Synthesis of New trans-Dehydrocrotonin Nitrogenated Derivatives and their Cytotoxic and DNA-Topoisomerase I Inhibitory Activities

Abstract

A new series of 19-nor-clerodane diterpene derivatives was synthesized from the natural trans-dehydrocrotonin obtained from stem barks of Croton cajucara (Euphorbiaceae), a native medicinal plant of the Brazilian Amazon. The new derivatives were obtained by changes in the ketone moiety of trans-dehydrocrotonin leading to nitrogenated derivatives which are: three substituted hydrazine diterpenes, oxime, and methyloxime. The cytotoxic effect of the diterpene derivatives was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma and K562 human leukemia cells. The cytotoxic activity of the hydrazine and oxime semi-synthetic derivatives was better than the one of the natural product trans-dehydrocrotonin. Moreover, all diterpenes were tested for their DNA topoisomerase I inhibitory activity, and the most effective one, in general, was observed to the phenyl-hydrazone derivative. Results indicated that the topoisomerase I inhibitory effect is correlated with the cytotoxic activity.

Keywords:
Croton cajucara; diterpenes; trans-dehydrocrotonin; cytotoxic activity; DNA-topoisomerase I

Introduction

Medicinal plants are the dominant form of medicine in most countries, and the World Health Organization estimates that around 80% of the world population in developing countries relies on traditional plant medicines for primary healthcare needs.¹

Many Euphorbiaceae plants are well known in different parts of the world as toxic and/or medicinal, and Croton is a large genus of this family widely distributed in tropical and subtropical regions of both hemispheres. Croton cajucara Benth, commonly known as "sacaca", is a medicinal plant largely grown in the Brazilian Amazon. Both stem bark and leaves are extensively used in the form of tea or pills for the treatment of several diseases.^2,3 It is widely known in the Amazonian traditional phytotherapy for the treatment of diarrhea and gastrointestinal disorders,³ liver diseases and weight loss,⁴ diabetes⁵ and high blood cholesterol levels.⁶ The terpenoid classes are the predominant special metabolite constituents of this genus, and the clerodane diterpenes, the prevalent type of Croton.^3,7-10

Several clerodane diterpenes isolated from different species had been tested for their biological activities, and presented potentially useful medicinal properties, such as antimicrobial,^11-14 anti-inflammatory,^15,16 antiprotozoal,^17-21 and antitumoral.^22-26

The trans-dehydrocrotonin (1) is a nor-clerodane diterpene isolated from Croton cajucara as major component.²⁷ Experimental laboratory animals and in vitro studies have shown that 1 has insecticidal,²⁸ antigenotoxic,²⁹ antiulcerogenic,^3,30-33 anti-inflammatory,³⁴ anti-leishmanial³⁵ and antitumor effects.^36-40

Our studies involving the Brazilian medicinal plants with potential cytotoxic activities led us to prepare a group of new clerodane diterpene derivatives starting from 1 (Figure 1). Not only did we consider the fact that 1 is the principal and abundant component of C. cajucara,²⁷ but also the previous in vivo and in vitro results of anti-tumor activities.^36,37 Clerodane 2, a natural component of C. cajucara stem bark, was also evaluated against representative neoplastic cell lines.

Figure 1
trans-Dehydrocrotonin (1) and trans-crotonin (2) clerodane diterpenes isolated from stem bark of Croton cajuacara.

In the present work, we report the synthesis of five new trans-dehydrocrotonin nitrogenated derivatives, and their in vitro evaluation of cytotoxic activity. The new derivatives were prepared from clerodane 1, previously isolated from the stem bark of Croton cajucara,^3,27 and they were also evaluated against Ehrlich carcinoma and K562 human leukemia cells. Furthermore, the inhibitory action of the new derivatives, as well as the isolated natural compounds 1 and 2, was evaluated for DNA-topoisomerase I inhibition (Topo I).

Results and Discussion

Synthesis and characterization

Clerodane 1 was isolated from the stem bark of methanol extract of Croton cajucara, as previously described.^3,27 The synthesis of the diterpene derivatives involved reactions of the C-2 carbonyl group in the A ring. The structure of the clerodane skeleton was maintained to assure its natural feature in an attempt to preserve or enhance the cytotoxic activities previously observed.^37,38 Thus, to construct a sensible series of trans-dehydrocrotonin derivatives for SAR purpose, some new semi-synthetic derivatives were prepared.

Those derivatives were performed including nitrogenated moieties at the ketone carbon (C-2) of A ring. The new compounds were the hydrazone derivatives, such as the unsubstituted hydrazone (3), methyl-hydrazone (4) and phenyl-hydrazone (5), as well as the oxime (6), and the methylated oxime (7). The synthetic route of the semi-synthetic clerodanes 3-7 is outlined in Scheme 1.

Scheme 1
Reagents and reaction conditions to prepare the semi-synthetic clerodanes 3-7.

Derivatives 3, 4 and 5 were obtained from the treatment of 1 using hydrazine hydrate, methyl hydrazine and phenyl hydrazine, respectively, at room temperature, at different time reactions (6, 21 and 24 h, respectively), in ethanol as solvent, following the previously reported procedure.⁴¹ The corresponding hydrazones were obtained in 80, 60 and 75% yield for 3, 4 and 5, respectively. The oxime derivative (6) was prepared from 1 using hydroxylamine hydrochloride in basified ethanol with NaOH, for 10 min under reflux, thus yielding 80%. The reaction between 6 and methyl iodide, and Ag₂O, at low temperature,⁴² furnished the corresponding methylated oxime (7) in 60% yield. All the compounds were purified by silica gel chromatographic column using CHCl₃, hexane and ethyl acetate as eluents. These new semi-synthetic diterpene derivatives 3-7 were characterized by infrared, ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, and then briefly discussed.

The infrared spectra showed the absence of carbonyl moiety signal at 1666 cm^-1 observed to trans-dehydrocrotonin²⁷ and, the presence of absorption in the range of 1622-1599 cm^-1 assigned to C=N group of hydrazone derivatives, 3, 4 and 5. ¹H NMR spectra of 3, 4 and 5 presented a similar pattern, detaching the chemical shifts in the range of d 6.51-6.98, and signals corresponding to aromatic hydrogens of phenyl-hydrazone derivative (5) were observed. The N−H signal was observed in the range of d 5.00-6.00. The ¹³C NMR spectra of 3, 4 and 5 presented coherent changes to respect 1 in accordance to the moiety nature attached to C-2. Typical values of imino function were observed for C-2 in the range of d 151.9-152.0, and smaller variations in the carbons C-1, C-3 and C-4, as expected.

The infrared spectra of 6 and 7 derivatives showed absorptions of 1635 and 1610 cm^-1, respectively, assigned to C=N moiety, instead of C=O absorption of trans-dehydrocrotonin (1666 cm^-1).²⁷ The ¹H NMR chemical shifts observed for 6, the oxime derivative, and for methyl-oxime (7) were coherent with the structure proposal, in which H-3 appeared in d 6.13 for 7, presenting deshielding effect when compared to 6 (d 5.89), due to the stereo-spatial position of the methyl group. The signal corresponding to the hydroxyl-oxime of 6 appeared in d 8.05 as a wide singlet. ¹³C NMR chemical shifts were in accordance to the proposal structures; d 156.2 and 158.7 were attributed to C-2 at 6 and 7, respectively; and, shielding effect (γ effect) was observed at C-3, with ∆d 6.5 and ∆d 10.0 when compared to 1, for 6 and 7, respectively.

Cytotoxic effects

The cytotoxic action of special metabolites isolated from C. cajucara Benth stem bark extracts against tumor cells^36,37 led us to continue studying the possible action of the new semi-synthetic derivatives of trans-dehydrocrotonin diterpene (1). The antiproliferative effect of 1-7 against the ascitic Ehrlich carcinoma and human leukemia K562 cells was evaluated. Assays were performed using the MTT method⁴³ with quercetin and vincristine as positive controls for Ehrlich and K562 leukemia, respectively.

In vitro assays using Ehrlich carcinoma cells with 4, 5, 6 and 7 nitrogenated derivatives showed a more significant antiproliferative activity when compared to 1,the major active component of C. cajucara, previously evaluated by Grynberg et al.,³⁶ with dose dependent responses over 48 h culture period. IC₅₀ values 45.78 ± 4.35, 16.78 ± 1.42, 21.88 ± 1.96 and 43.43 ± 3.96 µmol L^-1 were also compared to quercetin (IC₅₀ = 44 µmol L^-1), as shown in Table 1. Interestingly, the unsubstituted hydrazone derivative 3 did not present any significant activity until the maximum concentration of 50 µmol L^-1 used in the assays, thus suggesting a positive contribution of hydrophobic effect.

Table 1
IC50 values for nor-clerodane diterpene derivatives against Ehrlich carcinoma and K562 leukemia cells

Derivative	IC50 ± SD / (µmol L-1)
	Ehrlich	K562
1	> 50a	> 50
2	> 50b	> 50
3	> 50	24.74 ± 2.3
4	45.78 ± 4.35	7.85 ± 1.49
5	16.78 ± 1.42	13.08 ± 1.12
6	21.88 ± 1.96	> 50
7	43.43 ± 3.96	40.72 ± 5.63
Quercetin	44.0 ± 3.9	-
Vincristine	-	0.060 ± 0.001

• ^a
  1, IC₅₀ = 166 µmol L^-1;³⁷
• ^b
  2, IC₅₀ = 164 µmol L^-1.³⁷ SD: standard deviation.

The inhibitory effect on K562 cells in vitro, after 96 h in culture, was not observed for 1 nor 2 until the dose of 50 µmol L^-1.³⁷ However, the new nitrogenated derivatives and the unsaturated alcohol presented cytotoxic effect against K562, also with dose dependent responses, with IC₅₀ = 24.74 ± 2.3 (3), 7.85 ± 1.49 (4), 13.08 ± 1.12 (5), and 40.72 ± 5.63 µmol L^-1 (7), as shown in Table 1.

DNA-topoisomerase I inhibitory effect

The conversion of supercoiled plasmid DNA to relaxed DNA by human topoisomerase I (Topo 1) was examined in the presence of 1, 2 and the new semi-synthetic diterpene derivatives. The activity of the compounds on Topo I was observed through relaxation assays using pBR322 plasmid DNA. Campothecin, a well-known Topo 1 enzyme inhibitor, was used as a positive control.^44,45

Results were observed through the alteration of the electrophoretic mobility of pBR322 plasmid DNA, combining Topo I action and the drugs at 200 µmol L^-1. After this development, the results were analyzed with ethidium bromide in UV light, and recorded by photographing with a digital camera. As shown in Figure 2, the mobility of supercoiled closed circular double-stranded plasmid DNA increased on Topo I mediated relaxation, when subjected to electrophoresis with ethidium bromide (line 3). In the presence of 200 µmol L^-1 campothecin (positive control), the relaxation effect was not observed (line 1). The relaxation inhibitory effect was also observed in the presence of Topo I with 1, 3, 4, 5, 6, and 7. When the assays were performed at 20 µmol L^-1, only 4 did not show inhibitory effect on Topo I. These results must justify the cytotoxic activities observed.

Figure 2
Effect of trans-dehydrocrotonin derivatives in 200 µmol L^-1 on Topo I. Line 1: 0.25 µg DNA pBR322 + 1U Topo I + campothecin 200 µmol L^-1; line 2: 0.25 µg DNA pBR322; line 3: 0.25 µg DNA pBR322 + 1U Topo I; line 4: 0.25 µg DNA pBR322 + 1U Topo I + 1; line 5: 0.25 µg DNA pBR322 + 1U Topo I + 6; line 6: 0.25 µg DNA pBR322 + 1U Topo I + 7; line 7: 0.25 µg DNA pBR322 + 1U Topo I + 3; line 8: 0.25 µg DNA pBR322 + 1U Topo I + 4; line 9: 0.25 µg DNA pBR322 + 1U Topo I + 5.

Conclusions

In summary, new clerodane diterpene derivatives were synthesized from trans-dehydrocrotonin, the major bioactive metabolite of the Amazonian medicinal plant Croton cajucara, by simple processes and with good yields. In vitro studies of cytotoxic activities against Ehrlich carcinoma and K562 leukemia cells showed that the nitrogenated derivatives were more active than the natural products 1 and 2, especially phenyl and methyl-hydrazone. The study of a possible mechanism of action showed the strong inhibitory effect of the nitrogenated derivatives, except 4, on DNA-topoisomerase I, in the relaxation assay.

Experimental

Chemistry

Reagents and apparatus

The melting points were determined on a Quimis hot stage instrument. Infrared spectra (KBr pellets or CHCl₃) were recorded on a PerkinElmer 1605 spectrometer and expressed in cm^-1. The microanalyses were carried out with a Carlo Erba EA-1110 CHNS-O Elemental Analyser. ¹H and ¹³C NMR spectra were obtained on a Bruker AC 200 spectrometer (200 and 50.3 MHz), using CDCl₃ or DMSO-d₆ as solvent, and TMS as internal standard. Elemental analyses were performed on a PerkinElmer 2400 CHN in the Laboratory of Environmental Science at the State University of Northern Rio de Janeiro (UENF). Thin layer chromatography (TLC) analyses were performed on silica gel 60 F254 plates. All the reagents were purchased from Merck or Sigma-Aldrich.

Vegetal material and the isolation of 1 and 2

Plant material, Croton cajucara Benth was collected in Jacundá-PA (Amazon region, Brazil), and botanically authenticated by Dr Nelson A. Rosa, Museu Paraense Emílio Goeldi. A voucher specimen No. 247 is deposited at the Herbarium of the same museum. The methanolic extraction from the powdered bark of Croton cajucara and the isolation of 1 and 2 was carried out as previously reported.^3,27

General method for the preparation of hydrazone derivatives (3-5)

For the solution of 1 (0.30 mmol) in ethanol (1 mL), 1.2 mmol of corresponding hydrazine was added; the reaction mixture was stirred for 6 h at room temperature for hydrazine hydrate, and for 21 and 24 h under reflux for methyl and phenyl-hydrazine, respectively. At these times, the hydrazine excess had been evaporated, and the crude products were purified by flash chromatography on silica gel (EtOAc:hexane, 10:90) for 4 and 5, and by recrystallization from EtOH and powdered charcoal for 3.

2-Hydrazone-dehydrocrotonin (3)

80% yield; mp 119-121 ºC; IR (KBr) ν_max / cm^-1 3433, 2927-2876, 1695, 1378; ¹H NMR (200 MHz, CDCl₃) δ 1.11 (3H), 1.66 (1H, β), 1.84 (1H, α), 1.94 (3H, br s), 2.15 (1H, α), 2.22 (1H), 2.35-2.39 (1H), 2.48 (1H, β), 3.14 (1H, br s), 3.44 (2H), 5.40 (1H), 5.86 (1H), 2.35-2.39 (1H), 6.38 (1H, s), 7.42 (2H); ¹³C NMR (50 MHz, CDCl₃) δ 17.4, 21.6, 27.5, 28.5, 30.1, 38.8, 40.9, 41.6, 51.8, 45.5, 72.0, 108.8, 124.1, 125.2, 140.2, 143.8, 151.9, 160.1, 177.4. Anal. calcd. for C₁₉H₂₄N₂O₃: C, 69.49; H, 7.37; N, 8.53. Found: C, 69.73; H, 7.28; N, 8.72.

2-Methylhydrazone-dehydrocrotonin (4)

60% yield; mp 135-136 ºC; IR (KBr) ν_max / cm^-1 3296, 2960-2864, 1746, 1318; ¹H NMR (200 MHz, DMSO-d₆) δ 1.10 (3H), 1.59 (1H, β), 1.71 (2H), 1.81 (1H, α), 2.11 (1H, α), 2.20 (1H), 2.47 (1H, m), 2.57 (1H, β), 2.77 (3H), 2.86 (1H, br s), 5.58 (2H, br s), 5.86 (1H), 6.75 (1H, s), 7.79 (1H), 7.93 (1H); ¹³C NMR (50 MHz, CDCl₃) δ 17.6, 20.8, 25.5, 28.3, 30.2, 38.0, 40.9, 41.0, 44.1, 51.6, 71.7, 108.9, 124.1, 125.4, 140.6, 144.4, 177.4. Anal. calcd. for C₂₀H₂₆N₂O₃: C, 70.15; H, 7.65; N, 8.18. Found: C, 70.37; H, 7.43; N, 8.24.

2-Phenylhydrazone-dehydrocrotonin (5)

75% yield; mp 171-172 ºC; IR (KBr) ν_max / cm^-1 3328, 2958-2858, 1743, 1322; ¹H NMR (200 MHz, CDCl₃) δ 1.13 (3H, d, J 5.1 Hz), 1.62 (1H, β), 1.70 (2H), 1.77 (2H), 1.84 (3H, br s), 1.94 (1H, α), 2.18 (1H, α, d, J 3.3 Hz), 2.24-2.27 (1H), 2.57 (1H, br s, β), 2.33-2.70 (1H, dd, J 14.0, 6.3 Hz), 2.83 (1H, br s), 5.51 (1H), 5.59 (1H, sl), 6.48 (1H), 6.83 (2H, dd, J 0.7, 8 Hz), 6.96 (1H, d, J 6.3 Hz), 6.98 (2H, d, J 0.7 Hz), 7.58 (1H), 7.59 (1H); ¹³C NMR (50 MHz, CDCl₃) δ 17.4, 21.4, 26.0, 27.6, 30.0, 39.6, 40.4, 41.7, 45.9, 51.5, 72.3, 108.0, 113.2, 120.3, 122.3, 126.6, 129.1, 139.1, 144.3, 152.0, 153.1, 166.1, 177.4. Anal. calcd. for C₂₅H₂₈N₂O₃: C, 74.23; H, 6.98; N, 6.93. Found: C, 74.37; H, 6.72; N, 7.04.

The preparation of 2-oxime-dehydrocrotonin (6)

For the solution of 1 (0.5 mmol), HONH₂-HCl (0.8 mmol), 2 mL of ethanol in 0.4 mL of water was added under stirring NaOH (2.75 mmol). The mixture had been refluxed for 10 min, and when it reached the room temperature, it was added to an acid solution (0.3 mL HCl and 2 mL water). After that, the precipitate was filtered, washed with water and dried at room temperature. 80% yield; mp 197-198 ºC; IR (KBr) ν_max / cm^-1 3279, 2941-2860, 1751, 1635, 1463-1436, 1351, 1184; ¹H NMR (200 MHz, CDCl₃) δ 1.87 (1H, α), 1.12 (3H, d, J 5.1 Hz), 1.55 (1H), 1.63 (1H, β), 1.74 (1H), 1.82 (3H, br s), 2.15 (1H, α), 2.25 (2H), 2.44 (2H, t, J 8.3 Hz), 2.92 (1H, β), 3.44 (1H, d, J 5.1 Hz), 5.40 (1H, t, J 8.3 Hz), 5.89 (1H), 6.50 (1H, s), 7.43 (1H), 7.49 (1H), 8.05 (1H, br s); ¹³C NMR (50 MHz, CDCl₃) δ 17.6, 21.3, 28.5, 30.2, 38.4, 41.8, 46.3, 51.8, 72.1, 108.2, 119.2, 125.1, 140.0, 144.2, 153.2, 156.2, 177.4. Anal. calcd. for C₁₉H₂₃NO₄: C, 69.28; H, 7.04; N, 4.25. Found: C, 69.41; H, 6.98; N, 4.32.

The preparation of 2-O-methyloxime-dehydrocrotonin (7)

For the solution of 3 (0.15 mmol) in CH₂Cl₂ (2 mL), Ag₂O (0.22 mmol) and CH₃I (0.75 mmol) were slowly added. The solution was stirred at room temperature for 3 h. After that, CH₂Cl₂ was evaporated, and the residue was purified by flash chromatography on silica gel (hexane:AcOEt, 90:10), thus providing product 7 as an oil in 60% yield. IR (CHCl₃) ν_max / cm¹ 2929-2860, 1749, 1610, 1319;¹H NMR (200 MHz, CDCl₃) δ 1.23 (3H, br s), 1.66 (1H, β), 1.91 (1H, α), 2.00 (2H), 2.17 (1H, m, α), 2.44 (1H, t, J 7.6 Hz), 2.93 (1H, m, β), 3.17 (1H, br s), 5.05 (3H, br s), 5.42 (1H, br s), 6.13 (1H, br s), 6.53 (1H), 7.44 (1H), 7.49 (1H); ¹³C NMR (50 MHz, CDCl₃) δ 17.6, 22.0, 28.0, 30.0, 39.4, 39.7, 40.3, 44.0, 45.1, 51.6, 64.7, 72.3, 108.1, 116.6, 126.7, 140.0, 144.5, 158.6, 166.3, 177.2. Anal. calcd. for C₂₀H₂₅NO₄: C, 69.95; H, 7.34; N, 4.08. Found: C, 70.09; H, 7.21; N, 4.

Biological assays

Materials

The dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Sigma Co. (USA); quercetine and vincristine sulfate, used as positive control in cytotoxic assays, were obtained from Sigma-Aldrich Co. (USA) and Merck Co. (Germany), respectively.

Cell culture

Ehrlich carcinoma cells were maintained for 12-14 days in Swiss mice. The tumor cell cultures were initiated from mouse Ehrlich ascites with at least one in vitro passage prior to use. K562 cells were maintained in RPMI 1640 medium, containing 10% fetal calf serum (FCS), 100 U mL^-1 penicillin G and 100 µg mL^-1 streptomycin. The cell cultures were incubated at 37 ºC in a 5% CO₂ humidified atmosphere.

Drugs and cytotoxic assay

The stock solutions of the diterpene derivatives were prepared in DMSO. The drug cytotoxicity assays were performed as previously described by Mosmann,⁴³ using MTT for viable cell measurements. The aliquots of 5 × 10⁵ cells mL^-1 (Ehrlich) and 2 × 10⁴ cells mL^-1 (K562) were seeded in triplicate onto 96-microtiter flat well plates in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 µM 2-mercaptoethanol, 100 IU mL^-1 penicillin and 100 µg mL^-1 streptomycin. The drugs dissolved in DMSO at various concentrations were added to the culture and adjusted to a final DMSO concentration of 0.2% (v/v). The cultures were maintained under 5% CO₂ at 37 ºC. Cellular viability was determined in the presence or absence of diterpenes, using the standard MTT assay.⁴³ Quercetine and vincristine were used as positive control to Ehrlich and K562, respectively. All determinations were carried out in triplicate. After 48 h (Ehrlich) and 96 h (K562), at 37ºC under 5% CO₂, the cultures were incubated with MTT (5 mg mL^-1) for 3 h. The formazan produced by live cells were solubilized with acidic isopropanol, and the absorbance was measured at 570 nm. IC₅₀ values were obtained by linear regression analysis of the absorbance (percent) versus the log of drug concentration. The data are expressed as means ± standard deviation (SD) of 3 independent experiments. Statistical significance was assessed by the Student's t-test, p < 0.05 was considered significant difference.

DNA-topoisomerase I assay

Topo I inhibition was determined by relaxation assay, which was carried out as described in the TopoGEN screening kit. For Topo I, one unit of the enzyme was utilized to relax 0.125 µg of the supercoiled ϕ×174 plasmid DNA. The reaction mixture (10 µL) contained the drug, DNA, assay buffer, 1U of Topo I and water. The mixture was incubated at 37 ºC for 30 min, and the reaction was finalized by the addition of 1 µL of dye solution containing 25% bromophenol blue, 50% glycerol and 10% SDS. Reaction products were loaded onto a 1% agarose gel, containing ethidium bromide. Electrophoresis was carried out in tris-acetate-EDTA, pH 8.5, at 15 V, for 3.5 h; and then, it was photographed with a digital camera by illumination.

• This paper was prepared in honor of the late Professor Angelo da Cunha Pinto

Acknowledgments

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amaparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support and the fellowships.

Supplementary Information

Supplementary data (¹H and ¹³C NMR spectra) are available free of charge at http://jbcs.sbq.org.br as PDF file.

References

• ¹
  Sampson, J. H.; Phillipson, J. D.; Bowery, N. G.; O'Neill, M. J.; Houston, J. G.; Lewis, J. A.; Phytother. Res 2000, 14, 24.
• ²
  Salatino, A.; Salatino, M. L. F.; Negri, G.; J. Braz. Chem. Soc 2007, 18, 11.
• ³
  Maciel, M. A. M.; Pinto, A. C.; Arruda, A. C.; Pamplona, S. G. S. R.; Vanderlinde, F. A.; Lapa, A. J.; Cólus, I. M. S.; Echevarria, A.; Grynberg, N. F.; Farias, R. A. F.; Luna, A. M. C.; Rao, V. S. N.; J. Ethnopharmacol 2000, 70, 41.
• ⁴
  Grassi-Kassisse, D. M.; Wolf-Nunes, V.; Miotto, A. M.; Farias-Silva, E.; Souza-Brito, A. R. M.; Nunes, D. S.; Spadari-Bratfisch, R. C.; J. Pharm. Pharmacol 2003, 55, 253.
• ⁵
  Rodrigues, G.; Marcolin, E.; Bona, S.; Porawski, M.; Lehmann, M.; Marroni, N. P.; Arq. Gastroenterol 2010, 47, 301.
• ⁶
  Silva, R. M.; Santos, F. A.; Maciel, M. A. M.; Pinto, A. C.; Rao, V. S. N.; Planta Med 2001, 67, 763.
• ⁷
  Huang, W.; Li, G.; Wu, Y.; Ge, W.; Chung, H. Y.; Ye, W.; Li, Y.; Wang, G.; Heterocycles 2014, 89, 1585.
• ⁸
  Yang, L.; Zhang, Y.; Wu, Z.; Chen, N.; Jiang, S.; Li, Y.; Wang, G.; Chem. Lett 2016, 45, 1235.
• ⁹
  Li, R.; Morris-Natschke, S. L.; Lee, K.; Nat. Prod. Rep 2016, 33, 1166.
• ¹⁰
  Liu, C. P.; Xu, J. B.; Zhao, J. X.; Xu, C. H.; Dong, L.; Ding, J.; Yue, J. M.; J. Nat. Prod 2014, 77, 1013.
• ¹¹
  Gupta, V. K.; Tiwari, N.; Gupta, P.; Verma, S.; Pal, A.; Srivastava, S. K.; Darokar, M. P.; Phytomedicine 2016, 23, 654.
• ¹²
  Cavin, A. L.; Hay, A. E.; Marston, A.; Stoeckli-Evans, H.; Diallo, D.; Hostettmann, K.; J. Nat. Prod 2006, 69, 768.
• ¹³
  Huang, Z.; Jiang, M. Y.; Zhou, Z. Y.; Xu, D.; Z. Naturforsch. B: J. Chem. Sci 2010, 65, 83.
• ¹⁴
  Bayor, M. T.; Ayim, J. S. K.; Marston, G.; Phillips, R. M.; Shnyder, S. D.; Wheelhouse, R. T.; Wright, C. W.; Nat. Prod. Commun 2008, 3, 1875.
• ¹⁵
  Wu, T.; Cheng, Y.; Chen, C.; Ng, L.; Chou, L.; Huang, L.; Chen, Y.; Kuo, S.; El-Shazly, M.; Wu, Y.; Chang, F.; Liaw, C.; Molecules 2014, 19, 2049.
• ¹⁶
  Dai, S. J.; Liang, D. D.; Ren, Y.; Liu, K.; Shen, L.; Chem. Pharm. Bull 2008, 56, 207.
• ¹⁷
  Mambu, L.; Grellier, P.; Florent, L.; Joyeau, R.; Ramanitrahasimbola, D.; Rasoanaivo, P.; Frappier, F.; Phytochemsitry 2006, 67, 444.
• ¹⁸
  Harinantenaina, L.; Takahara, Y.; Nishizawa, T.; Kohchi, C.; Soma, G. I.; Asakawa, Y.; Chem. Pharm. Bull 2006, 54, 1046.
• ¹⁹
  Munro, T. A.; Duncan, K. K.; Xu, W.; Wang, Y.; Liu-Chen, L. Y.; Carlezon Jr, W. A.; Cohen, B. M.; Beguin, C. B.; Bioorg. Med. Chem 2008, 16, 1279.
• ²⁰
  Chang, H. L.; Chang, F. R.; Chen, J. S.; Wang, Y. P.; Wu, Y. H.; Wang, C. C.; Wu, Y. C.; Hwang, T. L.; Eur. J. Pharmacol 2008, 586, 332.
• ²¹
  Bautista, E.; Toscano, A.; Calzada, F.; Díaz, E.; Yepez-Mulia, L.; Ortega, A.; J. Nat. Prod 2013, 76, 1970.
• ²²
  Qiu, M.; Cao, D.; Gao, Y.; Li, S.; Zhu, J.; Yang, B.; Zhou, L.; Zhou, Y.; Jin, J.; Zhao, Z.; Fitoterapia 2016, 108, 81.
• ²³
  Maciel, M. A. M.; Pinto, A. C.; Brabo, S. N.; Silva, M. N.; Phytochemistry 1998, 49, 823.
• ²⁴
  Vieira-Júnior, G. M.; Dutra, L. A.; Ferreira, P. M.; Moraes, M. O.; Costa-Lotufo, L. V.; O'Pessoa, C.; Torres, R. B.; Boralle, N.; Bolzani, V. S.; Cavalheiro, A. J.; J. Nat. Prod 2011, 74, 776.
• ²⁵
  Jullian, V.; Bounduelle, C.; Valentin, L.; Acebey, L.; Duigou, A. G.; Prevostb, M. F.; Sauvain, M.; Bioorg. Med. Chem 2005, 15, 5065.
• ²⁶
  Williams, R. B.; Norris, A.; Miller, J. S.; Birkinshaw, C.; Ratovoson, F.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I.; J. Nat. Prod 2007, 70, 206.
• ²⁷
  Kubo, I.; Asaka, Y.; Shibata, K.; Phytochemistry 1991, 30, 2546.
• ²⁸
  Poersch, A.; Santos, F. V.; Maciel, M. A. M.; Câmara, J. K. P.; Dantas, T. N. C.; Cólus, I. M. S.; Mutat. Res 2007, 629, 14.
• ²⁹
  Souza-Brito, A. R. M.; Rodriguez, J. A.; Hiruma-Lima, C. A.; Haun, M.; Nunes, D. S.; Planta Med 1998, 64, 126.
• ³⁰
  Hiruma-Lima, C. A.; Spadari-Bratfisch, R. C.; Grassi-Kassisse, D. M.; Souza-Brito, A. R. M.; Planta Med 1999, 65, 325.
• ³¹
  Rodriguez, J. A.; Hiruma-Lima, C. A.; Souza-Brito, A. R. M.; Hum. Exp. Toxicol 2004, 23, 455.
• ³²
  Hiruma-Lima, C. A.; Tona, W.; Gracioso, J. S.; Almeida, A. B. A.; Batista, L. M.; Magri, L.; Paula, A. C. B.; Soares, F. R.; Nunes, D. S.; Souza-Brito, A. R. M.; Biol. Pharm. Bull 2002, 25, 425.
• ³³
  Carvalho, J. C. T.; Silva, M. F. C.; Maciel, M. A. M.; Pinto, A. C.; Nunes, D. S.; Lima, R. M.; Bastos, J. K.; Sarti, S. J.; Planta Med 1996, 62, 402.
• ³⁴
  Silva, R. M.; Oliveira, F. M.; Cunha, K. M. A.; Maia, J. L.; Maciel, M. A. M.; Pinto, A. C.; Nascimento, N. R. F.; Santos, F. A.; Rao, V. S. N.; Vasc. Pharmacol 2005, 43, 11.
• ³⁵
  Lima, G. S.; Castro-Pinto, D. B.; Machado, G. C.; Maciel, M. A. M.; Echevarria, A.; Phytomedicine 2015, 22, 1133.
• ³⁶
  Grynberg, N. F.; Echevarria, A.; Lima, J. E.; Pamplona, S. S. R.; Pinto, A. C.; Maciel, M. A.; Planta Med 1999, 65, 687.
• ³⁷
  Maciel, M. A. M.; Martins, J. R.; Pinto, A. C.; Kaiser, C. R.; Esteves-Souza, A.; Echevarria, A.; J. Braz. Chem. Soc 2007, 18, 391.
• ³⁸
  Corrêa, D. H. A.; Melo, P. D. S.; Carvalho, C. A. A.; Azevedo, M. B. M.; Durán, N.; Haun, M.; Eur. J. Pharmacol 2005, 510, 17.
• ³⁹
  Freire, A. C. G.; Assis, C. F.; Frick, A. O.; Melo, P. D. S.; Haun, M.; Aoyama, H.; Durán, N.; Sauer, M. M.; Kallás, E. G.; Ferreira, C. V.; Leuk. Res 2003, 27, 823.
• ⁴⁰
  Frungillo, L.; Martins, D.; Teixeira, S.; Anazetti, M. C.; Melo, P. D. S.; Durán, N.; J. Pharm. Sci 2009, 98, 4796.
• ⁴¹
  Newkome, G. R.; Fishel, D. L.; J. Org. Chem 1966, 31, 677.
• ⁴²
  Brehm, M.; Gӧckel, V. H.; Jarglis, P.; Lichtenthaler, F. W.; Tetrahedron: Asymmetry 2008, 19, 358.
• ⁴³
  Mosmann, T.; J. Immunol. Methods 1983, 65, 55.
• ⁴⁴
  Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D.; Biochim. Biophys. Acta 1998, 1400, 83.
• ⁴⁵
  Kim, D. H.; Lee, N.; Mini-Rev. Med. Chem 2002, 2, 611.

Publication Dates

• Publication in this collection
  Jan 2018

History

• Received
  27 Mar 2017
• Accepted
  27 June 2017