Introduction

Naphthoquinones are natural pigments that are widely distributed in plants, fungi and some animals (Premalatha et al., 2012; Zhou et al., 2012; Yang et al., 2013). Naphthoquinones are structurally characterized by the presence of two carbonyl groups at the 1,4-positions and, less frequently, at the 1,2- or 1,3-positions of the naphthalene, which has ring (A) and ring (B) (Fig. 1). These compounds, when they occur naturally, have hydroxyl and/or methyl groups as substituents (Glazunov and Berdyshev, 2012). They are privileged structures in medicinal chemistry due to their characteristics, structural properties and biological activities on prokaryotic and eukaryotic cells. In most cases, the biological activity of naphthoquinones is related to their redox and acid–base properties, both of which can be modulated synthetically.

Fig. 1
figure 1

Natural and synthetic naphthoquinones and derivatives tested (1–25)

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The synthesis of novel derivatives of 1,4-naphthoquinone is of particular interest since these compounds exhibit strong action as antimalarial, antibacterial, antifungal and anticancer agents (Koyama, 2010).

The mechanism of action of naphthoquinones could be due to their properties as oxidizing or dehydrogenation agents, in a similar way to hydrogen peroxide and superoxide radicals (Tran et al., 2004b; Pinto and de Castro, 2009). This behavior is related to the ability of quinones to accept one or two electrons to form highly reactive radical anion intermediates, which are responsible for the oxidative stress observed in the cells (Valderrama et al., 2008). However, several other mechanisms have been attributed to quinonoid compounds, including DNA intercalation, alkylation, induction of DNA strand breaks or the inhibition of special proteins or enzymes such as topoisomerases (Plyta et al., 1998).

Some naphthoquinones, such as juglone (5-hydroxy-1,4-naphthoquinone), lawsone (2-hydroxy-1,4-naphthoquinone) and plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), are present in plants, and their antibacterial effects on various species of aerobic and anaerobic organisms have been demonstrated (Lim et al., 2006; Sakunphueak and Panichayupakaranant, 2012; Bhattacharya et al., 2013).

Other naphthoquinones, such as toxins derived from naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), which are produced by Fusarium solani, can attack plants, fungi and bacteria (Rohnert et al., 1998). Alkannin, its enantiomer shikonin and its derivatives are active against Gram-positive and Gram-negative bacteria such as Staphylococcus aureus, Enterococcus faecalis/faecium, Escherichia coli and Pseudomonas aeruginosa (Shen et al., 2002; Al-Mussawi, 2010). Some compounds show strong activity against fungi such as Candida sp. and filamentous fungi (Pawar et al., 2014). These activities depend on the position(s) of the substituents in the naphthoquinone ring (Figs. 1, 2).

Fig. 2
figure 2

Naphthoquinone derivatives tested (26–50)

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In most studies, antibacterial and antifungal activities have been studied with not standardized methods. Best methods to achieve these activities are based by broth dilution antibacterial and antifungal susceptibility testing of bacteria that grow aerobically and yeasts. These methods are used in all microbiology laboratories around of the world. For this, the aim of our study was to test the antibacterial and antifungal activities of a number of naphthoquinone derivatives by these methods. Some compounds were commercially obtained, and other ones were synthesized in our laboratory to perform a structure–activity relationship study. The compounds are numbered in order to facilitate the discussion of their bioactivity rather than by their chemical nature.

Results and discussion

Fifty compounds were screened for activity against bacteria and yeast; 45 of these generated by parallel synthesis, and 5 purchased for comparison purposes.

Chemistry

Most of the naphthoquinones were prepared from 47, which can easily be obtained from 38 by reduction with tin(II)chloride (Guerrero-Vásquez et al., 2013). Compounds 39, 40 and 41 were also prepared from 38 by acetylation and methylation. Similarly, 37 was prepared using chlorohydroquinone and maleic anhydride rather than dimethoxybenzene and dichloromaleic anhydride (Bekaert et al., 1986). Compound 25 was obtained when the same reaction was carried out using 2-bromo-1,4-dimethoxybenzene (Scheme 1).

Scheme 1
scheme 1

General procedure for the preparation of naphthoquinone derivatives

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Oxidation of 47 with potassium superoxide produced compound 48 in good yield (92 %) (Lewis and Paul, 1977), whereas air oxidation in the presence of a solution of sodium hydroxide yielded 1 (93 %) (Lewis and Paul, 1977). Compound 1 was brominated with Br2 in acetic acid to produce 43 and 44, and acetylation of 43 afforded 46 (Horowska et al., 1988; Takeya et al., 1999; Tandon et al., 2005). Compound 42 was prepared by following the same strategy as for the preparation of 43 (Horowska et al., 1988; Takeya et al., 1999; Tandon et al., 2005).

Permethylation of 47 with dimethyl sulfate and potassium hydride gave 33 (Kawasaki et al., 1988). This compound was used to produce 32 by bromination with NBS (Bloomer and Zheng, 1998), 34 by formylation with POCl3 in chloroform and 3 by oxidation with ceric ammonium nitrate (CAN) (Scheme 2) (Terada et al., 1987; Kawasaki et al., 1988). Bromination of 3 produced 45 (Huot and Brassard, 1974). Compounds 16 and 17 were obtained by similar strategies employing bromochloromethane and potassium carbonate for the formation of the methylenedioxy derivatives and NBS for the bromination (Dallaeker et al., 1983).

Scheme 2
scheme 2

Procedure for the preparation of methoxylated derivatives of naphthoquinones

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Compounds 20, 34, 35 and 36 were obtained by the methodology described for the synthesis of the potent phytotoxic agent naphthotectone (Guerrero-Vásquez et al., 2013). Compounds 21–24 were obtained as described for the synthesis of speciosins G and P (Guerrero-Vásquez et al., 2014). Acyl derivatives of juglone were obtained using standard conditions; acetylation was carried out using acetic anhydride and pyridine, and the other 5-acetyl-juglone derivatives 5–15 were prepared with 4-dimethylaminopyridine (DMAP) as base in reactions with the corresponding acid chloride.

Compounds 2, 29, 39, 49 and 50 were obtained from Sigma-Aldrich

All derivatives were characterized by 1H and 13C nuclear magnetic resonance (NMR), infrared spectroscopy and high-resolution mass spectrometry (HRMS). The spectroscopic data for the compounds are consistent with the assigned structures (see “Experimental” section).

Biological evaluation

All of the compounds were screened by the broth microdilution method for their antibacterial activity against two Gram-positive bacteria, E. faecalis and S. aureus, and two Gram-negative bacteria, E. coli and P. aeruginosa. The compounds were also evaluated for their in vitro antifungal activity against Candida krusei, Candida parapsilosis and Cryptococcus neoformans. We did not test Candida albicans because this one has an antifungal susceptibility profile very similar to C. parapsilosis. The lowest concentrations of the compounds that prevented visible growth are listed in Table 1.

Table 1 Antimicrobial activity (MIC profiles) of the synthesized compounds
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It was determined that the solvent did not have antibacterial or antifungal activities against any of the test microorganisms. Gentamicin, doxycycline and amphotericin B were used as standard drugs, and these were also tested under similar conditions for comparison as positive controls. The minimum inhibitory concentrations (MIC) of the synthesized compounds against highly inhibited organisms are listed in Table 1.

Some of the tested compounds were very active against Candida sp., showed moderate antifungal activity against C. neoformans and were inactive or less active against all other test organisms. P. aeruginosa was the least susceptible microorganism as only 6 compounds (4, 6, 7, 37, 38 and 42) exhibited activity at ≥MIC 64 µg/mL. Only compound 42 showed moderate antibacterial activity against S. aureus at MIC 16 µg/mL. C. krusei was the most susceptible fungus in the study, with an MIC value of 2 µg/mL. This behavior is significant because C. krusei possesses an intrinsic resistance to many triazole antifungal drugs, especially fluconazole, which is the main drug used in antifungal therapy (Yadav et al., 2012).

SAR for naphthoquinone derivatives

In order to carry out a structure–activity relationship study, a number of naphthoquinone derivatives and related compounds were prepared and tested. These compounds comprise a variety of functionalization and structural modifications that allowed us to establish structural requirements for activity.

Influence of hydroxyl and methoxy groups at the C5 and/or C8 positions

Several authors have reported that the presence of hydroxyl groups in the B ring has a significant effect on activity, particularly in compounds with hydroxyl groups at the C5 and C8 positions. In these compounds, the antimicrobial and antifungal activities were twice as high as in those compounds with only one hydroxyl group (Tran et al., 2004b; Yakubovskaya et al., 2009a; Hughes et al., 2011). In our study, naphthazarin (1) showed antimicrobial activity against E. coli, E. faecalis and S. aureus with MIC values between 64 and 128 µg/mL. In yeasts, the MIC values were 32–128 µg/mL. Nevertheless, juglone (2) showed antifungal activity against C. krusei and C. parapsilosis with MICs of 32 and 128 µg/mL, respectively, while it was inactive against C. neoformans and all bacteria tested. Antifungal and antibacterial activities have been reported previously in these compounds. For example, naphthazarin was active against E. coli and S. aureus (Yakubovskaya et al., 2009b) and other fungi such as Saccharomyces carlsbergensis (Yakubovskaya et al., 2009b), while Tandon et al. (2004) did not find in vitro antifungal activity against C. albicans, C. neoformans, Sporothrix schenckii, Trichophyton mentagrophytes, Microsporum canis and Aspergillus fumigatus. Juglone is one of the most widely studied naphthoquinones. In some studies, this compound did not demonstrate good antimicrobial activity and it was also inactive against the bacteria P. aeruginosa, S. aureus and the fungus C. albicans (Pawar et al., 2012; Sreelatha et al., 2014a). However, juglone did exhibit very good activity against the filamentous fungi Aspergillus niger, Paecilomyces variotii, Trametes versicolor and Gloeophyllum trabeum (Yang et al., 2009). These results are consistent with ours, although the activity against filamentous fungi was not tested in this work.

Methoxylation at the C5 and/or C8 positions converted 1 to 5,8-dimethoxy-1,4-naphthoquinone (3), which was inactive against bacteria but very active against yeasts. In these microorganisms, compound 3 had an MIC ≤ 16 µg/mL, i.e., eight times lower than that of 1. This structural change is very important, since it demonstrates that the presence of hydroxyl groups in the naphthoquinone ring improves the cytotoxicity on normal cells, whereas the permethylated products of naphthazarin and its derivatives showed slightly lower cytotoxicity in this respect (Zhou et al., 2011). This compound was more active than compounds 1 and 2 against the yeasts tested.

Hydrophilicity versus lipophilicity

The level of activity may be influenced by excessive hydrophilicity or lipophilicity, as in compounds that contain substituents with 10 or more carbon atoms (Riffel et al., 2002). Among the series of compounds tested, from 5-acetyl-juglone (4) to 5-palmitoyl-juglone (15), the structures progressively changed by an increase by 1 carbon atom at the C5 position. This increase in the number of carbon atoms resulted in the loss of antimicrobial activity. In bacteria, 5-heptanoyl-juglone (9), with 7 carbons at the C5 position, was the first compound that was inactive. In yeast, 5-lauroyl-juglone (13) was inactive and this compound has 12 carbons in the same position. 5-Butanoyl-juglone (6) was the most active against bacteria and fungi. The activity of these compounds could be influenced by the partition coefficient or log P, which is a ratio of concentrations of non-ionized compounds between two solutions. The log P value is used in the study of quantitative structure–property relationships as a measure of lipophilicity (Leo et al., 1971). Log P values for the series of compounds tested were between 1.51 (4) and 7.16 (15). MIC values in the range 4–32 mg/L for yeasts correspond to log P values between 1.51 (4) and 2.26 (6). Log P values >3 were associated with inactive compounds and low activity compounds such as 7–15. This trend is consistent with other compounds in this study, where log P values of <1, e.g., compound 49 (0.99), and close to or >3, e.g., compounds 32–36, were inactive against all microorganisms tested.

Number of rings

Number of rings can affect antibacterial and antifungal activities. So, structures with more than two rings did not show any antimicrobial activity, including methylenedioxy-naphthalene (16), 2-bromo-methylenedioxy-naphthalene (17) and 1,4,8-trimethoxy-3-methyl-anthraquinone (18). This finding is supported by the results reported by Tran et al. (2004a) who found that addition of other rings led to a marked loss of activity. They also showed that compounds without a quinone structure were less active, as is the case with our compounds (from 19 to 31), where structures differed from the 1,4-naphthoquinone core.

Influence of carbonyl groups at the C1 and C4 positions

Previous studies showed that the biological activity of 1,4-naphthoquinone and its derivatives are affected and/or modulated by the presence of 1,4-substituents (Tran et al., 2004a). The presence of two carbonyl groups at the C1 and C4 positions is essential for antimicrobial activity, since the loss of both groups makes the molecule inactive (Verma and Hansch, 2004). Moreover, the presence of two free keto groups at the C1 and C4 positions leads to a greater inhibitory activity than at the C1 and C2 positions (Tran et al., 2004a). In our study, it was found that all compounds that lack the carbonyl groups at these positions were inactive on the seven microorganisms tested, i.e., compounds 32, 33, 34, 35 and 36. The addition or removal of other substituents in these inactive compounds did not improve their antimicrobial activity.

Halogen-substituted 1,4-naphthoquinones

The addition of halogen groups in the structure of naphthoquinone can produce a marked improvement in the antimicrobial and antifungal activity. The activity of these compounds has been explained as a short circuiting of the cell electron transfer normally executed by quinones (Holmes et al., 1964). The compounds with halogen groups in the nucleus had the lowest MIC values of all compounds in our study.

As far as the chloro-1,4-naphthoquinone derivatives are concerned, comparison of compound 1, which does not have substituents, with 2-chloro-5, 8-dihydroxy-1,4-naphthoquinone (37) showed increase in antimicrobial and antifungal activity by a factor of four against S. aureus and by a factor of sixteen against all yeasts tested. However, the presence in 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (38) of two chloro-substituents in the A ring led to a decrease in the antimicrobial and antifungal activity, except on C. neoformans, which gave the lowest MIC value (≤4 µg/mL) in our study for this microorganism. These compounds also showed activity against P. aeruginosa, albeit very low in this case (MIC 128 µg/mL).

Methoxylation at C5 and C8, as in the change from 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (40) to compound 38, did not improve the activity. In general, methoxylation led to lower antimicrobial activity, although the activity against Candida remained the same. Chlorination at positions C6 and C7, as in 6,7-dichloro-5,8-dimethoxy-1,4-naphthoquinone (41), led to the loss of activity.

Ethoxylation at the C5 and C8 positions, as in 2,3-dichloro-5,8-diacethoxy-1,4-naphthoquinone (39), improved the activity against S. aureus and C. krusei, but the activity against C. neoformans was four times lower. This finding demonstrates that ethoxylation improved the activities more markedly than methoxylation, although the most active compound against Candida sp. in the series tested was the monochloro derivative with hydroxyl groups at C5 and C8 (37) (MIC 2 µg/mL).

Regarding bromo 1,4-naphthoquinone derivatives, the presence of a bromo substituent at the C2 position, as in 2-bromo-5,8-dihydroxy-1,4-naphthoquinone (43), improved the activity against the Gram-positive bacteria and yeasts tested, albeit to a lesser extent than that observed for the monochloro derivative. The removal of the hydroxyl group at C8, as in 2-bromo-5-hydroxy-1,4-naphthoquinone (42), improved the activity against P. aeruginosa, S. aureus and C. neoformans, although the activities against other microorganisms assessed were similar. This compound showed the best antimicrobial activity against S. aureus of all compounds studied (MIC 16 µg/mL).

Methoxylation of monobromo derivatives at the C5 and C8 positions, as in 2-bromo-5,8-dimethoxy-1,4-naphthoquinone (45), markedly reduced the activity against all microorganisms studied and gave rise to considerably higher MIC values (64 µg/mL) compared to compound 43. However, ethoxylation of this compound gave 2-bromo-5,8-diacethoxy-1,4-naphthoquinone (46), which was inactive against bacteria but showed the same activity against yeast.

Dihalogenation at C2 and C3, as in 2,3-dibromo-5,8-dihydroxy-1,4-naphthoquinone (44), led to a slight increase in the antifungal activity but not in the antimicrobial activity.

In general, halogenated derivatives showed activity against all microorganisms assessed and they were more active against Candida sp., especially against C. krusei, with MIC values between 2 and 8 µg/mL (Ambrogi et al., 1970).

Ambrogi et al. observed that chloro derivatives showed stronger activity than bromo derivatives. Similar behavior was found in our study, and comparison of compounds 37 and 38 with 42 and 43 shows that the chloro derivatives are slightly more active against most of the microorganisms assessed. It has been demonstrated in several studies that the presence of a Cl group in ring A is essential for antifungal activity (Tandon et al., 2009; Tran et al., 2009).

In summary, chloro derivatives were more active than bromo derivatives, followed by compounds with methyl and hydroxyl groups, respectively.

Other modifications in the ring of 1,4-naphthoquinone

Finally, we decided to study other modifications in structure of 1,4-naphthoquinone. First, for comparison, the lack of the double bond in the A ring, as compound 47, enhanced antifungal activity with MIC ≤ 16 µg/mL, compared to 1, although 47 was inactive against all bacteria tested.

Addition of a hydroxyl group at C2 of the 1,4-naphthoquinone moiety, as in 2,5,8-trihydroxy-1,4-naphthoquinone (48), compared to 1, led to a compound that was inactive against Gram-negative bacteria and yeasts but more active against Gram-positive bacteria. This activity was very poor in general. These results are consistent with those obtained in a study carried out by Yakubovskaya et al. (2009b) who found significantly higher MIC values (>100 µg/mL) in E. coli and S. aureus. This behavior could occur because this hydroxyl derivative exhibited low cytotoxicity. The low activity of hydroxyl derivatives could be connected with deprotonation of this group under the conditions of the biological experiment or when it acts in cells (Pelageev et al., 2014).

The absence of hydroxyl groups at the C5 and C8 positions in lawsone (49), compared to 48, make this compound inactive against all microorganisms tested. Several authors have found similar results on using different methods for the evaluation of antibacterial and antifungal activities of lawsone, namely the disk diffusion (Tekin et al., 2015) and broth microdilution methods (Sreelatha et al., 2014b). This behavior could be attributed to the chemical structure of lawsone, which is a hydroxyquinone that can exist as a 1,2-diketone or a 1,4-quinone. Structural modification of the phenolic hydroxyl group could result in a reduction in activity, as observed in our study (Sreelatha et al., 2014b).

Replacement of the hydroxyl group at the C2 position of 49 by a methyl group gave 2-methyl-1,4-naphthalenedione (or menadione, 50), which has a low activity against bacteria but moderate activity against yeast. Compound 50 showed antimicrobial activity, whereas compound 49 was completely inactive. This is because menadione inhibited the growth of Candida species by stimulating the production of radical oxygen species (Ueno et al., 2008).

Of these compounds, only 47 had a moderate activity against yeasts. The antibacterial activity was very low.

Experimental

General

All reagents (obtained from Aldrich Chemical Co.) and solvents (HPLC grade) were used without further purification. NMR spectra were recorded at room temperature on Agilent Inova 500- and 400-MHz spectrometers. The 1H and 13C chemical shifts are referenced to the CDCl3 solvent at δ H 7.25 and δ C 77.0 ppm. Melting points were taken on a Kofler hot stage apparatus and are uncorrected. General IR spectra (KBr) were recorded on a PerkinElmer FTIR Spectrum 1000 spectrophotometer. High-resolution mass spectra (HRMS) were obtained on a WATERS SYNAPT G2 mass spectrometer (70 eV). Reactions were monitored by thin-layer chromatography (TLC) on silica gel (F245 Merck plates).

Procedure for the synthesis of compound 4

Compound 4 was prepared by acetylation of juglone according to a literature procedure with modifications (Greco et al., 2010). Juglone (50 mg, 1 eq.) was dissolved in the minimum amount of pyridine. Acetic anhydride (271 µL, 10 eq.) was added dropwise to the solution of juglone, and the reaction mixture was stirred at room temperature and monitored by TLC. Saturated aqueous copper sulfate was added, and liquid–liquid extraction was carried out to remove the pyridine. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography using 10 % AcOEt/hexane as eluent to give 5-acetyl-1,4-naphthoquinone (40 % yield).

Procedure for the synthesis of compounds 5–15

These derivatives were obtained according to the methodology previously described by Mathew et al. (2010) with some modifications. These acylation reactions were performed in dichloromethane with juglone (50 mg, 1 eq.) as the starting material. 4-Dimethylaminopyridine (68.4 µL, 3 eq.) and the corresponding acyl chloride (125 µL, 5 eq.) were added at 0 °C. The reaction mixture was stirred at room temperature and monitored by TLC. After completion of the reaction, the organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The resulting mixture was purified by column chromatography using 100 % chloroform as eluent to obtain compounds 5–15. New compounds are characterized.

5-O-Butanoyloxy-1,4-naphthoquinone ( 6 )

This compound was obtained as a yellow solid in 78 % yield; mp 72–75 °C; IR (KBr) νmax 3078, 2963, 1753, 1664, 1594, 1138 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.02 (1H, dd, J = 1.2, 7.8 Hz, H-8), 7.74 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.2, 8.0 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.71 (2H, t, J = 7.5 Hz, H-2′), 1.84 (2H, qt, J = 7.5, 7.4 Hz, H-3′), 1.08 (3H, t, J = 7.4 Hz, H-4′); 13C NMR (CDCl3, 100 MHz): δ = 184.0 (C, C-4), 183.4 (C, C-1), 171.7 (C, C-1′), 149.4 (C, C-5), 139.7 (CH, C-2), 137.1 (CH, C-3), 134.6 (CH, C-7), 133.3 (C, C-8a), 129.6 (CH, C-6), 124.7 (CH, C-8), 123.2 (C, C-4a), 35.8 (CH2, C-2′), 17.8 (CH2, C-3′), 13.5 (CH3, C-4′); HRESIMS m/z (pos) 245.0788 C14H13O4 [M + H]+ (calcd.: 245.0814).

5-O-Pentanoyloxy-1,4-naphthoquinone ( 7 )

This compound was obtained as a brown oil in 90 % yield; IR (KBr) νmax 3079, 2957, 1762, 1669, 1594, 1138 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.02 (1H, dd, J = 1.2, 7.8 Hz, H-8), 7.73 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.2, 8.0 Hz, H-6), 6.91 (1H, d, J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.73 (2H, t, J = 7.5 Hz, H-2′), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3′), 1.48 (2H, tt, J = 7.5, 7.4 Hz, H-4′), 0.98 (3H, t, J = 7.4 Hz, H-5′); 13C NMR (CDCl3, 100 MHz): δ = 184.3 (C, C-4), 183.7 (C, C-1), 172.2 (C, C-1′), 149.7 (C, C-5), 140.0 (CH, C-2), 137.4 (CH, C-3), 134.9 (CH, C-7), 133.6 (C, C-8a), 129.9 (CH, C-6), 125.0 (CH, C-8), 123.5 (C, C-4a), 34.0 (CH2, C-2′), 26.6 (CH2, C-3′), 22.4 (CH2, C-4′), 13.9 (CH3, C-5′); HRESIMS m/z (pos) 259.0936 C15H15O4 [M + H]+ (calcd.: 259.0970).

5-O-Heptanoyloxy-1,4-naphthoquinone ( 9 )

This compound was obtained as a brown oil in 56 % yield; IR (KBr) νmax 3079, 2929, 1768, 1667, 1596, 1135 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.03 (1H, dd, J = 1.2, 7.8 Hz, H-8), 7.75 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.2, 8.1 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.83 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.5 Hz, H-2′), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3′), 1.48–1.31 (6H, m, H-4′, H-5′, H-6′), 0.90 (3H, t, J = 7.1 Hz, H-7′); 13C NMR (CDCl3, 100 MHz): δ = 184.2 (C, C-4), 183.2 (C, C-1), 172.1 (C, C-1′), 149.6 (C, C-5), 139.9 (CH, C-2), 137.3 (CH, C-3), 134.8 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.9 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH2, C-2′), 31.5 (CH2, C-5′), 28.8 (CH2, C-4′), 24.4 (CH2, C-3′), 22.5 (CH2, C-6′), 14.0 (CH3, C-7′); HRESIMS m/z (pos) 287.1320 C17H19O4 [M + H]+ (calcd.: 287.1283).

5-O-Octanoyloxy-1,4-naphthoquinone ( 10 )

This compound was obtained as a brown oil in 49 % yield; IR (KBr) νmax 3038, 2924, 1768, 1668, 1596 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.03 (1H, dd, J = 1.2, 7.8 Hz, H-8), 7.75 (1H, dd, J = 8.1, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.2, 8.1 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.83 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.5 Hz, H-2′), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3′), 1.48–1.24 (8H, m, H-4′, H-5′, H-6′, H-7′), 0.89 (3H, t, J = 6.9 Hz, H-8′); 13C NMR (CDCl3, 100 MHz): δ = 184.2 (C, C-4), 183.6 (C, C-1), 172.1 (C, C-1′), 149.6 (C, C-5), 139.9 (CH, C-2), 137.3 (CH, C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.9 (CH, C-8), 123.4 (C, C-4a), 34.2 (CH2, C-2′), 31.7 (CH2, C-6′), 30.9 (CH2, C-4′), 29.0 (CH2, C-5′), 24.4 (CH2, C-3′), 22.6 (CH2, C-7′), 14.1 (CH3, C-8′); HRESIMS m/z (pos) 301.1422 C18H21O4 [M + H]+ (calcd.: 301.1440).

5-O-Nonanoyloxy-1,4-naphthoquinone ( 11 )

This compound was obtained as a yellow solid in 64 % yield; mp 56–61 °C; IR (KBr) νmax 3079, 2926, 1767, 1667, 1595, 1133 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.02 (1H, dd, J = 1.3, 7.8 Hz, H-8), 7.73 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.3, 8.0 Hz, H-6), 6.91 (1H, d, J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.5 Hz, H-2′), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3′), 1.48–1.24 (10 H, m, H-4′, H-5′, H-6′, H-7′, H-8′), 0.87 (3H, t, J = 6.9 Hz, H-9′); 13C NMR (CDCl3, 100 MHz): δ = 184.2 (C, C-4), 183.6 (C, C-1), 172.1 (C, C-1′), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2 (CH, C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.8 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH2, C-2′), 31.8 (CH2, C-7′), 29.23 (CH2, C-4′), 29.13 (CH2, C-6′), 29.11 (CH2, C-5′), 24.4 (CH2, C-3′), 22.6 (CH2, C-8′), 14.1 (CH3, C-9′); HRESIMS m/z (pos) 315.1586 C19H23O4 [M + H]+ (calcd.: 315.1596).

5-O-Decanoyloxy-1,4-naphthoquinone ( 12 )

This compound was obtained as a brown solid in 84 % yield; mp 44–49 °C; IR (KBr) νmax 3076, 2924, 1766, 1667, 1594, 1137 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.02 (1H, dd, J = 1.3, 7.9 Hz, H-8), 7.73 (1H, dd, J = 7.9, 7.9 Hz, H-7), 7.36 (1H, dd, J = 1.3, 7.9 Hz, H-6), 6.91 (1H, d, J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.7 Hz, H-2′), 1.80 (2H, tt, J = 7.7, 7.7 Hz, H-3′), 1.40–1.24 (12 H, m, H-4′, H-5′, H-6′, H-7′, H-8′, H-9′), 0.87 (3H, t, J = 6.7 Hz, H-10′); 13C NMR (CDCl3, 100 MHz): δ = 184.2 (C, C-4), 183.6 (C, C-1), 172.1 (C, C-1′), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2 (CH, C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.8 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH2, C-2′), 31.8 (CH2, C-8′), 29.41 (CH2, C-6′), 29.28 (CH2, C-7′), 29.24 (CH2, C-4′), 29.13 (CH2, C-5′), 24.4 (CH2, C-3′), 22.6 (CH2, C-9′), 14.1 (CH3, C-10′); HRESIMS m/z (pos) 329.1787 C20H25O4 [M + H]+ (calcd.: 329.1753).

5-O-Palmitoyljuglone ( 15 )

This compound was obtained as a yellow solid in 49 % yield; mp 66–69 °C; IR (KBr) νmax 3076, 2921, 1760, 1664, 1590, 1140 cm−1; 1H NMR (CDCl3, 400 MHz): δ = 8.03 (1H, dd, J = 1.1, 7.8 Hz, H-8), 7.74 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.1, 8.0 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.83 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.6 Hz, H-2′), 1.81 (2H, m, H-3′), 1.48–1.25 (24 H, m, H-4′, H-5′, H-6′, H-7′, H-8′, H-9′, H-10′, H-11′), 0.87 (3H, t, J = 6.4 Hz, H-12′); 13C NMR (CDCl3, 100 MHz): δ = 184.2 (C, C-4), 183.2 (C, C-1), 172.1 (C, C-1′), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2 (CH, C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.8 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH2, C-2′), 31.9 (CH2, C-10′), 29.58 (CH2, C-6′), 29.56 (CH2, C-8′), 29.4 (CH2, C-7′), 29.24 (CH2, C-4′), 29.31 (CH2, C-5′), 29.28 (CH2, C-9′), 29.14 (CH2, C-4′), 24.4 (CH2, C-3′), 22.6 (CH2, C-11′), 14.1 (CH3, C-12′); HRESIMS m/z (pos) 357.2024 C22H29O4 [M + H]+ (calcd.: 357.2060).

Procedure for the synthesis of compound 25

To a stirred solution of commercially available 1-bromo-2,5-dimethoxybenzene (500 mg, 2.3 mmol) in dichloroethane (15 mL) at 0 °C was added maleic anhydride (237 mg, 2.42 mmol) followed by AlCl3 (644 mg, 4.83 mmol) in small portions over 2 h 30 min. The reaction mixture was stirred at 0 °C and warmed slowly to room temperature and then stirred for 72 h. The reaction mixture was poured into ice water, and then, aqueous NaHCO3 5 % was added until the solid had dissolved. The resulting mixture was concentrated under reduced pressure to remove dichloroethane. The aqueous solution was acidified to pH 1 by addition of 5 % HCl. The mixture was stirred for 15 min and extracted with ethyl acetate (3 × 20 mL), and the organic extracts were combined and washed with water and brine and dried over anhydrous Na2SO4. The crude material was purified by silica gel column chromatography, eluting with hexane/ethyl acetate (1:1) with 0.5 % acetic acid to afford 25 as a yellow solid (348.4 mg).

4′-(4-bromo-2,5-dimethoxyphenyl)-4′-oxobut-2′-enoic acid ( 25 )

This compound was obtained as a yellow solid in 48 % yield; mp 153–154 °C; IR (KBr) νmax 3082, 3013, 2944, 1712, 1658, 1595, 1488, 1388, 1289, 1269, 1215 cm−1; 1H NMR (Pyridine-d5, 400 MHz): δ = 8.23 (1H, d, J = 15.6, H-3´), 7.47 (1H, s, H-6), 7.46 (1H, s, H-3), 7.31 (1H, d, J = 15.6, H-2´), 5.72 (1H, s, COOH), 3.75 (3H, s, C-2–O–CH 3), 3.74 (3H, s, C-5–O–CH 3); 13C NMR (Pyridine-d5, 100 MHz): δ = 191.2 (C, C-4′), 169.0 (C, C-1′), 154.0 (C, C-5), 153.0 (C, C-2), 140.8 (CH, C-3′), 133.4 (CH, C-2′), 128.2 (C, C-4), 118.9 CH, C-6), 118.7 (C, C-1), 113.9 (CH–C-3), 57.2 (CH3, C-2–O–CH3), 57.1 (CH3, C-5–O–CH3); HRESIMS m/z (pos) 314.9861, 316.9842 C12H12O5Br [M]+ (calcd.: 314.9864).

Biological evaluation

The compounds were subjected to an evaluation of their level activity using the broth microdilution method to estimate the minimal inhibitory concentration (MIC) according to the recommendations of (CLSI 2008; PA, 2009).

The microorganisms assessed were E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212, S. aureus, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 and C. neoformans.

A series of twofold dilutions from 1024 to 2 µg/mL (dissolved in DMSO up to 2 % final DMSO concentration) were prepared in a 96-well sterile microplate.

For bacteria In each well was introduced 50 µL of the dilution compound in Muller Hinton broth. Subsequently, 50 µL of an inoculum containing 5 × 106 CFU was added to each well. Gentamicin and doxycycline were used as the antibacterial references. The microplate was incubated at 37 °C for 24 h.

For yeasts 100 µL of an inoculum containing 1 × 103–5 × 103 CFU was added to each well. In each well was introduced 100 µL of the dilution compound in RPMI 1640 with l-glutamine and without sodium bicarbonate (Sigma-Aldrich). Amphotericin B was used as the antifungal reference. The microplate was incubated at 37 °C for 48 h in Candida sp. and 72 h in C. neoformans.

MIC was considered as the weakest concentration at which turbidity could not be observed with the naked eye.

The effect of DMSO at a concentration of 2 % was checked and eliminated; at these concentrations, DMSO has no apparent effect on the microbial and fungal growth. The wells used as a negative control were prepared using the inoculum alone.

All experiments were repeated three times, and the results are expressed as average values.

Conclusions

Several known and new naphthoquinone derivatives have been tested for antibacterial and antifungal activity. In bacteria, compound 42 displayed the highest activity against S. aureus at MIC 16 µg/mL. Compound 37 was the most active against Candida spp. with MIC of 2–4 µg/mL, similar to amphotericin B. Structure–activity relationships of these compounds showed that halogen substituents at the C2 and/or C3 positions are the functional groups that have the most influence on the antibacterial and antifungal activities. The carbonyl groups at C-1 and C4 enhance the antimicrobial activity. The presence of side chains with more than ten carbon atoms increases the lipophilicity of these compounds. These chains led to a progressive decrease in the activity until they became inactive. The two rings of the naphthoquinone core are essential for the antimicrobial activity of these compounds. The presence of additional rings in the structure or the lack of either ring results in inactive compounds. The presence of hydroxyl groups at the C-5 and/or C-8 positions is critical for antibacterial activity. The replacement of these groups with other functional groups leads to the loss of activity.