Novel 2-(R-phenyl)amino-3-(2-methylpropenyl)-[1,4]-naphthoquinones: synthesis, characterization, electrochemical behavior and antitumor activity

Abstracts

Novel 2-(R-phenyl)amino-3-(2-methyl-propenyl)-[1,4]-naphthoquinones (R = H, 4-OMe, 4-Ferrocenyl, 4-Me, 3-Me, 4-I, 3-I, 4-CN, 3-CN, 4-NO2 and 3-NO2) derived from nor-lapachol [2-hydroxy-3-(2-methylpropenyl)-1,4-naphthoquinone] were obtained in good yields. Their structures were proposed on the basis of a single crystal X-ray diffraction study (R = OMe, 2b), ¹H and 13C NMR studies and calculations using the B3LYP functional and the 6-311+G(2d,p) basis set. The half-wave potentials of the aminonaphthoquinones and ¹H NMR chemical shifts of the 3-propenyl hydrogen in 2a-k show good correlation with the substituent Hammett constants on the phenylamino ring. The antitumor assays showed promising activity for substrate methoxy-nor-lapachol 1 and the 4-ferrocenyl derivative 2c.

Nor-lapachol; arylamine; aminonaphthoquinone; electrochemistry; B3LYP; 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-NO2 e 3-NO2) derivadas do nor-lapachol [2-hidroxi-3-(2-metilpropenil)-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 ¹H e 13C 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.

ARTICLE

Novel 2-(R-phenyl)amino-3-(2-methylpropenyl)-[1,4]-naphthoquinones: synthesis, characterization, electrochemical behavior and antitumor activity

Acácio I. FranciscoI; Annelise CasellatoI, ^#; Amanda P. Neves^I; J. Walkimar de M. Carneiro^I; Maria D. Vargas^{*, I}; Lorenzo do C. Visentin^II; Alviclér Magalhães^III; Celso A. Câmara^IV; Claudia Pessoa^V; Letícia V. Costa-Lotufo^V; José D. B. Marinho Filho^V; Manoel O. de Moraes^V

^IInstituto de Química, Universidade Federal Fluminense, 24020-141 Niterói-RJ, Brazil

^IIInstituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil

^IIIInstituto de Química, Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil

^IVInstituto de Química, Universidade Federal Rural de Pernambuco, 52171-900 Recife-PE, Brazil

^VDepartamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270 Fortaleza-CE, Brazil

ABSTRACT

Novel 2-(R-phenyl)amino-3-(2-methyl-propenyl)-[1,4]-naphthoquinones (R = H, 4-OMe, 4-Ferrocenyl, 4-Me, 3-Me, 4-I, 3-I, 4-CN, 3-CN, 4-NO₂ and 3-NO₂) derived from nor-lapachol [2-hydroxy-3-(2-methylpropenyl)-1,4-naphthoquinone] were obtained in good yields. Their structures were proposed on the basis of a single crystal X-ray diffraction study (R = OMe, 2b), ¹H and ¹³C NMR studies and calculations using the B3LYP functional and the 6-311+G(2d,p) basis set. The half-wave potentials of the aminonaphthoquinones and ¹H NMR chemical shifts of the 3-propenyl hydrogen in 2a-k show good correlation with the substituent Hammett constants on the phenylamino ring. The antitumor assays showed promising activity for substrate methoxy-nor-lapachol 1 and the 4-ferrocenyl derivative 2c.

Keywords:Nor-lapachol, arylamine, aminonaphthoquinone, electrochemistry, B3LYP, antitumor activity

RESUMO

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-NO₂ e 3-NO₂) derivadas do nor-lapachol [2-hidroxi-3-(2-metilpropenil)-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 ¹H e ¹³C 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 fungicide⁵ and antimalarial⁶ activities. The presence of an amino group in quinones has led to interesting biologically active compounds.^7-12

Biological activity of quinones is often related to their electrochemical behavior.¹³ The 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.¹⁴ The 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.^16,17 We reported recently¹⁸ the synthesis of a series of 2-arylamino-1,4-naphthoquinones from 2-methoxy-1,4-naphthoquinone and arylamines in the presence of MgCl₂^•6H₂ 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-(2-methyl-propenyl)-1,4-naphthoquinone]⁴ and lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone].¹⁹Nor-lapachol is obtained from lapachol by the Hooker oxidation²⁰ and has been used as a substrate for the synthesis of several active compounds.^9-12 The incorporation of polyamines to this quinone, for example, has led to significant increase in the DNA topoisomerase II-α inhibition, compared to the original naphthoquinone.^13,14 Furthermore, arylamino derivatives of nor-α and nor-β lapachones present potent antitumor¹¹ and trypanocide activities.⁹

Herein is the first report on the synthesis and characterization of the novel 2-arylamine derivatives of nor-lapachol 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 activation²¹ and their cytotoxicity²² has been reported, we also investigated the redox properties of these compounds by cyclic voltammetry.

Results and Discussion

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 MgCl₂^•6H₂ O in methanol under reflux.¹⁸ The 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 ¹H NMR and infrared spectra of compounds 2a-k are consistent with their composition and structure. The ¹H NMR spectra exhibit signals in the δ 7.5-8.2 ppm region as double dublets and triple dublets, attributed to the four naphthoquinone aromatic hydrogens H5-H8. Attributions were made on the basis of ¹H x ¹H-COSY experiments, J values and multiplicity. All expected resonances were observed in the ¹³C NMR spectra of compounds 2a-k. The carbonyl peaks appear around δ 183 and 180, and those attributed to C2 bound to the nitrogen at about δ 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 ¹H NMR spectra of 2a-k is directly influenced by the nature of the R substituent group in the phenylamino ring [5.80 (4-OMe) < δ_H(18) < 6.08 (4-NO₂)].

X-ray structure / Theoretical calculations

The structure of 2-(4-methoxy-phenyl)amino-3-(2-methylpropenyl)-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.³⁶ The naphthoquinone ring system of 2b is approximately planar, the dihedral angle between the naphthoquinone plane and the arylamine phenyl ring being 47.3°(1). The torsion angles around the fragments involved in the H-bond are: C(2)-N(1)-C(11)-C(12), -158.94(17)º, C(1)-C(2)-N(1)-C(11) -154.47(16)º and N(1)-C(2)-C(1)-O(2) -3.2(2)°. The planar unsaturated side chain is twisted about 54° with respect to the ring system. This is the first reported structure of an amine derivative of nor-lapachol.

The packing of 2b involves molecules that interact through classical and non-classical hydrogen bonds, forming a 1D infinite network along the crystallographic [100] direction (Figures 4 and 5). The carbonyl O1ⁱ atom makes a classical and a non-classical hydrogen bonds with H1 to the N1 atom (amino group) and with H12 to the C12 atom (C11-C16 phenyl ring) of a neighboring molecule, forming a six-membered ring [symmetry code: i = x-1, y, z].

In addition the other C=O group interacts via O2ⁱⁱ 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.³⁷ Energies and molecular properties were obtained from a single-point calculation on the optimized geometries using the 6-311+G(2d,p) basis set³⁸ and the B3LYP functional.³⁹ 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-methylpropenyl group bonded to position 3 of the naphthoquinone ring rotated by 180º 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 ¹H NMR chemical shifts using the GIAO approach⁴⁰ confirmed that electron-attracting groups yield higher δ_H(18) values than electron-donor groups. Calculations including solvent (chloroform) show essentially the same behavior. Interestingly, the δ_H(18) value for the alternative conformation with the 2-methylpropenyl side chain rotated by 180º 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 ¹H NMR spectra of 2a do not show broadening of H18 down to -90 ºC in CD₂Cl₂.

UV-Vis spectra

The UV-Vis spectra of the compounds obtained in CHCl₃ 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 π-π^* transitions and the low-energy band in the visible region between 456 and 512 nm is attributed predominantly to π phenyl-π^* naphthoquinone transitions. Electron-donor substituents blue shift the latter band, whereas electron-attracting groups red shift it.

Cyclic voltammetry

The redox behavior of compounds 2a-k was evaluated by cyclic voltammetry (CV) at room temperature in acetonitrile/Bu₄NPF₆ (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 studied⁴¹ 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.¹⁸

Good correlation of the E_1/2(1) potentials with the σ_p and σ_p^- Hammett constants⁴² was obtained (Figures 6 and 7, respectively) except for the Fc group (Figure 7) for which the low σ_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 σ_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 assay³⁵ and the results, summarized in the 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 IC₅₀ determination of previously selected compounds (1 and 2c). Only methoxy-nor-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.

Experimental

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. ¹H and ¹³C NMR spectra were recorded with a Varian Unit Plus 300 MHz spectrometer in CDCl₃; coupling constants are reported in Hertz (Hz) and chemical shifts in parts per million (ppm) relative to internal standard Me₄Si. The hydrogen signals were attributed through coupling constant values and ¹H × ¹H - 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 TBABF₄ 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.²³ Geometries were fully optimized using the B3LYP functional²⁴ with the standard 6-31G(d) basis set²⁵ 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 model²⁶ (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.

Synthesis of the compounds 2a-k

Compounds 2a-k were synthesized by the same procedure we reported recently for the synthesis of 2-arylamine-1,4-naphthoquinones derived from 2-hydroxy-1,4-naphthoquinone and were obtained in yields that varied from 71% (2c) to 84% (2h).¹⁸ [2-methoxy-3-(2-methylpropenyl)-[1,4]-naphthoquinone] (155 mg, 0.64 mmol) 1 in MeOH (6.00 mL) was heated under reflux in the presence of 4-toluenesulfonic acid (17.2 mg, 0.1 mmol) and MgCl₂^•6H₂ O (20.3 mg, 0.1 mmol) as catalysts for 10 min. to dissolve most of 1. After addition of the respective arylamine (0.96 mmol), the reactions were monitored by TLC (1:9 ethyl acetate/hexane) and were stopped when 1 was no more observed. The resulting solids were filtered, washed with water and cold MeOH and dried under vacuum. The melting points and elemental analysis data are indicative of pure compounds. In contrast, the analogous reactions of methoxylapachol under the same conditions yielded the corresponding 1-aza-1,2-dihydro-5,10-anthraquinones in low yields described previously.¹⁹

2-(Phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2a)

Yield: 78%. mp 131-133 ºC. Anal. Calc. for C₂₀H₁₇NO₂: C 79.14; H 5.47; N 4.67%; found: C 79.19; H 5.65; N 4.62%. ¹H NMR (300 MHz, CDCl₃): δ 8.13 (ddd, 1H, J 6.51, J 1.51, J 0.49 Hz), 8.10 (ddd, 1H, J 6.51, J 1.51, J 0.49 Hz), 7.73 (td, 1H, J 6.51, J 6.51, J 1.51 Hz), 7.65 (td, 1H, J 6.51, J 6.51, J 1.51 Hz), 7.23 (br t, 1H, J 7.52 Hz), 7.07 (tt, 1H, J 7.52, J 2.05, J 1.13 Hz), 7.91 (m, 1H), 6.89 (dt, 1H, J 7.93, J 2.05, J 1.34 Hz), 5.87 (m, 1H), 1.38 (d, 3H, J 1.54 Hz), 1.24 (d, 3H, J 1.32 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 188.2, 184.3, 140.2, 139.5, 137.9, 134.8, 133.6, 132.6, 130.8, 127.9, 126.7, 124.2, 120.1, 118.9, 117.0, 100.2, 25.6, 20.6. IR (KBr) ν_max/cm^-1: 3278, 3102, 3051, 2902, 1670, 1590, 1567. UV-Vis (CHCl₃) λ_max/nm: 290 (log ε 4.15), 463 (3.37).

2-(4-Methoxy-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2b)

Yield: 80%. mp 167-169 ºC. Anal. Calc. for C₂₁H₁₉NO₃: C 75.66; H 5.74; N 4.20%; found: C 75.56; H 5.69; N 4.22%. ¹H NMR (300 MHz, CDCl₃): δ 8.10 (m, 2H), 7.72 (td, 1H, J 7.49, J 7.49, J 1.54 Hz), 7.63 (td, 1H, J 7.70, J 1.54 Hz), 5.80 (m, 1H), 6.85 (br d, 2H, J 9.25 Hz), 6.77 (br d, 2H, J 9.25 Hz), 3.80 (s, 3H), 1.40 (d, 3H, J 1.54 Hz), 1.25 (d, 3H, J 1.32 Hz). ¹³C NMR (75 MHz, CDCl₃): 183.7, 182.9, 156.5, 140.4, 138.8, 134.4, 133.0, 132.1, 130.8, 130.4, 126.0, 124.5, 118.4, 115.4, 114.7, 112.9, 55.4, 25.3, 20.2. IR (KBr) ν_max/cm^-1: 3288, 3100, 3031, 2889, 1668, 1600, 1577. UV-Vis (CHCl₃) λ_max/nm: 289 (log ε 4.20), 511 (3.45).

2-(4-Ferrocenyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2c)

Yield: 71%. mp 125-126 ºC. Anal. Calc. for C₃₀H₂₅FeNO₂: C 73.93; H 5.17; N 2.87%; found: C 73.84; H 5.11; N 2.81%. ¹H NMR (300 MHz, CDCl₃): δ 8.16 (ddd, 1H, J 7.40, J 1.57, J 0.51 Hz), 8.13 (ddd, 1H, J 7.40, J 1.57, J 0.51 Hz), 7.79 (td, 1H, J 7.40, J 7.40, J 1.57 Hz), 7.69 (td, 1H, J 7.40, J 7.40, J 0.51 Hz), 7.55 (dd, 2H, J 6.12, J 2.09 Hz), 7.17 (dd, 2H, J 6.12, J 2.09 Hz), 5.82 (m, 1H), 4.61 (dd, 2H, J 2.37, J 1.98 Hz), 4.31 (dd, 2H, J 2.37, J 1.98 Hz), 4.04 (s, 5H), 1.35 (d, 3H, J 1.61 Hz), 1.24 (d, 3H, J 1.47 Hz). ¹³C NMR (75 MHz, CDCl₃): 184.1, 181.8, 155.8, 139.4, 138.1, 131.7, 133.5, 131.3, 130.2, 129.6, 125.8, 124.3, 118.2, 114.9, 114.5, 112.3, 69.2, 68.7, 66.3, 24.9, 19.8. IR (KBr) ν_max/cm^-1: 3281, 3098, 2880, 1670, 1599, 1571, 1108, 994. UV-Vis (CHCl₃) λ_max/nm: 294 (log ε 4.29), 510 (3.64).

2-(4-Methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2d)

Yield: 81%. mp 141-142 ºC. Anal. Calc. for C₂₁H₁₉NO₂: C 79.47; H 6.03; N 4.41%; found: C 79.43; H 6.01; N 4.38%. ¹H NMR (300 MHz, CDCl₃): δ 8.13 (m, 2H), 7.74 (td, 1H, J 7.57, J 1.47 Hz), 7.66 (td, 1H, J 7.57, J 1.47 Hz), 7.05 (br d, 2H, J 8.06 Hz), 6.80 (br d, 2H, J 8.06 Hz), 5.85 (m, 1H), 2.34 (s, 3H), 1.40 (d, 3H J 1.47 Hz), 1.26 (d, 3H, J 1.22 Hz). ¹³C NMR (75 MHz, CDCl₃): 182.1, 181.3, 157.4, 141.4, 137.5, 133.9, 132.9, 132.0, 131.0, 130.9, 125.6, 123.9, 119.1, 114.3, 114.1, 112.5, 49.3, 25.1, 20.8. IR (KBr) ν_max/cm^-1: 3272, 3101, 3045, 2900, 1668, 1587, 1565. UV-Vis (CHCl₃) λ_max/nm: 286 (log ε 4.00), 505 (3.25).

2-(3-Methyl-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2e)

Yield: 84%. mp 151-152 ºC. Anal. Calc. for C₂₁H₁₉NO₂: C 79.47; H 6.03; N 4.41%; found: C 80.01; H 6.03; N 4.53%. ¹H NMR (300 MHz, CDCl₃): δ 8.12 (m, 2H); 7.74 (td, 1H, J 7.48, J 7.48, J 1.51 Hz), 7.66 (td, 1H, J 7.48, J 7.48, J 1.51 Hz), 7.70 (m, 2H); 7.06 (br d, 2H, J 8.30 Hz); 5.84 (m, 1H); 6.80 (br d, 2H, J 8.30 Hz); 2.34 (s, 3H); 1.40 (d, 1H, J 1.22 Hz); 1.26 (d, 1H, J 1.33 Hz. ¹³C NMR (75 MHz, CDCl₃): 181.9, 181.2, 157.3, 141.5, 137.2, 133.8, 133.5, 132.4, 131.9, 131.0, 130.5, 129.5, 125.3, 123.4, 118.9, 114.0, 113.8, 112.1, 50.3, 24.5, 20.3. IR (KBr) ν_max/cm^-1: 3277, 3106, 3049, 2906, 1669, 1588, 1570. UV-Vis (CHCl₃) λ_max/nm: 287 (log ε 4.15), 507 (3.31).

2-(4-Iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2f)

Yield: 73%. mp 111-112 ºC. Anal. Calc. for C₂₀H₁₆INO₂: C 55.96; H 3.76; N 3.26%; found: C 55.91; H 3.71; N 3.24%. ¹H NMR (300 MHz, CDCl₃): δ 8.13 (dd, 1H, J 7.52, J 1.58 Hz), 8.10 (dd, 1H, J 7.52, J 1.39 Hz), 7.74 (td, 1H, J 7.52, J 7.52, J 1.39 Hz), 7.66 (td, 1H, J 7.52, J 7.52, J 1.58 Hz), 7.54 (br d, 2H, J 8.71 Hz), 6.65 (br d, 2H, J 8.71 Hz), 5.90 (m, 1H), 1.47 (d, 3H, J 1.53 Hz), 1.24 (d, 3H, J 1.33 Hz). ¹³C NMR (75 MHz, CDCl₃): 184.3, 183.0, 140.3, 139.8, 137.7, 136.8, 134.9, 133.4, 132.8, 130.7, 126.5, 124.6, 118.9, 117.7, 100.3, 87.2, 25.7, 20.8. IR (KBr) ν_max/cm^-1: 3327, 3065, 2994, 1664, 1594, 1566. UV-Vis (CHCl₃) λ_max/nm: 290 (log ε 4.01), 460 (3.47).

2-(3-Iodo-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2g)

Yield: 75%. mp 117-118 ºC. Anal Calc. for C₂₀H₁₆INO₂: C 55.96; H 3.76; N 3.26%; found: C 55.92; H 3.74; N 3.23%. ¹H NMR (300 MHz, CDCl₃): δ 8.11 (dd, 1H, J 7.61, J 1.64 Hz), 8.08 (dd, 1H, J 7.61, J 1.45 Hz), 7.72 (td, 1H, J 7.61, J 1.45 Hz), 7.63 (td, 1H, J 7.61, J 1.64 Hz), 7.40 (m, 1H), 6.90 (m, 3H), 5.89 (m, 1H), 1.44 (d, 3H, J 1.49 Hz), 1.23 (d, 3H, J 1.28 Hz). ¹³C NMR (75 MHz, CDCl₃): 184.1, 182.9, 140.5, 140.1, 137.3, 136.1, 134.6, 133.1, 132.4, 130.1, 126.9, 126.3, 124.3, 123.2, 118.1, 116.9, 100.1, 87.1, 25.2, 20.5. IR (KBr) ν_max/cm^-1: 3331, 3069, 2998, 1664, 1595, 1568. UV-Vis (CHCl₃) λ_max/nm: 284 (log ε 4.30), 456 (3.40).

2-(4-Cyano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2h)

Yield: 79%. mp 161-163 ºC. Anal. Calc. for C₂₁H₁₆N₂O₂ : C 76.81; H 4.91; N 8.53%; found: C 76.79; H 4.87; N 8.50%. ¹H NMR (300 MHz, CDCl₃): δ 8.14 (dd, 1H, J 1.59, J 7.46 Hz), 8.12 (dd, 1H, J 1.49, J 7.46 Hz), 7.77 (td, 1H, J 7.46, J 7.46, J 1.49 Hz), 7.70 (td, 1H, J 7.46 Hz, J 7.46, J 1.59 Hz), 7.52 (br d, 2H, J 8.58 Hz), 6.90 (br d, 2H, J 8.58 Hz), 6.03 (m, 1H), 1.50 (d, 3H, J 1.48 Hz), 1.24 (d, 3H, J 1.27 Hz). ¹³C NMR (75 MHz, CDCl₃): 184.4, 182.8, 141.9, 141.4, 138.6, 135.1, 133.2, 132.0, 130.7, 127.0, 126.7, 121.7, 120.3, 119.2, 118.8, 106.1, 100.3, 25.9, 21.0. IR (KBr) ν_max/cm^-1: 3282, 3079, 2972, 2927, 2218, 1664, 1594, 1567. UV-Vis (CHCl₃) λ_max/nm: 290 (log ε 4.19), 471 (3.48).

2-(3-Cyano-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2i)

Yield: 82%. mp 173-174 ºC. Anal Calc. for C₂₁H₁₆N₂O₂ : C 76.81; H 4.91; N 8.53%; found: C 76.77; H 5.01; N 8.55%. ¹H NMR (300 MHz, CDCl₃): δ 8.15 (ddd, 1H, J 5.25 Hz, J 1.60 Hz, J 0.46 Hz), 8.12 (ddd, 1H, J 5.25 Hz, J 1.60 Hz, J 0.46 Hz), 7.76 (td, 1H, J 5.25, J 5.25, J 1.60 Hz), 7.69 (td, 1H, J 5.25, J 5.25, J 1.60 Hz), 7.34 (m, 2H), 7.11 (m, 2H), 5.95 (m, 1H), 1.47 (d, 3H, J 1.37 Hz), 1.25 (d, 3H, J 1.14 Hz). ¹³C NMR (75 MHz, CDCl₃): 183.7, 182.4, 140.8, 138.7, 138.2, 134.7, 132.8, 132.7, 130.2, 128.3, 126.7, 126.5, 126.2, 126.0, 124.9, 118.4, 118.1, 111.5, 25.3, 20.5). IR (KBr) ν_max/cm^-1: 3281, 3081, 2970, 2925, 2218, 1661, 1591, 1564. UV-Vis (CHCl₃) λ_max/nm: 284 (log ε 4.02), 475 (3.19).

2-(4-Nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2j)

Yield: 84%. mp 205-207 ºC. Anal. Calc. for C₂₀H₁₆N₂O₄ : C 68.96; H 4.63; N 8.04%; found: C 68.86; H 4.55; N 8.03%. ¹H NMR (300 MHz, CDCl₃): δ 8.14 (m, 4H), 7.87 (br s, 1H), 7.78 (td, 1H J 7.59, J 7.59, J 1.47 Hz), 7.71 (td, 1H, J 7.59, J 1.57 Hz), 6.91 (br d, 2H, J 9.08 Hz), 6.08 (m, 1H), 1.27 (d, 3H, J 1.17 Hz), 1.53 (d, 3H, J 1.43 Hz). ¹³C NMR (75 MHz, CDCl₃): 184.0, 182.3, 143.4, 142.4, 141.4, 138.0, 134.7, 132.7, 132.9, 130.7, 126.6, 123.6, 120.4, 120.8, 118.3, 99.8, 25.6, 20.7. IR (KBr) ν_max/cm^-1: 3283, 3084, 2974, 2911, 1593, 1663, 1500, 1332. UV-Vis (CHCl₃) λ_max/nm: 281 (log ε 4.37), 469 (3.63).

2-(3-Nitro-phenyl)amino-3-(2-methylpropenyl)-1,4-naphthoquinone (2k)

Yield: 83%. mp 210-211 ºC. Anal. Calc. for C₂₀H₁₆N₂O₄ : C 68.96; H 4.63; N 8.04%; found: C 68.49; H 4.66; N 8.01%. ¹H NMR (300 MHz, CDCl₃): δ 8.15 (ddd, 1H, J 7.57, J 1.46, J 0.49 Hz), 8.13 (ddd, 1H, J 7.57, J 1.46, J 0.49 Hz), 7.91 (ddd, 1H, J 8.06, J 1.93, J 0.98 Hz), 7.82 (br s, 1H), 7.77 (td, 1H, J 7.57, J 7.57, J 1.46 Hz), 7.69 (td, 1H, J 7.57, J 1.46 Hz), 7.67 (br s, 1H), 7.40 (br t, 1H, J 8.06 Hz), 7.21 (dd, 1H, J 8.06, J 1.95 Hz), 6.01 (m, 1H), 1.43 (d, 3H, J 1.46 Hz), 1.24 (d, 3H, J 0.98 Hz). ¹³C NMR (75 MHz, CDCl₃): 183.8, 182.4, 147.3, 141.1, 138.6, 134.7, 132.8, 132.7, 130.2, 128.1, 127.1, 126.5, 126.2, 118.7, 118.4, 117.9, 116.3, 25.4, 20.6). IR (KBr) ν_max/cm^-1: 3279, 3080, 2970, 2908, 1597, 1661, 1498, 1330. UV-Vis (CHCl₃) λ_max/nm: 275 (log ε 4.39), 470 (3.59).

X-ray crystallography

The X-ray diffraction data for 2b were collected at 295 K from a Enraf-Nonius Kappa-CCD²⁷ diffractometer with graphite monochromatized Mo K_α radiation. The cell parameters were obtained and refined using PHICHI²⁸ and EvalCCD²⁹ programs. Intensities for (1) were corrected by Lorentz polarization and absorption with the SADABS³⁰ program. The structure was solved by SHELXS-97 Direct Methods,²⁹ and refined with SHELXL-97,³² contained within the WinGX-32 crystallography program.³³ The 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 Csp², and refined as riding on their respective C atoms, with U_iso(H) = 1.2Ueq(Csp² ). H atoms bonded to C atoms in the methyl group were located geometrically and with the C-H distances fixed at 0.96Å for Csp³ and with U_iso(H) = 1.5Ueq(Csp³). 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-3³⁴ for Windows was used to draw the Figures.

Antitumor assays

The compounds (1-5 mg mL^-1) were tested for cytotoxic activity against four cancer cell lines: SF-295 (Central Nervous System), HCT-8 (colon), MDAMB-435 (breast) and HL-60 (human leukemia). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol L^-1 glutamine, 100 U mL^-1 penicillin, and 100 μg mL^-1 streptomycin at 37 ºC with 5% CO₂. Each compound was dissolved in DMSO and diluted with water to obtain a concentration of 1 mg mL^-1. They were incubated with the cells for 72 h. The negative control received the same amount of DMSO (0.5% in the highest concentration). Doxorubicin (0.1-0.58 μg mL^-1) was used as a positive control. The cell viability was determined by reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-phenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as described by Mosmann.³⁵

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.

Supplementary Information

Supplementary data associated with this paper are available free of charge at http://jbcs.sbq.org.br, as a PDF file and contain the results of the theoretical calculations, crystallographic data, NMR spectra (¹H and APT), cyclic voltammograms and antitumor assays of compounds 2a-k. Crystallographic data for the structural analysis of the three complexes have been deposited with the Cambridge Crystallographic Data Center, CCDC 734112. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: [phone]; e-mail: [e-mail]).

Acknowledgments

The authors thank Prof. A. V. Pinto and Dr. M. C. F. R. Pinto for providing nor-lapachol and the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support. Pronex-FAPERJ (grant number E-26/171.512/2006) is acknowledged. J. W. D. Carneiro, M. D. Vargas, A. P. Neves and C. A. Camara are recipients of CNPq research fellowships. A. I. Francisco and A. Casellato were benefited with CAPES fellowships. We also thank the X-ray diffraction laboratory (LDRX) of Universidade Federal Fluminense for data collection.

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Received: August 11, 2009

Web Release Date: November 12, 2009

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

1. 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 180º 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 δ_H18 value for the alternative conformation (with the 2-methyl-propenyl group rotated by 180º, 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 90º (entry 2b-2 in Table S1) yielded essentially the same δ_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.

Table S1 - Click to enlarge

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.

2. Crystallographic Data

2.1. Computing details

Data collection: COLLECT (Nonius, 1998); cell refinement: PHICHI (Duisenberg, 2000); data reduction: EVALCCD (Duisenberg, 2003); program(s) used to solve structure: SHELXS97 (Sheldrick, 1997); program(s) used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: WinGX publication routines (Farrugia, 1999).

2.2. 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_α radiation. The cell parameters were obtained and refined using PHICHI and EvalCCD programs. Intensities for (2b) were corrected by Lorentz polarization and absorption with the SADABS program. The structure was solved by SHELXS-97 Direct Methods, and refined with SHELXL-97, contained within the WinGX-32 crystallography program. The 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 Csp², and refined as riding on their respective C atoms, with U_iso(H) = 1.2Ueq(Csp²). H atoms bonded to C atoms in the methyl group were located geometrically and with the C-H distances fixed at 0.96Å for Csp³ and with U_iso(H) = 1.5Ueq(Csp³). The positional parameters of H(1) bonded to N(1) was obtained from a Fourier difference map and refined freely with an isotropic displacement parameter; the distance for N(1)-H(1) is 0.87(2). X-ray data are listed in Table S2.

Crystallographic data for the structural analysis of compound 2b have been deposited with the Cambridge Crystallographic Data Center, CCDC 734112. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: [phone]; e-mail: [e-mail]).

The packing of 2b involves molecules that interact intra- and intermolecularly through classical and non-classic hydrogen bonds, forming a 1D infinite network along the [100] crystallographic direction (Figures S2 and ^S3). The naphthoquinone carbonyl O1ⁱ interacts via classical and non-classic hydrogen bonds with H(1) to N(1) (imine group) and with H(12) to C(12) [C(11)-C(16) phenyl ring] of a neighboring molecule forming a six membered ring [symmetry code: i = x-1, y, z]. In addition the other carbonyl group interacts via O2ⁱⁱ with H(5) to C(5) forming a ten membered ring in association with N(1)-H(1)^...O(1). The H-bond geometric parameters are listed in Table S4. Table S5 gathers the atomic coordinates and equivalent isotropic displacement parameters.

^{Figure S3 - Click to enlarge}

3. ¹H NMR and APT Spectra (CDCl₃) of Compounds 2a-k

Figure S4 - Click to enlarge

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4. Variable Temperature ¹H NMR Spectra of Compound 2a

Figure S26 - Click to enlarge

4. Cyclic Voltammograms (100mV) of Compounds 2a-k

5. 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.

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Present address: Instituto de Química, Universidade Federal do Rio de Janeiro, 22640-000 Rio de Janeiro-RJ, Brazil

Publication Dates

• Publication in this collection
  18 Feb 2010
• Date of issue
  2010

History

• Received
  Augu
• Accepted
  Nove