Crystal structure of Staudtienic acid, a diterpenoid from Staudtia kamerunensis Warb. (Myristicaceae)

This work presents the crystal structure of a natural diterpenoid, Staudtienic acid, isolated from the stem bark of Staudtia kamerunensis Warb (Myristicaceae). This work confirmed the results previously obtained from NOE spectroscopy.


Chemical context
Diterpenoids are secondary metabolites mainly obtained from plant species but that can also originate from marine species (Hanson, 2009).
The biological importance of diterpenoids is notable.For example, Clerodanes' diterpenoids are compounds isolated from several plant sources that have been reported to possess very good antifeedant activity against certain plant worms such as the tobacco cutworm (Spodoplera litura) and certain locusts such as the African desert locust (Schisticerea gregoria).They are reported to possess antiviral, antitumour and antibiotic properties, among many others (Merritt & Ley, 1992).Diterpenoids isolated from Salva multicaulis were reported with significant antituberculosis activity (Ulubelen et al., 1999).Paclitaxel, a diterpenoid isolated from the Taxus genus and which is used in cancer therapy, is recognised as one of the most successful so far in that category (Zhu & Chen, 2019); forskolin, found in the roots of Coleus forskohlii, is a good cardioprotective compound (Jagtap et al., 2011).
Natural product research has gained more attention due to the failure of other drug discovery techniques to supply lead compounds to face the threatening rise of resistance among cancerous strains, bacteria cells, etc. (Butler, 2004).The extraction of various compounds in different parts of specific plant species has allowed the isolation of compounds with interesting biological activities, such as diterpenoids, which have been associated with many therapeutic functions: analgesic, anti-inflammatory, anticarcinogenic, etc. (Sun et al., 2006).
The objectives of this study were, on one hand, to chromatographically separate and describe the secondary metabolites responsible for the pharmaceutical activities observed during the use of Staudtia kamerunensis Warb.(Myristicaceae) in folkloric medicine.Pharmacological studies on the plant highlighted its antibacterial properties.Analysis against twelve Gram-negative and Gram-positive microbial strains of the ethyl acetate extract of the stem bark of the specie revealed significant antimicrobial activity, with the lowest MIC being 15.625 mg mL À 1 (Tonga et al., 2022).Crystals of Staudtienic acid, isolated from the stem bark, were analysed by single crystal X-ray diffraction to confirm the structure and the stereochemistry of the compound that had been previously published by Noumbissie and collaborators (Noumbissie et al., 1992).Phytochemists have widely used single-crystal X-ray diffraction to confirm the structures of compounds that were previously characterized using spectroscopic methods (Ahmad et al., 2016;Simard et al., 2014;Adelekan et al., 2008).

Structural commentary
Plant material was processed as described below, resulting in the title diterpenoid.The title compound crystallizes in the orthorhombic space group P2 1 2 1 2 (No. 18) with two independent molecules in the asymmetric unit (Fig. 1).Staudtienic acid comprises two fused six-membered rings attached to a benzene.A representation of the molecule with Mercury (Mercury 2022.3.0;Macrae et al., 2020) gives the spatial disposition of the two cyclohexane rings.Ring A (the ring with the carboxylic acid) adopts a chair conformation, and ring B adopts a half-chair conformation.The half-chair conformation is probably adopted to avoid steric hindrance between the methyl groups and the carboxylic acid.The crystal structure confirmed the NOE data of the compound previously described by Noumbissie and collaborators (Noumbissie et al., 1992).
The most likely reason for this is probably the presence of the OH groups bonded to C1 that are involved in hydrogen bonding.These may cause small distortions resulting in deviations in the angles around C2.
The propenyl moiety of Molecule A is disordered over two positions (see Fig. 1 for numbering), with the major disorder component residing on C19A [66.9 (11) %] and the minor disorder component residing on C19B [33.1 (11) %].Atom H1 was also found to be disordered over two positions, and each atom was assigned a 50% occupancy over O1 and O2.This approach was used as the data were insufficient to determine more precisely the positions of the H atoms from the electrondensity map, to place it onto residual electron density, and to refine it to obtain a better percentage occupancy.

Supramolecular features
Several forms of hydrogen bonding are present in this structure, as shown in Table 1.The packing of the structure is consolidated by classical O-H� � �O hydrogen bonds as well as by two C-H� � �O intermolecular hydrogen bonds and two

Figure 1
The two independent molecules of the title compound shown with 50% probability ellipsoids.
C-H� � �� interactions.Each crystallographically independent molecule is connected to another of the same type related by the symmetry operation 1 À x, 1 À y, z.In terms of graph sets (Etter et al., 1990), this motif is represented by the symbol R 2 2 (8) (see Fig. 2).The two dimers are then connected by C-H� � �O hydrogen bonds, forming an infinite column along the c-axis.The columns are connected to similar columns by van der Waals interactions with molecules related by 1 2 + x, The most illustrative view of the packing arrangement of the title compound is the view down the c axis shown in Fig. 3.

Database survey
The structure was thoroughly validated using PLATON (Spek, 2020).A search was also performed in the Cambridge Structural Database (CSD Version 5.44 2023.1,March 2023 update; Groom et al., 2016), and it was established that this structure had not been previously published or deposited.
A search in the CSD for organic compounds with the backbone of two fused rings attached to a benzene revealed 1390 hits.When the propenyl substituent of the benzene ring is introduced, the number of hits is reduced to four [refcodes LATPOP (Zhu et al., 2022), LAPHOA (Hitchcock et al., 2005), MIXFUX (Ye et al., 2019) and OVAQIN (Green et al., 2016)].When the carboxylic functional group search is introduced at position C4 of the cyclohexane, the number of search hits is reduced to zero.

Synthesis and crystallization
Plant Material.The stem bark of this plant species was collected from Minkam Mengale Menkom, a location in the South region of Cameroon.A voucher specimen is in deposit there under the number 49184 HNC.
Extraction and Isolation.Extraction and isolation were carried out as described by Noumbissie and collaborators (Noumbissie et al., 1992).Briefly, after collection, the sample was dried and ground to yield a powder of 15.79 Kg.A Soxhlet apparatus was used to extract the stem bark using ethyl acetate, and the liquor obtained was evaporated using a rotary evaporator under reduced pressure.A residue of mass 550.12 grams was obtained, and 250 g was partitioned using benzene (C 6 H 6 ) to obtain 41.96 g (yield 41.86, 16.74%) of the sample.The latter was loaded with 500 g of silica, and elution was done with a gradient of polarity starting with benzene, then a mixture of benzene and petroleum ether, then petroleum ether.The final sample was obtained from a mixture of 60% petroleum ether in benzene (yield: 80.22 mg, 0.19%)

Refinement
The systematic absences in the diffraction data were uniquely consistent with the space group P2 1 2 1 2 (No. 18) determined by XPREP (Bruker, 2014), which yielded chemically reasonable and computationally stable results of refinement (Sheldrick, 2015a,b).
A successful solution by Intrinsic phasing methods (SHELXT; Sheldrick, 2015a) provided all non-hydrogen atoms from the E-map.The remaining hydrogen atoms were located in an alternating series of least-squares cycles and visualization of the difference-Fourier map.All non-hydrogen atoms were refined with anisotropic displacement coefficients.All hydrogen atoms connected to C atoms were included in the structure-factor calculation at idealized positions and were allowed to ride on the neighbouring atoms with relative The ring motif of the hydrogen bond between a pair of one of the independent molecules.Hydrogen bonds are denoted as red dotted lines.The graph-set symbol is indicated in the centre of the hydrogen bonding ring; only heteroatoms have been labelled.Symmetry codes: (i) À x + 1, À y + 1, z.

Figure 3
The packing of the title compound viewed down the c axis.Hydrogenbonding contacts are shown in light blue.isotropic displacement coefficients.Two symmetry-independent molecules are in the asymmetric unit as shown in Fig. 1.
The compound crystallized in a chiral space group and the Flack x parameter was determined using 1758 quotients (Parsons et al., 2013).However, since the data were collected using Mo radiation, the absolute structure could not be reliably determined.In addition, the crystals used for the data collection diffracted weakly.Attempts were made to find crystal structures of similar compounds where the structural chirality was already known, but these attempts were unsuccessful, as no hits could be found in the CSD.
The final least-squares refinement of 420 parameters against 8260 reflections resulted in residuals R (based on F 2 for I�2�) and wR (based on F 2 for all data) of 0.0522 and 0.1339, respectively.The final difference-Fourier map was basically featureless.The structure was validated using PLATON (Spek, 2020).Crystal data, data collection and structure refinement details are summarized in Table 2.

Special details
Geometry.All esds (except the esd in the dihedral angle between two l.s.planes) are estimated using the full covariance matrix.The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry.An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s.planes.

Table 2
Experimental details.