3 ; coupling constants are reported in Hertz (Hz) and chemical shifts in parts per million (ppm) relative to internal standard Me 4 Si.The hydrogen signals were attributed through coupling constant values and 1 H × 1 H -COSY experiments.Electronic spectra were taken on a Diode Array 8452A (Hewlett Packard-HP) spectrophotometer using spectroscopic grade solvents (Tedia Brazil) in 10 -3 and 10 -4 mol L -1 solutions.Cyclic voltammograms were obtained on an Epsilon-BAS potentiostat-galvanostat from 1 × 10 -3 mol L -1 solutions in chloroform containing 0.1 mol L -1 of TBABF 4 as supporting electrolyte, at room temperature and under argon atmosphere.A standard three component system was used: a carbon-glassy working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode for organic media.Ferrocene was used as an internal standard (E 1/2 0.40 V vs NHE).Density functional calculations were carried out using the Gaussian03W molecular orbital package. 23Geometries were fully optimized using the B3LYP functional 24 with the standard 6-31G(d) basis set 25 Solvent effects (chloroform) were estimated by single-point calculations on the gas-phase optimized geometries by mean of the continuum solvation model using the conductor-like polarisable continuum model 26 (CPCM) at the same level.NMR absolute chemical shifts were calculated using the GIAO (Gauge Independent Atomic Orbital) method with the B3LYP/6-311+G(2d,p) approach on the B3LYP/6-31G(d) optimized geometry.The electronic spectra were calculated using the TD (Time Dependent) methodology available in Gaussian.The PBE1PBE functional together with the 6-311+G(2d,p) basis set was employed.

X-ray crystallography
The X-ray diffraction data for 2b were collected at 295 K from a Enraf-Nonius Kappa-CCD 27 diffractometer with graphite monochromatized Mo K a radiation.The cell parameters were obtained and refined using PHICHI 28 and EvalCCD 29 programs.Intensities for (1) were corrected by Lorentz polarization and absorption with the SADABS 30 program.The structure was solved by SHELXS-97 Direct Methods, 29 and refined with SHELXL-97, 32 contained within the WinGX-32 crystallography program. 33The positional parameters of the H atoms bonded to C atoms in the phenyl rings were obtained geometrically, with the C-H distances fixed in 0.93Å for Csp 2 , and refined as riding on their respective C atoms, with U iso (H) = 1.2Ueq(Csp 2 ).H atoms bonded to C atoms in the methyl group were located geometrically and with the C-H distances fixed at 0.96Å for Csp 3 and with U iso (H) = 1.5Ueq(Csp 3 ).The positional parameters of atom H1 bonded to N1 was obtained from a Fourier difference map and refined freely with an isotropic displacement parameter; the distance for N1-H1 is 0.87(2).X-ray data are listed in Table 3 and ORTEP-3 34 for Windows was used to draw the Figures.

Conclusions
The eleven novel aminonaphthoquinones 2a-k, obtained from methoxy-nor-lapachol and various arylamines, were synthesized in good yields and showed interesting electrochemical behavior due to the nature of the substituents in the phenylamino ring, presenting a good correlation with Hammett parameter, which confirms that the reaction with electron-donor or electron-attracting groups follow a single mechanism.Unfortunately, because the arylamine derivatives of nor-lapachol were not active against the tested tumor cells, correlation between structure, electrochemical data and antitumor activity could not be attempted.

Theoretical Calculations
Starting from the experimental structure for 2b (4-Me), the geometries of 2a-2e and 2h-2i were fully optimized with the B3LYP/6-31G(d) method.Energies and molecular properties were obtained from a single-point calculation using the 6-311+G(2d,p) basis set and the B3LYP functional.To confirm that the most stable conformation in the gas-phase is similar to that found in the solid state the geometry of an alternative conformation for 2b, with the 2-methyl-propenyl group bonded to the position 3 of the naphthoquinone ring rotated by 180 o was also optimized.This alternative conformation was found 0.4 kcal mol -1 less stable than the solid state conformation.The barrier for conversion between the two conformers calculated at the 6-31G(d) level is 5.5 kcal mol -1 .
The orientation of the 2-phenylene ring is stabilized by an intramolecular hydrogen bond with one carbonyl group of the naphthoquinone ring and was, therefore, not further investigated.NMR absolute chemical shifts were calculated using the GIAO (Gauge Independent Atomic Orbital) method with the B3LYP/6-311+G(2d,p) approach on the B3LYP/6-31G(d) optimized geometry.Absolute energies and GIAO nuclear magnetic shielding tensors for H (18) are given in Table S1.Interestingly, the d H18 value for the alternative conformation (with the 2-methyl-propenyl group rotated by 180 o , entry 2b-4 in Table S1) is 1.67 ppm shifted highfield as compared to the same hydrogen in the most stable conformation.Calculation for 2b on a geometry with the methoxy group rotated by 90 o (entry 2b-2 in Table S1) yielded essentially the same d H18 value as that obtained for the most stable conformation.To verify the effect of the solvent on the chemical shifts some calculations were repeated with the solvent chloroform, using the CPCM approach.These calculations revealed that the solvent has only marginal influence on the relative chemical shifts.
The electronic spectra were calculated using the TD (Time Dependent) methodology available in Gaussian.The PBE1PBE functional together with the 6-311+G(2d,p) basis set was employed.All calculations were carried out with the G03W package of molecular orbital calculation.(Farrugia, 1997); software used to prepare material for publication: WinGX publication routines (Farrugia, 1999).

Experiment X-ray diffraction
The X-ray diffraction data for (2b) were collected at 295 K from a Enraf-Nonius Kappa-CCD diffractometer with graphite-monochromatized Mo K a radiation.The cell parameters were obtained and refined using PHICHI and

Antitumor Assays
The antitumor screening of compounds 2a-k was carried out against three cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast) through an MTT assay ref and the results are summarized in Table S6.

Figure 3 .
Figure 3.View of the ORTEP plot for 2b with labeled atoms and 50% probability ellipsoids.

Figure 4 .
Figure 4. View of the intramolecular interaction.

Table 3 .
Crystal data and structure refinement for compound 2b

Table S2 .
Crystal data and structure refinement for 2b.

Table S6 .
Screening of the cytotoxic activity (growth inhibition %) of compounds 2a-k and 1