1 Introduction

Zanthoxylum atchoum (Aké Assi), P.G. Waterman (Rutaceae) is an endemic straggling shrub distributed in humid forests of southern Ivory Coast. The plant is used to treat amenorrhea in traditional medicine. There has been no report of other phytochemical study and biological value of Zanthoxylum atchoum so far. In our continuous search for chemical bioactive constituents of Ivorian Zanthoxylum species [1], we investigated the methanol extract of Z. atchoum.

The present study deals with the isolation and structural elucidation of two new benzophenanthridine alkaloids, 6-nitronitidine (1) and 6-nitro-8-methoxy-7,8-dihydronitidine (2), and of two new indolopyridoquinazoline alkaloids, 3-hydroxydehydroevodiamine (3) and its zwitterionic form 3-hydroxylatedehydroevodiamine (4), along with 18 (5–22) known compounds.

This is the first report of a nitro group in the biosynthesis of new natural benzo[c]phenantridine alkaloids. We also report in the ¹H NMR experiences an unusual hydrogen–deuterium exchange of the 8-methoxy hydrogens (CD₃OD solvent) in compound 2. The antibacterial activities of compounds 1-4 were evaluated against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis.

2 Experimental

3 Results and discussion

The root bark of Z. atchoum was extracted successively with petroleum ether and methanol. The extracts were chromatographed on silica gel to afford 22 compounds 1–22. Structural elucidation of these compounds was performed using spectroscopic methods, especially 1D and 2D-NMR, HR-EI MS, UV and IR.

Compound 1 was isolated as a yellow powder. The UV spectrum showed absorptions at 271, 293, 309, 328 and 390 nm, which are characteristic of the benzo[c]phenanthidine skeleton [4]. The HRESIMS spectrum displayed the molecular ion [M + H]⁺ at m/z 394,1348 (calcd for 394.1347), which, in combination with ¹H and ¹³C NMR spectroscopy and IR data, indicated molecular formula C₂₁H₁₇N₂O_6.

The ¹H-NMR spectrum (Table 1) of 1 revealed only ten singlet signals due to six aromatic proton resonances at δ_H 8.17 (1H, s, H-1), 7.68 (1H, s, H-4), 8.63 (1H, s, H-5), 9.82 (1H, s, H-8), 7.93(1H, s, H-9), and 7.41(1H, s, H-12), one methylenedioxy group at 6.33 (2H, s, O–CH₂–O) and three methyl groups assignable to two methoxy groups at 4.07 (3H, s, 10-OCH₃), 4.12 (3H, s, 11-OCH₃) and one N-methyl group at 4.93. In agreement with the above, the ¹³C NMR of compound 1 showed resonances for 21 C-atoms, including 11 quaternary carbons, six methines, one methylene and three methyl groups (Table 1).

The ¹H NMR and ¹³C NMR signals of compound 1 were closely related to those of nitidine (8), except for the absence of one aromatic proton and the presence of one more quaternary carbon, suggesting that 1 was certainly an ortho-pentasubstituted benzo[c]phenanthidine [4]. Total assignments of protons H-1, H-4, H-5, H-8, H-9, H-12 and of the methyl groups were clearly obtained from the NOESY and HMBC experiments (Fig. 1). The location of the N-methyl group and of proton H-1 were secured by the NOE interaction between H-1 at δ_H 8.17 and N-Me at δ_H 4.93; in addition, both protons and the deshielded proton at δ_H 9.82 (H-8) showed long-range connectivity with the signal at δ_C 138.0 (C-14). The HMBC interactions between the protons at δ_H 6.33, 8.17 and 7.68 (H-4) and carbons at δ_C 153.5 (C-2) and 152.9 (C-3) suggested that the methylenedioxy group was connected to C-2 and C-3 on the ring D. Furthermore, the NOESY spectrum displayed a significant cross-peak between H-4 and the proton at δ_H 8.63, which was unequivocally assigned to H-5, and the proton H-9 at δ_H 7.93 was assigned by its cross-peak between H-8. Thus H-12 was assigned to δ_H 7.41, confirming its long-range correlations with C-8a at δ_C 122.0 and C-13 at δ_C 119.0. The location of the two methoxy groups at C-10 and C-11 were established by HMBC correlations of protons at δ_H 4.07 to C-10 (δ_C 155.0) and 4.12 to C-11 (δ_C 160.0), and were confirmed by the NOESY correlations between H-9/Me-10 and H-12/Me-11. The HMBC correlations between H-5 and C-6 (δ_C 144.0) suggested that a heteroatom, presumably a nitro group, was certainly attached to C-6. This was in accordance with the upfield shift of C-6 and consequently inferred that 1 was a 2,3,6,10,11-ortho-pentasubstituted benzophenanthridine alkaloid. The IR spectrum of 1 showed two strong bands at 1521 and 1351 cm^–1 which can be ascribed to N-O stretching of the nitro group. In addition, the presence of the nitro group was confirmed by the fragmentation MS/MS of the molecular ion [M]⁺ at m/z 393, which gave a series of ions at m/z 346, 332, 318 and 301, from which the loss of a nitro group was respectively deduced as follows: [M–NO₂–H]⁺, [M–NO₂–CH₃]⁺, [M–NO₂–OCH₃ + 2H]⁺ and [M–NO₂–OCH₃–CH₃]⁺. Thus, compound 1 was characterized as 2,3-(methylenedioxy)-6-nitro-10,11-(dimethoxy)-7-methylbenzo[c]phenanthridinium, to which we gave the trivial name 6-nitronitidine. This is the first occurrence of a natural benzylisoquinoline alkaloid with a nitro group on the C ring of the benzo[c]phenanthidine nucleus isolated from Zanthoxylum atchoum. The presence of the nitro group seems to be scarce in the plant kingdom, regardless of special phenanthrene derivatives mentioned in the Aristolochia genus [5]. Furthermore, several examples of C-6 substituted on the C ring of benzophenanthridines do exist, but it concerns 6-O-alkyl compounds in which the C ring is not completely aromatic [6,7].

Compound 2 was obtained as a yellow amorphous solid. Its molecular formula was deduced as C₂₂H₂₀N₂O₇ by positive-ion mode HRESIMS [M + H]⁺ at m/z 425.1349 (calcd for 425.1349). Comparison of the NMR data (Table 1) of 2 to those of 1 indicated a slight modification of the iminium bond CN at C-8. Instead of the aromatic methine proton at C-8, the signals detected were those of the methine proton at δ_H 5.25 (1H, s, H-8) and of a methoxy group at δ_H 3.50 (3H, s, 8-OCH₃) in the ¹H-NMR spectrum. The location of this methoxy group was confirmed by the NOE correlation between the –OMe and H-8. Compound 2 seems to be the pseudo base of 1 and was characterized as 6-nitro-8-methoxy-7,8-dihydronitidine (Fig. 2).

This kind of product is well known among the quaternary benzo[c]phenanthridine alkaloid-free bases and detailed structural and NMR studies have already been reported by Dostal et al. [8,9]. Surprisingly enough, the signal of this methoxy group at δ_H 3.5 was gradually vanished within 24 h, whereas the signal of the residual solvent (MeOH) at δ_H 3.38 was increased (Fig. 3). Fig. 3 shows the ¹H-NMR spectrum of 2, in which t₁ and t₃ represent respectively the beginning and the end of the experiments. It is remarkable that the protons of the 8-OCH₃ group were H–D exchanged during the ¹H-NMR experiments by the deuterated solvent (CD₃OD) (Fig. 3). This situation made very difficult the NMR interpretation because of the bewildering assignment of the 8-OCH₃ or OH group in benzophenanthridine pseudo-base compounds. In our study, deuterium retention of the 8-OCH₃ to 8-OCD₃ was confirmed by mass spectra with 3 amu more than in 1.

Compound 3 was isolated as a yellow amorphous solid. Our EI mass spectra evidenced the positive ion [M]⁺ at m/z 318, which is consistent with the molecular formula C₁₉H₁₆N₃O₂. The IR spectrum of 3 implied the presence of hydroxyl and NH groups (3000–3063 cm⁻¹) and of the amide carbonyl group (1692 cm⁻¹). The UV spectrum showed absorptions bands at 250, 330 and 380 nm, with a bathochromic shift observed in alkaline medium at 417 nm, suggesting the presence of a phenol group. The ¹H-NMR spectrum of 3 showed four aromatic protons of an 1,2-ortho-disubstituted phenyl ring at δ_H 7.85 (1H, d, J = 8.2 Hz, H-9), 7.30 (1H, t, J = 7.7, H-10), 7.56 (1H, t, J = 7.7 Hz, H-11), 7.65 (1H, d, J = 8.4 Hz, H-12), three aromatic proton signals at δ_H 7.96 (1H, d, J = 9.7 Hz, H-1), 7.55 (1H, dd, J = 9.7, 2.8 Hz, H-2) and 7.72 (1H, d, J = 2.8 Hz, H-4), revealing the presence of an ABX system in a 1,3,4-trisubstituted phenyl ring, one methyl singlet group at δ_H 4.40 (3H, s, N-CH₃), and two methylene groups at δ_H 4.58 (2H, t, J = 6.7 Hz, H-7) and 3.33 (2H, t, J = 6.7 Hz, H-8). All these data readily help us to identify a typical 7, 8-dihydropyridoquinazoline nucleus [10,11]. The ¹³C-NMR spectrum of 3 showed 19 carbon signals due to eight quaternary carbons, seven methines, two methylenes, N-methyl and carbonyl groups. The ¹H and ¹³C NMR spectra (Table 2) of 3 were similar to those of dehydroevodiamine (5) [12], except for the increase of chemical shift of C-3. According to the preceding data, 3 was suggested to be a 3-hydroxy analog of 5 and similar to those described by Li et al., 2001 [13]. The protons H-9 and H-1 were assigned by the NOESY spectrum, which showed respectively cross-peaks between H-9/H-8 and H-1/N–CH₃ (Fig. 4). The location of the hydroxy at C-3 was confirmed by the HMBC experiment, which showed correlations between the protons H-1, H-2 and H-4 and C-3 (δ_C 159.8) (Fig. 4). Thus, on the basis of spectral evidence, the structure of compound 3 was elucidated as a salt of 3-hydroxy-8,13-dihydro-14-methyl-5-oxo-7H-indolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-14-ium, to which we gave the trivial name of 3-hydroxydehydroevodiamine.

Compound 4 was obtained as an orange amorphous solid. A molecular ion peak at m/z [M]⁺ 317 suggested its molecular formula C₁₉H₁₅N₃O₂, which was one proton less than in that of 3.

Furthermore, the NMR data of 4 were similar to those of 3. However, the ¹³C NMR spectrum of 4 showed the presence of a deshielding C-3 at δ_C 170.4 instead of 159.8; this means that 4 is the zwitterionic 3-phenolate form of 3, named 3-phenolatedehydroevodiamine (Fig. 2).

The known compounds (5–23) were readily identified, from their spectral data and by comparison with reported corresponding compounds in the literature, as dehydroevodiamine (5), evodiamine (6) [14], decursinol (7) [15], asarinin (8) [16], oxynitidine (9), norchelerythrine (10) [17], tridecanonchelerythrine (11) [18], nitidine (12), chelerythrine (13), methoxychelerythrine (14), fagaridine (15), methoxyfagaridine (16), arnottianamide (17), pellitorine (18), sesamin (19), skimmianine (20), stigmasterol (21), and lupeol (22). Compounds 12–23 were previously found in other Ivorian Zanthoxylum species [1]. We describe the structural elucidation and configuration of 13b αH of compound 6 which displayed negative values: [α]_D = –522° (c 0.54 in CH₃OH) and–642° (c 0.67 in CHCl₃) for optical rotation, like that of the synthesized one [19]. Nevertheless, the isolated evodiamine described in literature was optically inactive or displayed positive optical rotation values [20–22].

The isolated new compounds (1–4) were evaluated for their antibacterial activities against four pathogenic microorganisms, E. coli, P. aeruginosa, S. aureus, and E. faecalis; the results are summarized in Table 3. Among them, compound 2 was the most active one, with MICs of 4, 8, 16, and 32 μg·mL⁻¹ against S. aureus, E. faecalis, P. aeruginosa, and E. coli, respectively (Table 3). Compound 1 exhibited mild activity against these four bacteria with MICs of 32 μg·mL⁻¹. This result suggested that the pseudo base of these isolated nitro benzophenanthridine is more active than the ammonium quaternary 1. A similar antimicrobial effect was observed with sanguinarine and chelerythrine and their pseudo bases [23].

4 Conclusions

This work constituted the first phytochemical study of Z. acthoum. The methanolic extract of the roots led to the isolation of 11 benzophenanthridines, four indolopyridoquinazoline, one furanoquinoline alkaloids, two lignans, one coumarin, one amid, one triterpene, and one phytosterol. Among them, 6-nitronitidine (1), 6-nitro-8-methoxy-7,8-dihydronitidine (2), 3-hydroxydehydroevodiamine (3), and 3-phenolatedehydroevodiamine (4) were characterized as new compounds. This paper reports the first natural nitro group on the benzophenanthridine nucleus. This work allowed us to understand the fickle behavior of benzophenanthridine bases towards solvents, in particular deuterium retention of the OCH₃ to OCD₃, which made difficult the interpretation of the spectra. The antibacterial activities against two gram-positive (S. aureus and E. faecalis) and two gram-negative (E. coli and P. aeruginosa) strains for compounds 1–4 were tested. Compound 2 showed strong antibacterial activity against S. aureus at the concentration of MIC₅₀ 4 μg·mL⁻¹.

Acknowledgements

The authors thank Prof. C. Lavaud and Dr. J.-M. Nuzillard for profitable scientific exchanges, and are grateful to CNRS UMR 7312 France and Government of Ivory Coast for financial support. The authors would like to pay a tribute to the Prof. Aké Assi L. (University FHB Cocody-Abidjan) for the identification of the plant.