1,2-Dimethoxy-12-methyl-13H-[1,3]benzodioxolo [5,6-c]phenanthridine

1. Introduction

Benzo[c]phenanthridines are a small subclass of isoquinoline alkaloids widely distributed in higher plant families, notably Papaveraceae and Rutaceae, and shown to possess diverse pharmacological effects including antimicrobial, anti-inflammatory, and antitumour properties [1,2]. Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae), whose common English name is Usambara prickly ash, is a tree native to East Africa where it is used medicinally among indigenous communities for malaria and other infections, rheumatism, and gastrointestinal ailments [3,4,5]. Extracts of Z. usambarense have demonstrated antiplasmodial activity in experimental models [5].

Previous phytochemical investigations on Z. usambarense have reported several benzo[c]phenanthridine alkaloids including nitidine and chelerythrine [6], as well as norchelerythrine and oxychelerythrine [7]. To the best of our knowledge, dihydrochelerythrine (Figure 1) has not previously been reported from Z. usambarense. However, it has been isolated from the stem bark of Z. nitidum [8,9], the root bark of Z. zanthoxyloides [10], and roots of Z. paracanthum [11] and Z. flavum [12]. Dihydrochelerythrine has demonstrated antiproliferative [10,13] and antifungal activities [14].

While investigating photoactivatable dihydroalkaloids for cancer cell imaging and chemotherapy, Ling et al. [15] semi-synthesized dihydrochelerythrine (1) from chelerythrine and reported its crystal structure in the monoclinic space group P2₁/c. In contrast, the present study reports the isolation of (1) from the stem bark of Z. usambarense and its single-crystal X-ray structure in the orthorhombic space group Pbca. This represents both the first report of dihydrochelerythrine from Z. usambarense and the first description of this orthorhombic Pbca polymorph. This finding is significant due to potential utility of Z. usambarense as a natural source of dihydrochelerythrine.

2. Results

2.1. Structural Commentary

Dihydrochelerythrine (1) crystallized in the orthorhombic space group Pbca with Z = 8. The benzophenanthridine nucleus is fully resolved with all non-hydrogen atoms refined anisotropically and all hydrogen atoms freely refined. Although the fused tetracyclic framework formed by rings A-B-C-D is approximately planar, ring C is not aromatic and is consequently puckered. Least-squares planes fitted through the 10 C and 2 O atoms of rings A and B and through the 6 C and 2 O atoms of ring D show that these are both planar (RMSD values 0.021 and 0.016 Å, respectively); the interplanar angle due to the twist about the C(10)–C(14) bond (Figure 2) in ring C is 12.60°, which is a similar value to two of the torsion angles involving this bond, e.g., ∡ C(9)-C(10)-C(14)-C(15) is 11.70° and ∡ C(11)-C(10)-C(14)-C(13) is 14.32°. In the puckered ring C, the sp³-hybridized C(12) carbon and N(1) nitrogen atoms lie 0.621 and 0.527 Å below / above the mean aromatic planes of rings AB and ring D, respectively. These displacements generate a shallow envelope conformation in ring C, distort π-conjugation, produce a racemic mixture of chiral conformers, and contribute to a herringbone packing motif. This non-planarity contrasts with the more planar iminium framework observed in salts of oxidized benzophenanthridine analogues such as chelerythrine [16].

2.2. Supramolecular Features

The crystal structure exhibits a herringbone packing arrangement (Figure S1). The molecules associate via π–π stacking between adjacent benzophenanthridine ring systems, with centroid–centroid separations consistent with typical aromatic stacking in benzo[c]phenanthridine alkaloids. Additional stabilization arises from weak C-H···O contacts involving both methoxy and methylenedioxy oxygen atoms, which link the molecules into layers extending along the c axis generating the signature three-dimensional herringbone packing framework.

2.3. Database Survey

A search of the Crystallography Open Database using “exact match” command returned one card entry (COD 1558616) for dihydrochelerythrine reported by Ling et al. [15]. A similar search of the Cambridge Structural Database yields the single entry WUZTOD. In contrast to the earlier reported semisynthetic form which has a monoclinic crystal structure with a P2₁/c space group, the present natural isolate adopts the orthorhombic Pbca space group, implying distinct polymorphic differences between the two crystal forms. Surprisingly, the two crystals contain conformationally-equivalent molecules (Figure S2) with the methyl group on O(4) lying approximately in the fused plane of the A-B-C-D rings but with the methyl groups on O(3) and N(1) pointing out of the plane (Figure 2).

3. Discussion

Polymorphism in organic crystal structures frequently results from flexible organic molecules adopting different conformations, and depending on the crystallization conditions (e.g., speed, choice of solvent), different solid forms may be obtained. We note that fine needles were initially obtained before the final block form was obtained, which may correspond to another polymorphic form. Unusually, the molecules of (1) in the orthorhombic form reported here exhibit a very similar conformation to those found in the monoclinic polymorph reported previously suggesting that this is the most stable conformation for this molecule. In this instance, the two polymorphic forms arise from different packing arrangements of the molecules (cf. Figure S1 and Figure S3). Furthermore, there is likely to be a lack of a distinct minimum in the energy landscape as the crystal structures of both polymorphs are held together solely via weak non-covalent interactions. Thus, it would be interesting to see if there are other forms predicted by crystal structure prediction. The existence of polymorphic forms has implications regarding any pharmaceutical applications of this material.

4. Materials and Methods

4.1. Plant Material

The stem bark of Zanthoxylum usambarense was collected in January 2024 in the outskirts of the Nairobi National Park, Kenya (1.3333° S, 36.7° E). The species was authenticated by comparing the collected specimen with archived herbarium material of Zanthoxylum usambarense preserved at the University of Nairobi (UoN) Herbarium. Authentication was conducted by a senior taxonomist in the Department of Botany, UoN.

4.2. Extraction and Isolation

The dried and ground bark (300 g) was extracted twice by maceration in 1,000 mL of methanol-dichloromethane (1:1) for 72 h with continuous stirring on a magnetic stirrer. The crude extract was filtered and concentrated at 50 ℃ under reduced pressure to afford a dark brown residue (20 g). The extract was adsorbed onto 25 g of silica gel, dry-packed on a column containing 180 g of silica gel, eluted with a hexane-ethyl acetate-methanol gradient and collected in approximately 10 mL fractions. Dihydrochelerythrine was obtained from the hexane-ethyl acetate fractions as colourless needles (227 mg, 1.2%). Recrystallization from acetone afforded colourless blocks, from which crystals suitable for SCXRD were obtained by slow evaporation at room temperature. The identity of the isolated compound was supported by liquid chromatography-mass spectrometric (LC-MS) analysis and single-crystal X-ray diffraction.

4.3. Liquid Chromatography-Mass Spectrometry Analysis

LC-MS analysis was performed using a Shimadzu LCMS-8040 system equipped with an electrospray ionization (ESI) interface. Chromatographic separation was achieved on a Kinetex XB-C18 column (2.6 μm, 150 × 3 mm) fitted with a Phenomenex SecurityGuard Ultra cartridge. The column oven was maintained at 40 °C and the injection volume was 5 μL. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B), delivered at a flow rate of 0.2 mL/min. Gradient elution was used: 10% B, 0−0.5 min; 10−50% B, 0.5−5 min; 50−90% B, 5−8 min; 90% B, 8−11 min; 90−10% B, 11−12 min; and 10% B, 12−20 min. Mass spectra were acquired in full-scan mode with polarity switching. The nebulizing and drying gas flow rates were 2.0 and 15 L/min, respectively, while the desolvation-line and heating-block temperatures were 250 and 400 °C, respectively.

Dihydrochelerythrine eluted at 12.07 min and gave an ion at m/z 350 in positive-ion mode assigned to [M+H]⁺ (Figure S4), consistent with the molecular formula C₂₁H₁₉NO₄ and previously reported LC-MS data for dihydrochelerythrine [22].

4.4. X-Ray Data Collection and Structure Refinement

Single-crystal X-ray diffraction (SCXRD) data were collected on an Agilent Oxford Diffraction SuperNova diffractometer equipped with a microfocus Cu Kα radiation source (λ = 1.54184 Å) and a HyPix-Arc 100 hybrid pixel detector. A piece of (1) was sliced off a much larger block-shaped crystal, mounted on a 20 μm nylon loop (Hampton Research) (Figure S5) and cooled to 150 K using an Oxford Instruments Cryojet5^®. A complete sphere of data to a resolution of 0.84 Å was obtained in under 45 min using 0.5° ω-scan frames. Data reduction, including indexing, integration, scaling, and absorption correction, was performed using the CrysAlis^Pro (Version 1.171.42.60a; Rigaku Oxford Diffraction).

The structure was solved initially using the ‘What is this?’ (WIT) feature in CrysAlisPro [17], with intrinsic phasing in SHELXT [18], followed by full-matrix least-squares refinement on F² using SHELXL [19] within Olex2 [20]. As (1) has conformational chiral forms, atoms in the asymmetric unit were chosen to chiral match the conformation reported in [15]. The positional and anisotropic displacement parameters for the non-hydrogen atoms were refined freely. All hydrogen atoms were located in the difference Fourier maps and were freely refined with isotropic displacement parameters. Molecular and crystal structure illustrations were prepared using Mercury (Cambridge Crystallographic Data Centre, CCDC) [21], with displacement ellipsoids for non-hydrogen atoms shown at the 50% probability level, while hydrogen atoms are shown in their refined positions.

Crystal data for C₂₁H₁₉NO₄ (M_r = 349.37): orthorhombic, space group Pbca, a = 14.35758(7) Å, b = 8.45782(4) Å, c = 27.38114(13) Å, V = 3325.00(3) Å^3, Z = 8, T = 150 K, ρ_calc = 1.396 g cm⁻³, μ(Cu Kα) = 0.791 mm⁻¹, F(000) = 1472. Crystal size: 0.715 × 0.396 × 0.277 mm³. A total of 72,954 reflections were collected, of which 3567 were independent (R_int = 0.0359, R_sigma = 0.0106). Refinement on F² converged to R₁ = 0.0324 and wR₂ = 0.0876 for reflections with I ≥ 2σ(I), and R₁ = 0.0341 and wR₂ = 0.0890 for all data, with goodness-of-fit = 1.074. The largest residual electron-density peak and hole were +0.24 and −0.16 e Å⁻³, respectively.