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Article

Antiprotozoal Aminosteroid Alkaloids from Buxus obtusifolia (Mildbr.) Hutch.

by
Justus Wambua Mukavi
1,
Monica Cal
2,3,
Marcel Kaiser
2,3,
Pascal Mäser
2,3,
Njogu M. Kimani
4,
Leonidah Kerubo Omosa
5 and
Thomas J. Schmidt
1,*
1
University of Münster, Institute of Pharmaceutical Biology and Phytochemistry (IPBP), PharmaCampus, Corrensstraße 48, D-48149 Münster, Germany
2
Swiss Tropical and Public Health Institute (Swiss TPH), Kreuzstrasse 2, CH-4123 Allschwil, Switzerland
3
University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
4
Department of Physical Sciences, University of Embu, Embu P.O. Box 6-60100, Kenya
5
Department of Chemistry, Faculty of Science & Technology, University of Nairobi, Nairobi P.O. Box 30197-00100, Kenya
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(23), 4558; https://doi.org/10.3390/molecules30234558
Submission received: 29 October 2025 / Revised: 20 November 2025 / Accepted: 21 November 2025 / Published: 26 November 2025
(This article belongs to the Collection Natural Products as Leads or Drugs against Neglected Tropical Diseases)
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Abstract

Human African Trypanosomiasis (HAT; sleeping sickness) and Malaria are life-threatening protozoan infections in tropical regions, with limited treatment options. As part of our ongoing efforts to discover new aminosteroid alkaloids from the Buxaceae family with antiprotozoal activity, which might serve as leads to new drugs against these infections, we investigated the dichloromethane extract from the leaves of Buxus obtusifolia (Mildbr.) Hutch. collected in Kenya, a species native to Kenya and Tanzania. To the best of our knowledge, and based on the most recent comprehensive literature review, this study represents the first phytochemical investigation of this plant. The alkaloid-enriched fraction yielded a total of 24 aminosteroid alkaloids, including 18 hitherto undescribed compounds (2, 3, 5–9, 11, 12, 15–19, and 21–24), along with six known compounds, two of which (1 and 4) are described as constituents of a natural source for the first time. Obtusiaminocyclin (24) represents the first Buxus alkaloid with a novel carbocyclic steroid skeleton with a cyclopropane ring comprising C-9, C-19 and C-11 accompanied by an unprecedented amino bridge between C-3 and C-10. The structures of the isolated compounds were determined using UHPLC/+ESI-QqTOF-MS/MS and NMR spectroscopy. The total crude extract, the alkaloid-enriched fraction, CPC subfractions and all isolated compounds were tested for in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense (Tbr, responsible for East African HAT) and Plasmodium falciparum (Pf, responsible for tropical Malaria) as well as cytotoxicity against mammalian cells (L6 cell line). Deoxycyclovirobuxeine-B (12) (IC50 = 0.8 µmol/L, SI = 108) and 29-trimethoxybenzoyloxy-obtusibuxoline (5) (IC50 = 0.5 µmol/L, SI = 11) showed the highest activities with good selectivity indices against Tbr and Pf, respectively. Consequently, our findings provide valuable aminosteroid candidates that can serve as promising leads in our ongoing search for new drugs against HAT and Malaria.

1. Introduction

Human African Trypanosomiasis (HAT), also known as sleeping sickness, and Malaria are vector-borne protozoan infections that affect millions of people, particularly in sub-Saharan Africa and other developing nations [1,2].
HAT is caused by two genetically different protozoan parasites that are transmitted to humans by the bites of infected tsetse flies (Glossina spp.); Trypanosoma brucei gambiense (Tbg), which causes the chronic form in West and Central Africa, currently accounts for around 92% of recorded cases, and T. b. rhodesiense (Tbr) causes acute illness in East and Southern Africa [3]. While the number of reported cases has steadily decreased since 2010, in 2024 the World Health Organization (WHO) reported a total of only 583 cases of HAT. However, this figure is likely underestimated due to reduced surveillance and lack of reported data in some endemic regions [3]. Although worldwide efforts to manage this disease have greatly decreased its occurrence, underdiagnosis and intermittent outbreaks still pose problems. While eradication of HAT as a public health issue seems within reach, continued research into new chemotherapeutic agents is still crucial, especially in light of the limitations of existing therapies, which are often toxic, costly, complex to administer, or restricted by the disease stage [4,5,6].
Malaria is caused by various species of Plasmodium, with P. falciparum (Pf) being responsible for the most devastating and deadly infections. Infected female Anopheles mosquitoes transmit the protozoan parasite into the human body, where the parasite first infects the liver and then multiplies in red blood cells, resulting in a number of symptoms, including organ failure or death [5,7]. Based on a 2024 World Health Organization report, Malaria infections increased by 11 million in 2023, reaching an estimated 263 million cases and 597,000 deaths. More than 95% of both cases and fatalities occurred in the WHO African Region [5]. Despite the availability of artemisinin-based combination therapies (ACTs) and recent advances such as Malaria vaccines, the emergence of artemisinin resistance and decreased sensitivity to partner drugs highlight the urgent need for novel antimalarial compounds with new mechanisms of action [5,8,9].
In the previous studies of our group on steroidal alkaloids from the aerial parts of the European Buxus sempervirens, several constituents were identified, some of which exhibited significant activity against Pf and Tbr [10,11,12,13]. In an initial study, Althaus et al. isolated O-tigloylcyclovirobuxeine-B from the dichloromethane leaf extract of B. sempervirens as the principal compound responsible for the extract’s antiplasmodial activity [10]. In a more detailed follow-up study by Szabó et al., 25 alkaloids were systematically isolated from the aerial parts of this plant and assessed for antiprotozoal activity. Several compounds exhibited potent activity against Pf and Tbr, while others showed moderate to low activity [12]. Very recently, aminosteroid alkaloids isolated from Pachysandra terminalis, another member of the Buxaceae family studied by Schäfer et al. of our group, demonstrated notable activity against both parasites, further expanding the scope of bioactive scaffolds within the family [14]. These findings underscore the importance of isolating and evaluating further Buxus alkaloids for their antiprotozoal potential.
In this context, the present study investigated the antiprotozoal potential of amino-nortriterpenoid alkaloids (termed aminosteroids in this article for simplicity) isolated from Buxus obtusifolia, an evergreen shrub whose root decoction is used in the folk medicine of the Giriama community in Kenya against chest complaints [15]. The species is currently classified as vulnerable, highlighting the importance of its conservation. A comprehensive literature review revealed no prior pharmacological or phytochemical investigations on this species, but as a close relative to Buxus sempervirens, it promises to contain further potent antiprotozoal alkaloids. Through targeted extraction, fractionation, and structural analysis, this study focused on identifying novel compounds with in vitro activity against Pf and Tbr. This work further broadens the structural diversity of antiprotozoal aminosteroid alkaloids and contributes valuable candidates to our ongoing search for new therapeutic hits or leads against Malaria and HAT.

2. Results and Discussion

2.1. In Vitro Antiprotozoal Activity of the Crude Extracts and Fractions

To determine the optimal plant part and extraction solvent, 5 g of the air-dried twigs and leaves were separately extracted on a small-scale using dichloromethane (CH2Cl2) and methanol (CH3OH). The crude extracts were evaluated for their in vitro efficacy against Trypanosoma brucei rhodesiense (Tbr), and Plasmodium falciparum (Pf) as well as their cytotoxicity against L6 rat skeletal myoblasts (Cytotox. L6). The results (Table 1) revealed that the dichloromethane extract of the leaves, had the strongest activity and selectivity index (SI) against Pf (IC50 = 1.1 ± 0.11 µg/mL, SI = 24). These findings are consistent with earlier reports on Buxus sempervirens, which showed that leaf extracts exhibit significant and selective in vitro activity against Plasmodium falciparum [11,16]. Based on these first biological findings, the CH2Cl2 extract of the leaves was chosen for further fractionation and isolation of active constituents. For the large-scale extraction, about 680 g of the powdered plant material were exhaustively extracted with CH2Cl2 in a Soxhlet apparatus set up. In order to separate the alkaloids and lipophilic compounds from the crude extract, an acid-base extraction was carried out. The resulting alkaloid fraction as well as the lipophilic and hydrophilic residuals were subsequently evaluated for antiprotozoal and cytotoxic activities. As indicated in Table 1, the alkaloid fraction demonstrated enhanced antiprotozoal potency against both parasites compared to the previously tested crude extract. Additionally, the alkaloidal fraction showed stronger activity against Tbr and Pf relative to the lipophilic fraction as expected, whereas the hydrophilic residue was inactive. These findings point to the alkaloids as the main contributors to the antiprotozoal effects of the CH2Cl2 leaf extract. Consequently, the alkaloid fraction was further fractionated using centrifugal partition chromatography (CPC), followed by isolation of pure compounds.

2.2. Isolation and Characterization of Aminosteroids from Buxus Obtusifolia

The fractionation and isolation procedure is summarized in Scheme 1. A portion of the alkaloid fraction (5 g) was subjected to CPC fractionation using a biphasic solvent system composed of iso-hexane/ethyl acetate (8/2; v/v, upper phase) and CH3OH/H2O/propan-2-ol (8/2/1; v/v/v, lower phase). This process yielded 16 subfractions, which were monitored by TLC and UHPLC/+ESI-QqTOF-MS/MS (henceforth abbreviated LC/MS) and subsequently evaluated for their in vitro efficacy against Tbr and Pf, as well as for cytotoxicity (Table 2). Fractions 3 through 16 exhibited promising antiplasmodial activity, with IC50 values ranging from 0.9 ± 0.1 to 1.9 ± 0.5 μg/mL. Among these, fraction 15 was the most potent. Furthermore, subfractions 3 and 4 demonstrated significant activity against Tbr (IC50 < 1 μg/mL) with greater selectivity indices (SI = 34 and 21, respectively) compared to the parent alkaloidal fraction. The antiprotozoal activity observed in the CPC subfractions was consistent with previous findings from our group [13], which showed that most CPC subfractions derived from the leaf CH2Cl2 extract of B. sempervirens exhibited greater potency against Pf than against Tbr. As similarly observed in that study [13], the majority of CPC subfractions in the present work showed a clear preference for inhibiting Pf compared to mammalian cytotoxicity (all SI > 10).
Scheme 1 provides a summary of the isolation of 24 pure aminosteroid alkaloids from various CPC subfractions using preparative HPLC. Structural elucidation and identification of the isolated compounds (see Figure 1) were accomplished through LC/MS analysis and comprehensive NMR spectroscopic techniques. A total of 15 compounds (1–15) with a 9β,10β-cyclo-5α-pregnane skeleton, and seven compounds (17–23) featuring the 9 (10→19) abeo-5α-pregnane framework, both structural motifs typical of Buxus alkaloids, were successfully isolated. In addition to these compounds, an unprecedented C-4 demethylated aminosteroid (16) and a novel Buxus alkaloid featuring a previously unknown carbocyclic core structure (24) were isolated and their structures fully elucidated. Since Buxus obtusifolia had not been previously subjected to chemical investigation, all compounds reported herein are described for the first time as constituents of this species. Compounds 2, 3, 5–9, 11, 12, 15–19, and 21–24 are newly identified and have not been previously described in the literature. Compounds 1 and 4, which were formally reported only as hydrolysis products of N-benzoylcycloprotobuxoline-C isolated from B. sempervirens [17], are herein described for the first time as natural products. Given the limited spectroscopic data available in the literature for compound 1, its complete NMR assignments are presented in Table 3 and Table 4. The identification of known compounds cycloprotobuxoline-C (1) [17], cycloprotobuxoline-D (4) [17], cyclonataminol (10) [18], cyclovirobuxeine-A (13) [19], cyclovirobuxeine-B (14) [20,21], and O2-natafuranamine (20) [18] was accomplished through their high resolution mass spectrometry in combination with 1D- and 2D-NMR spectroscopic analyses, which were in full agreement with previously published data.
Compound 2 was isolated as a yellow gum and its molecular formula was determined by its LC/MS quasimolecular ions at m/z 217.1949 [M + 2H]2+ and 433.3773 [M + H]+, as C27H48N2O2, which differed from that of the known compound 1 (cycloprotobuxoline-C) by containing an additional oxygen atom (Supplementary Materials, Figures S1–S3). The 1H- and 13C-NMR data of compound 2 (Table 3 and Table 4, respectively; spectra shown in Supplementary Materials, Figures S4 and S5) were generally very similar to those of compound 1, with notable differences confined to the side chain region. For instance, in the 13C-NMR spectrum of compound 2, δC20 was shifted downfield from 67.6 ppm to 82.8 ppm, δC21 from 11.5 ppm to 16.0 ppm, and δC33/34 from 43.6/35.9 ppm to 57.1/51.8 ppm. These localized downfield shifts suggested N-oxidation of the tertiary amine [22,23]. Compound 2 was, therefore, elucidated as cycloprotobuxoline-C N20-oxide. Notably, this compound was observed in the LC/MS chromatogram of the crude extract, confirming that it is a genuine natural metabolite rather than an artifact generated during isolation. The occurrence of this compound alongside its non-oxidized analog 1 suggests the possibility of an oxidative branch in the Buxus alkaloid biosynthetic pathway. However, N-oxides are uncommon in Buxus alkaloids, and literature search only revealed (-)-N-oxide-pseudocyclobuxine-D, reported by Vachnadze et al. from Buxus colchica [24].
Compound 3 was obtained as a white gum, and its molecular formula, determined from its LC/MS molecular adduct ions at m/z 217.2010 [M + 2H]2+ and 433.3840 [M + H]+, was identical to that of 2 (C27H48N2O2; Supplementary Materials, Figures S11–S13). The 1H- and 13C-NMR spectroscopic data (Table 3 and Table 4, respectively; Supplementary Materials, Figures S14 and S15) also showed great similarity between 3 and 1. However, unlike the methylene group at C-16 in 1, compound 3 exhibited signals corresponding to a hydroxy methine group at δH = 4.30 ppm (ddd, J = 9.6, 7.5, 2.2 Hz)/δC = 77.1 ppm, which showed an HMBC correlation with C-20. In addition, the methylene group at C-15 appeared downfield shifted at δC15 = 47.9 ppm compared to 36.5 ppm in compound 1, confirming the presence of an electron withdrawing group at C-16. The NOE correlations of H-16 with H-15β (δH = 2.03 ppm), H-20 (δH = 3.58 ppm) and, most importantly, CH3-18 (β, δH = 1.08 ppm) confirmed the relative configuration at this position (H-16β, OH-16α). Based on these data, compound 3 was, thus, unambiguously identified as 16α-hydroxycycloprotobuxoline-C.
The molecular formula of compound 5, which was obtained as a colorless gum, was determined by the LC/MS as C37H58N2O6, from its quasimolecular ions at m/z 314.2334 [M + 2H]2+ and 627.4483 [M + H]+ (Supplementary Materials, Figures S23–S25). Sharing the same core nortriterpenoid framework as compound 1, the 1H- and 13C-NMR spectroscopic data (Table 3 and Table 4, respectively; Supplementary Materials, Figures S26–S28) of compound 5 showed replacement of the C-29 methyl group by a methylene bearing an oxygen substituent (δH29 = 4.62 ppm (d, J = 13.0 Hz); 4.17 ppm (d, J = 13.0 Hz)/δC29 = 64.9 ppm), and additional aromatic and methoxy signals that were assigned to a trimethoxybenzoate moiety. The HMBC correlations from the C-29 methylene protons to the ester carbonyl confirmed attachment of the trimethoxybenzoate group at this position. It is important to note that 13C-NMR and X-Ray diffraction studies on Buxus alkaloids have shown that the C-4α methyl (C-29) undergoes preferential oxidation compared to the C-4β methyl (C-30), indicating the need to revise previous β-configuration of hydroxymethylene or other derivatives attached to C-4 unless evidence to the contrary is available [25,26]. Furthermore, the NOESY spectrum of compound 5 showed strong correlations of the C-30 methyl protons with both C-2 and C-19 protons. In light of these data, the stereochemical configuration at C-4 was assigned. To the best of our knowledge, benzoates and benzamides occurring frequently, trimethoxybenzoate esters have not previously been reported among the alkaloids of Buxus species. This structural modification expands the chemical diversity observed within Buxus alkaloids; accordingly, compound 5 was assigned the new trivial name 29-trimethoxybenzoyloxy cycloprotobuxoline-C.
Compound 6, whose molecular formula was established from its LC/MS molecular adduct ions at m/z 202.1918 [M + 2H]2+ and 403.3722 [M + H]+ as C26H46N2O (Supplementary Materials, Figures S35–S37), was obtained as a yellow gum. The signals of the 1H- and 13C-NMR spectra (Table 3 and Table 4, respectively; Supplementary Materials, Figures S38 and S39) were, for the most part, in close agreement with those of derivatives 1–3, indicating the same nortriterpenoid core and substitution pattern in the side-chain at C-17. The key difference was the presence of only two N-methyl singlets (δH33/34 = 2.90 s, 3H; 2.72 s, 3H) instead of three. An upfield shift in C-3 (δC3 67.2 ppm) indicated a primary amino group at this position, replacing the secondary (monomethylamino) group observed in the previous derivatives (δC3 76.7–76.8 ppm). The configuration of the amino substituent was unchanged in comparison to 1–3, as deduced from NOESY correlations between H-3 and H-5, together with a large coupling constant (J = 10.5 Hz) between H-3 and the equatorial H-2. Based on these data, compound 6 was identified as N3-demethylcycloprotobuxoline-C.
For compound 7, isolated as a white gum, the molecular formula was determined as C26H46N2O2 by its LC/MS, which showed molecular adduct ions at m/z 210.1910 [M + 2H]2+ and 419.3691 [M + H]+ (Supplementary Materials, Figures S45–S47). Most signals in the 1H- and 13C-NMR spectra (Table 3 and Table 4, respectively; Supplementary Materials, Figures S48 and S49) were generally similar to those of 6. The principal difference was observed at C-16 of 7, where the methylene group present in compound 6 was replaced by a hydroxy methine. As already observed with compound 3, it was shown that the OH group occupies the α-position at C-16. Accordingly, compound 7 was identified as 16α hydroxy-N3-demethylcycloprotobuxoline-C.
Compounds 8a and 8b were obtained as a mixture inseparable by preparative HPLC. The LC/MS analysis (Figure 2A–C) revealed two partly separated peaks with identical molecular masses (at m/z 216.1896 [M + 2H]2+/431.3678 [M + H]+ and m/z 216.1897 [M + 2H]2+/431.3671 [M + H]+, respectively) and the same molecular formula (C27H46N2O2). Compared to compound 1, the 1H- and 13C-NMR spectroscopic data of the two compounds, (Table 3 and Table 4, respectively; Supplementary Materials, Figures S56–S59), indicated the presence of a monomethylamino group at C-20 and a formamide group at C-3. The NMR spectra of 8a and 8b were largely superimposable, differing primarily in the chemical shifts in the C-3 methine and the amide-linked N-methyl group, C-31. Furthermore, MS/MS fragmentation patterns confirmed identical core structures in both compounds (Figure 2B, C), with characteristic neutral losses of the C-3 methylformamide, C-2 hydroxyl, and C-20 methylamino substituents. Analysis of the 1H–1H coupling constants between H-2 and H-3 showed identical J values in both 8a and 8b, ruling out epimerization or configurational isomerism at C-3 (Figure 2D).
Instead, the observation of distinct 1H and 13C resonances for the N3-methyl and C-3 methine groups strongly supported the presence of rotational isomers (rotamers) arising from restricted rotation about the C–N amide bond at C-3. In the 13C-NMR of 8a (trans rotamer) the amide methyl was more shielded (δC31 = 29.4 ppm) and the C-3 more deshielded (δC3 = 74.2 ppm) compared to 8b (cis rotamer; δC31 = 33.7 ppm; δC3 = 66.6 ppm) consistent with reported shielding/deshielding effects in N-methylated amides [27]. The presence of the axial hydroxyl group at C-2 may influence the rotamer equilibrium and stabilization through intramolecular H-bonding or steric interactions, enhancing the NMR distinction between the two forms [28]. Based on the integrals in the 1H-NMR spectrum, the proportion of the two compounds was calculated to be 80% (trans rotamer) and 20% (cis rotamer). To the best of our knowledge, this represents the first report of a C-3 N-methyl formamide substitution and spectroscopic evidence for hindered amide rotation in a Buxus alkaloid. Based on the above-mentioned data, and the relation to compound 4, compounds 8a and 8b were assigned as cylcoprotobuxoline-D N3-trans- and cylcoprotobuxoline-D N3-cis-formamide, respectively.
Compounds 9a and 9b were also obtained as a mixture. LC/MS analysis (Supplementary Materials, Figures S67–S69) showed identical molecular masses (at m/z 231.1955 [M + 2H]2+/461.3808 [M + H]+ and m/z 231.1940 [M + 2H]2+/461.3775 [M + H]+, respectively) and the same molecular formula (C28H48N2O3). Most signals in the 1H- and 13C-NMR spectra (Table 3 and Table 4, respectively; Supplementary Materials, Figures S70–S73) closely resembled those of 8a/8b. In contrast to a monomethylated amine in 8a/8b, 9a/9b displayed signals of two N-methyl groups of a dimethylamino group at C-20 (δH = 2.81/2.96 ppm, (s, 6H)/δC = 36.6/43.7 ppm) and a hydroxylated methine in position 16 (δH = 4.30 ppm (ddd, J = 9.5, 7.6, 2.2 Hz)/δC = 77.2 ppm), both showing HMBC correlations with C-20 and adjacent carbons. Thus, in contrast to compounds 8a/8b, the structures of 9a/9b possess a tertiary amine (dimethylamino group) at C-20 instead of a secondary amine (monomethylamino group) and a hydroxyl group at C-16 instead of a methylene. As with 8a and 8b, the two analogues differed mainly in the chemical shifts in the C-3 methine and the amide-linked N-methyl carbon, supporting their assignment as rotational isomers about the C–N amide bond at C-3. Thus, compounds 9a and 9b were identified as the formamide derivatives of compound 3 and hence consistently named 16α-hydroxycycloprotobuxoline-C N3-trans-formamide and 16α-hydroxycycloprotobuxoline-C N3-cis-formamide.
The molecular formula of compound 11, obtained as a white gum, was established from its LC/MS quasimolecular ions at m/z 216.1937 [M + 2H]2+ and 431.3721 [M + H]+, as C27H46N2O2 (Supplementary Materials, Figures S81–S83). The complete 1H- and 13C-NMR data of 11 (Table 5 and Table 6, respectively; Supplementary Materials, Figures S84–S86) was generally similar to those of 3. The key difference was the presence of two conspicuous downfield resonances (δH = 5.71 ppm (d, J = 10.7 Hz)/δC = 126.4 ppm) and (δH = 5.57 ppm (ddd, J = 10.7, 6.2, 3.2 Hz)/δC = 130.9 ppm) attributable to a double bond, specifically Δ6,7 in a Buxus alkaloid [12,18]. Structurally, compound 11 was identified as the 6,7-dehydro derivative of 3 but also an analogue of the known compound 10 (cyclonataminol), from which it differs in that it bears a monomethylamino moiety at C-3 instead of a dimethylamino group. Consequently, compound 11 was named N3-demethyl cyclonataminol.
Compound 12 was obtained as a yellow gum, and its molecular formula (C27H46N2) was determined by LC/MS analysis which displayed quasimolecular ions at m/z 200.1954 [M + 2H]2+ and 399.3785 [M + H]+ (Supplementary Materials, Figures S93–S95). In contrast to the previous compounds all bearing a hydroxy group at C-2, the 1H- and 13C-NMR data of 12 (Table 5 and Table 6, respectively; Supplementary Materials, Figures S96–S99) displayed signals of a methylene group at this position (δH2 = 1.91 ppm, m; 2.11 ppm, m/δC2 = 21.6 ppm). This assignment was confirmed by the 1H/1H COSY correlations between H-2 and both H-1 and H-3 as well as all other spectral features. The structure of compound 12 was thus established as a derivative of cyclovirobuxeine-B (compound 14) [12,29], differing by the presence of a methylene group at C-16 instead an OH substituent. Based on these findings, compound 12 was named deoxycyclovirobuxeine-B.
The molecular formula of compound 15, obtained as a colorless gum, was determined as C27H46N2O by LC/MS analysis which showed quasimolecular ions at m/z 208.1943 [M + 2H]2+ and 415.3736 [M + H]+ (Supplementary Materials, Figures S106–S108). The structural similarity of this alkaloid to the above-described compounds was indicated by its 1H- and 13C-NMR data (Table 5 and Table 6, respectively; Supplementary Materials, Figures S109–S111), which showed the typical resonances for the fully saturated nortriterpenoid (norcycloartane) core, including a tertiary C-methyl, a cyclopropyl methylene, three quaternary C-methyls and no olefinic protons, along with a methylamino moiety attached to C-20. However, of interest was the observation of a set of distinctive AB doublets centred at δH = 3.56 ppm, (d, J = 11.1 Hz) and 3.90 ppm, (d, J = 11.1 Hz) assignable to C-29 methylene protons, while another pair of AB doublets at δH = 4.40 ppm (d, J = 8.6 Hz) and 5.03 ppm (d, J = 8.6 Hz) corresponded to methylene protons (H-31) adjacent to the C-3 nitrogen [30,31]. HMBC correlations from H-3 to both methylene pairs confirmed the presence of a tetrahydro-1,3-oxazine ring in ring A. Comparison with literature data indicated a close relationship between compound 15 to deoxycyclobuxoxazine A [32], from which it differs only by the replacement of the C-20 dimethylamino group with a methylamino group. Compound 15 was therefore named N20-demethyl deoxycyclobuxoxazine A.
The LC/MS analysis of 16 (Supplementary Materials, Figures S119–S121) displayed a singly charged quasimolecular ion at m/z 374.3115 [M + H]+ indicating the presence of a monobasic alkaloid. The molecular formula of 16 was thereby established as C24H39NO2. Its 1H- and 13C-NMR spectra (Table 5 and Table 6, respectively; Supplementary Materials, Figures S122–S125) revealed signals characteristic of the C-19 cyclopropane methylene, a C-3 monomethylamino substituent, and two quaternary C-methyl groups at C-18 and C-28. Additional resonances included a sharp singlet germinal to a carbonyl carbon at δH = 2.16 ppm, (s, 3H)/δC = 31.5 ppm, assigned to C-21, and a methoxy singlet (δH = 3.18 ppm, (s, 3H)/δC = 57.5 ppm) attributed to C-16, whose position was confirmed by HMBC correlations between the methoxy protons and the C-16 methine carbon. In contrast to the 9,19-cyclo-5α-pregnane derivatives possessing a quaternary C-4 (1–15), compound 16 exhibited a methylene group at C-4 (δH = 1.97 ppm, m; 1.18 ppm, (d, J = 11.8 Hz)/δC = 35.5 ppm), which was confirmed by its vicinal correlations with both H-3 and H-5 in the COSY spectrum, as well as its HMBC correlations with C-2, C-3, C-5 and C-10 (Supplementary Materials, Figures S128–S131). To the best of our knowledge, such a compound featuring an unsubstituted C-4 methylene within an otherwise norcycloartanoid carbon skeleton has not been previously described among the Buxus alkaloids. The vast majority of Buxus alkaloids reported to date feature either a 4,4-dimethyl substitution (often hydroxylated or esterified) or sometimes an exomethylene moiety (C = CH2) at this position. It is worth noting, that the presence of this compound in the crude extract confirmed it as a true natural plant metabolite. All spectral data led to the unambiguous assignment of the structure 16. For this unusual new aminosteroid alkaloid with a previously unknown carbon scaffold, we chose the generic name obtusibuxeine A.
The LC/MS spectrum (Supplementary Materials, Figures S134–S136) of compound 17 showed quasimolecular ions at m/z 297.1921 [M + 2H]2+ and 593.3679 [M + H]+ corresponding to the molecular formula, C35H48N2O6 and indicating the presence of 13 double bond equivalents in the molecule. The intensity of the singly charged adduct was clearly higher than that of the doubly charged adduct, suggesting two nitrogen atoms with significantly different basicities. The observed fragmentation pattern revealed some key structural features of compound 17. The fragment ion at m/z = 575 [M − (H2O)]+ arose from the loss of a hydroxyl group as a water molecule, while the subsequent fragment at 533 m/z [M − (CH3COOH)]+ was attributed to the loss of an acetate group as an acetic acid unit. Furthermore, the fragment signals at m/z = 72 and 105 indicated presence of an N,N-dimethylamino side chain at C-17 [C4H10N]+ and a benzoyl ion [C7H5O]+ corresponding to a benzamide moiety at C-3, respectively [33,34]. Based on the NMR spectroscopic analysis (Table 5 and Table 6; Supplementary Materials, Figures S137–S144) the 13C-NMR spectrum exhibited resonances characteristic to a 9(10→19) abeo-5α-pregnane framework [12,35,36], including signals for the allylic C-19 methylene carbon (δC19 = 45.2 ppm), and olefinic carbons at C-1 (δC1 = 133.8 ppm), C-2 (δC2 = 130.5 ppm) and C-11 (δC11 = 126.1 ppm). In the 1H-NMR spectrum, the C-29 methylene protons appeared as a set of AB doublets at δH29 = 3.70 ppm and 3.62 ppm, (d, J = 8.6 Hz), while C-10 appeared as a quaternary carbon at δC10 = 81.1 ppm. A combination of COSY and HMBC correlations indicated an ether linkage between C-29 and C-10 in accordance with previous findings observed in the related compound O10-natafuranamine [18]. Additional aromatic signals were assigned to a benzamide moiety, which was located at C-3 based on the HMBC cross signal of the H-3 (δH3 = 4.71 ppm) with the carbonyl carbon (δC1″ = 170.3). Two downfield methine protons geminal to an acetoxy group (δH = 5.05 ppm) and an OH group (δH = 4.02 ppm) were due to H-6 and H-7, respectively. Furthermore, the position of the acetoxy group at C-6 was confirmed by the cross peaks in the COSY (H-6 to H-5 and H-7) and HMBC (H-6 to C-1′) spectra, respectively. A multiplet signal at δH = 4.33 ppm was consistent with a C-16 methine proton, geminal to an OH group. The assignments at various stereo centres were based on the NOESY correlations, together with chemical shifts, coupling constants and biogenetic comparisons with literature data [18]. The a-orientation of the oxygenated C-4 methyl (C-29 methylene) was based on the NMR and X-ray diffraction studies by Sangre et al. and Guilhem, respectively [25,26]. Consistent with the 9,19-cyclo-5α-pregnanes, the C-16 hydroxyl group was assigned an α-orientation based on NOE correlations of H-16 with both H3-18 and H-20. The methine proton at C-6 exhibited as a double doublet (dd) showing one large coupling constant (11.3 Hz) with the axial H-5 and another small coupling constant (1.7 Hz) with H-7, indicating different spatial orientations for H-5 relative to H-6 and a similar orientation for H-6 and H-7. The measured dihedral angles of −178.00 between H-5 and H-6, and 77.80 between H-6 and H-7 were consistent with the observed coupling constants (see Figure 3, right 3D model, B). Additionally, NOE correlations were observed between H-7 and H2-15, which corroborated only with an a-oriented C-7 OH group, ruling out β-orientation (Figure 3, right vs. left 3D model, B vs. A). Furthermore, the observed NOE between H-7 and the a-oriented H3-28 was confirmed to be possible with both orientations of H-7 (Figure 3, right vs. left 3D model, B vs. A), that is, not to contradict the postulated configuration. Taken together, the spectral data led us to unambiguously elucidate structure 17 shown in Figure 1 and structure 17-B in Figure 3 for this new compound, which was generically named O10-obtusifuranamine-A.
The molecular formula of compound 18 was established from the LC/MS spectrum (Supplementary Materials, Figures S146–S148) as C40H50N2O6 on the basis of the quasimolecular ions (m/z 328.1977 [M + 2H]2+, 655.3817 [M + H]+) and thus included 17 double bond equivalents. The molecular mass of 18 differed from that of 17 by 62 Da and exhibited identical MS/MS fragmentation, suggesting replacement of the acetate group by a benzoate moiety. Consistent with the LC/MS data, evaluation of NMR spectra (Table 5 and Table 6; Supplementary Materials, Figures S149–S157) indicated that most signals were identical to those of compound 17. The key difference was additional aromatic signals, which were assigned to a benzoate group bound to the oxygen at C-6. The stereochemical assignments for various stereo centers were established in a similar manner to that used for 17. Therefore compound 18 was identified as the benzoate analog of 17 and named O10-obtusifuranamine-B.
Compound 19 had a molecular formula of C40H50N2O5 as indicated by the LC/MS quasimolecular ions at m/z 320.1993 [M + 2H]2+ and 639.3802 [M + H]+ (Supplementary Materials, Figures S159–S161), representing a difference of -16 Da from compound 18. Comprehensive comparison of its NMR data (Table 5 and Table 6; Supplementary Materials, Figures S162–S170) with those of compound 18 revealed a high degree of similarity in the observed signals. The key deviation occurred in the chemical shifts at position C-16, where the hydroxymethine group of 18 was replaced by a methylene group. This conclusion was supported by the COSY correlations of the C-16 methylene protons with adjacent protons, as well as the HMBC correlation from H-20 (δH20 = 3.44 ppm) to C-16 (δC16 = 26.0 ppm). Compound 19 was thus elucidated as 16-deoxy-O10-obtusifuranamine-B.
Compound 21 afforded the molecular formula of C35H48N2O5 as indicated by its LC/MS quasimolecular ions at m/z 289.1940 [M + 2H]2+ and 577.3728 [M + H]+, which differed from compound 17 by 16 Da (Supplementary Materials, Figures S172–S174). Based on its NMR spectroscopic data (Table 5 and Table 6; Supplementary Materials, Figures S175–S183), compound 21 was confidently assigned to the Buxus alkaloids class characterized by a 9(10→19) abeo-5α-pregnane backbone. In analogy to 17, compound 21 showed characteristic NMR resonances corresponding to a C-3 benzamide moiety, a C-6 acetoxy substituent, a quaternary C-10, a hydroxylated C-16, an allylic C-19 methylene group, and a dimethylamino side chain. However, in contrast to compounds 17–19, compound 21 lacked an ether linkage between C-29 and C-10. Instead, it exhibited distinct NMR signals corresponding to two geminal methyl groups at C-4 (δH29 = 1.09 ppm, (s, 3H)/δC29 = 28.2 ppm; δH30 = 1.08 ppm, (s, 3H)/δC30 = 18.9 ppm). The C-6 methine proton (δH6 = 4.99 ppm), geminal to the C-6 acetoxy group, exhibited COSY cross peaks to H-5 (δH5 = 2.67 ppm) and an oxygenated methine assigned to C-7 (δH7 = 3.89 ppm). The latter showed vicinal coupling with H-8 (δH8 = 2.26 ppm). Analysis of the HMBC spectrum confirmed the presence of an epoxide moiety between C-7 and C-10, as evidenced by a distinct three bond (3JCH) correlation from H-7 to C-10. The epoxidated abeo-pregnane type Buxus alkaloids reported to date have featured epoxide linkages between C-29/C-10, as observed in compounds 17–19, C-29/C-2 and C-1/C-10 as in compound 20, or at C-29/C-6 or C-9/C-11 positions [18,34,37]. Compound 21, therefore, represents the first example of this structural type bearing an epoxide bridge between C-7 and C-10. As already demonstrated for compound 17, the substituents at C-6, C-7 and C-16 were confirmed to adopt the α-orientation. To further validate the α-configuration of the epoxide, 3D molecular models were generated and various interproton distances measured to support the proposed stereochemistry. As depicted in Figure 4 (right vs. left 3D model, B vs. A), the epoxide bridge between C-7 and C-10 must adopt the α-configuration, as only this spatial arrangement positions H-6 sufficiently close to H-8 and H-19b, to account for the observed NOE correlations. Additionally, the NOE correlations of H-7 with both H-15a and H3-28 were similarly substantiated, further supporting the proposed stereochemistry. Based on these data, compound 21 was assigned the structure depicted in Figure 1 and structure 21-B in Figure 4 and was named obtusiepoxamine A.
The molecular formula of compound 22 was determined as C35H50N2O5 on the basis of the LC/MS quasimolecular ions at m/z 290.2016 [M + 2H]2+ and 579.3887 [M + H]+ (Supplementary Materials, Figures S185–S187). The NMR spectroscopic data of compound 22 (Table 5 and Table 6; Supplementary Materials, Figures S188–S197), suggested the presence of another Buxus alkaloid with a 9(10→19) abeo-5α-pregnane skeleton. This structure closely resembles those of compounds 17–19 with the key distinction being the presence of a double bond between C-1 and C-10, rather than between C-1 and C-2 as observed in the latter. This structural assignment was supported by two multiplets corresponding to geminal methylene protons at δH = 2.18 ppm and 2.34 ppm (δC = 30.0 ppm in the 1H/13C-HSQC spectrum), which displayed vicinal correlations with the methine protons at C-1 and C-3 in the COSY spectrum, thereby confirming their position at C-2. Furthermore, the quaternary carbon at C-10 resonated significantly downfield (δC = 136.8 ppm), consistent with the presence of a C-1/C-10 double bond [33,38]. As observed in 21, compound 22 also exhibited signals corresponding to two geminal methyl groups at C-4 (δH29 = 1.08 ppm, (s, 3H)/δC29 = 28.9 ppm; δH30 = 1.00 ppm, (s, 3H)/δC30 = 22.1 ppm). The configuration of the stereocentres at C-6, C-7 and C-16 was confirmed by 3D molecular modelling, which showed full agreement with all NMR spectroscopic data. Based on these spectroscopic analyses, the structure of this new alkaloid was unambiguously established and designated as obtusidienolamine-A.
For compound 23, the molecular formula was determined to be C35H50N2O4, based on its LC/MS quasimolecular ions at m/z 282.1999 [M + 2H]2+ and 563.3801 [M + H]+ (Supplementary Materials, Figures S198–S200). The majority of signals for compound 23 in the NMR spectra (Table 5 and Table 6; Supplementary Materials, Figures S201–S209) were similar to those observed for compound 22. The only structural difference was observed at C-16, where the hydroxy methine group present in compound 22 was replaced by a methylene group. Consequently, compound 23 was identified as 16-deoxyobtusidienolamine-A.
The LC/MS spectrum of compound 24 (Supplementary Materials, Figures S211–S213) displayed a singly charged molecular adduct ion at m/z 368.2660 [M + H]+, corresponding to the molecular formula C24H33NO2 and indicating the presence of nine double bond equivalents.
The NMR spectroscopic data (Table 7; Supplementary Materials, Figures S214–S224) revealed the typical structural characteristics associated with a nortriterpenoid Buxus alkaloid. The 1H-NMR spectrum showed multiple methylene and methine resonances, along with signals corresponding to five methyl groups (δH18 = 1.14 ppm, δH21 = 1.85 ppm, δH28 = 0.85 ppm, δH29 = 1.34 ppm, and δH30 = 1.05 ppm), while no signals attributable to methylamino groups were observed. An olefinic methine proton resonance at δH = 6.51 ppm (q, J = 6.1 Hz), correlated with a carbon signal at δC = 131.0 ppm in the 1H/13C-HSQC spectrum suggesting a geminal position to a methyl group, and was assigned to C-20. The 13C-NMR in combination with APT and DEPT-135 spectra, revealed 24 carbon signals, comprising five methyl groups, six methylene groups, five methine carbons (including one olefinic at δC = 131.0 ppm). Additionally, eight quaternary carbons were observed among which were, one olefinic at δC = 148.5 ppm and two carbonyl carbons at δC = 206.6 and 211.3 ppm. The two carbonyl groups and one double bond accounted for three degrees of unsaturation, thereby implying the existence of six rings within the molecule.
The positions of the two carbonyl groups at C-6 (δC = 206.6 ppm) and C-16 (δC = 211.3 ppm) were unequivocally established by HMBC correlations from H-5, H-7, and H-8 to C-6 and from H-15 and H-20 to C-16, respectively (Figure 5). Further analysis of the NMR data deduced the presence of an unprecedented C-3/C-10 amino bridge, consistent with the absence of a methylamino substituent in the molecule. This conclusion was supported by HMBC correlations (3JCH) from H-3 (δH3 = 3.81 ppm) to C-1 (δC = 30.0 ppm), C-5 (δC = 62.1 ppm), C-10 (δC = 81.4 ppm), and C-30 (δC = 23.8 ppm). Additionally, COSY correlations between H-3 and H-2, and between H-2 and H-1 provided further evidence for this connectivity (Figure 5). The cyclopropane methylene protons exhibited downfield chemical shift values and elevated coupling constants (δH19a = 1.18 ppm, (dd, J = 10.9, 6.3 Hz) and δH19b = 1.30 ppm, (d, J = 6.6 Hz)) compared to those observed in the 9,19-cyclo-5α-pregnane (1–16) derivatives, indicating a different mode of ring closure and thus a unique position of the cyclopropane moiety. Additionally, COSY cross-peaks between H-11 (δH11 = 1.58 ppm/δC = 22.6 ppm) and both C-19 and C-12 methylene groups, and HMBC correlations (3J) from H2-19 to C-8 (δC = 40.4 ppm), C-10 (δC = 81.4 ppm), and C-12 (δC = 30.4 ppm) further confirmed the position of the cyclopropane ring within the molecule. Furthermore, H2-1, H-8, H2-19 and H2-12, all showed 2J or 3J correlations with the quaternary C-9 (δC = 22.6 ppm). These diagnostic correlations unequivocally confirmed that the cyclopropane ring is closed between C-9, C-19 and C-11. Until now, all previously known Buxus alkaloids bearing a cyclopropane moiety possess the norcycloartanoid carbon skeleton, wherein the cyclopropane ring typically comprises C-9, C-10 and C-19. An exception of this pattern is spirofornabuxine, which features a unique rearranged spirocyclic scaffold [39].
The relative stereochemistry of 24 was established through analysis of its NOESY spectrum (Figure S224), complemented by and biosynthetic considerations of Buxus steroidal alkaloids. The NOE correlations of H2-19 with H-8 and H3-18 suggested a β-orientation of that the cyclopropane ring. Furthermore, the correlations between H-5, and methyl groups H3-28 and H3-29 suggested a “syn” spatial relationship, which, based on biosynthetic grounds, is consistent with an α-orientation. Nearly all previously reported Buxus alkaloids, as well as all other compounds characterized in this study possess a nitrogen substituent at C-3 in the β-configuration. Only a limited number of Buxus alkaloids featuring a 3α-amino substituent have been documented [40,41]. In compound 24, the amino-substituent was assigned the more common β-orientation on the basis of the NOE correlations between the a-oriented H3-29 with H-2a. This structural arrangement is only feasible if the amino bridge between positions C-3 and C-10 adopts a β-orientation rather than an α-orientation (Figure 5, right vs. left 3D model, B vs. A). Additionally, a distinct NOE correlation between H3-30 (β) and H-7b was observed, which would not be possible in the stereoisomer bearing an α-oriented amino group. Compound 24 was thus unambiguously assigned the structure depicted in Figure 1 and structure 24-B in Figure 5.
Notably, this structurally unique alkaloid was detected in the LC/MS chromatogram of the crude extract, confirming its presence as a native component of Buxus obtusifolia with a novel structural framework. It is likely that compound 24, like all other Buxus alkaloids, is biosynthetically derived from cycloartenol, the common precursor of all Buxus alkaloids [42,43]. However, its formation may involve a previously uncharacterized pathway, potentially involving a rearrangement through an abeo-pregnane type intermediate, as observed in compounds 17–23. This novel Buxus alkaloid was named obtusiaminocyclin.

2.3. Antiprotozoal Activity of the Aminosteroids Isolated from Buxus Obtusifolia

The isolated compounds were assessed for their in vitro antiplasmodial and antitrypanosomal properties against Trypanosoma brucei rhodesiense (Tbr), and Plasmodium falciparum (Pf), respectively. In order to determine their selectivity toward the target parasites, their cytotoxicity against L6 rat skeletal myoblasts was also evaluated (Table 8). As the alkaloids were isolated as mono- or bis-trifluoroacetate salts (depending on the number of basic amino groups), the molecular masses of the salts were used to calculate the molar IC50 values. It is important to note that sodium trifluoroacetate was separately tested and found to be inactive against the target parasites in a recent study by our research group [14]. Among the 9,19-cyclo-5α-pregnanes (1–16), the new compounds 6 and 12 demonstrated the highest antitrypanosomal activity with IC50 values of 0.9 µmol/L and 0.8 µmol/L and SI values of 53 and 108, respectively. Albeit with moderate potency, the N-oxide derivative (compound 2), demonstrated 2-fold the antitrypanosomal activity (IC50 = 3.8 µmol/L vs. IC50 = 6.7 µmol/L) and 3-fold the antiplasmodial activity (IC50 = 5.2 µmol/L vs. IC50 = 28 µmol/L) as compared to the unoxidized parent compound 1.
The 9(10→19) abeo-5α-pregnane derivatives (17–23) generally exhibited low antitrypanosomal activities with the exception of compounds 19 (IC50 = 2.0 µmol/L) and 21 (IC50 = 2.2 µmol/L), which showed moderate activities against Tbr. It is worth noting that cyclovirobuxeine-B (14), previously reported by our research group to possess significant antitrypanosomal activity [12,21], demonstrated considerably reduced antiprotozoal activity in this study compared to its newly characterized derivative, deoxycyclovirobuxeine-B (12).
Remarkable antiplasmodial activities were recorded for compounds 4 (IC50 = 1.1 µmol/mL) and, especially 5 (IC50 = 0.5 µmol/L) with the trimethoxy benzoate moiety. Additionally, compound 6 (IC50 = 3.0 µmol/L) exhibited moderate antiplasmodial activity, and the highest selectivity index against Pf (SI = 17). The rest of the alkaloids showed low antiplasmodial activity with IC50 values ranging from 5.4 to 49 µmol/L.
The in vitro antiprotozoal activity data enabled a preliminary structure–activity relationship (SAR) analysis of the isolated compounds. Overall, the 9,19-cyclo-5α-pregnanes (1–16) exhibited stronger antiprotozoal activity and more favourable selectivity indices compared to the 9(10 → 19) abeo-5α-pregnane analogues (17–23). Furthermore, within the 9,19-cyclo-5α-pregnane series, it was observed that di- or monomethylation at both the C-3 amino group and C-20 significantly influence the antiprotozoal potency. These observations are consistent with previous findings reported by our group in the study of B. sempervirens alkaloids [12]. Notably, the most active compound identified in the present study (compound 5) features a bulky trimethoxy benzoate substituent at C-29 position, while the most potent compounds against both Pf and Tbr in the study on B. sempervirens, namely O-benzoyl-cycloprotobuxoline-D and C-16 cyclomicrophyllidine-B contained unsubstituted benzoate groups at either C-2 or C-16, respectively. Interestingly, a recent study by Schäfer et al. on Pachysandra terminalis, a closely related species within the Buxaceae family afforded an aminosteroid alkaloid bearing a benzoate group at C-4 (3α,4α-Diapachsanaximine A). This compound showed very strong activities against both protozoan parasites [14]. Given the proposed similarity in the mechanisms of action against these two distinct parasites [14], it is evident that the presence of a benzoate moiety on either ring A or D (whether substituted or unsubstituted) in combination with N-methylation at C-3 and C-20 appears to be a critical structural feature contributing to antiprotozoal activity.

3. Materials and Methods

3.1. Plant Material

The leaves of B. obtusifolia (Mildbr.) Hutch. were collected from the Gongoni forest Kenya (04°24′38.2″ S 039°28′34.6″ E) in May 2022. The plant material was identified by Mr. Patrick Mutiso, a taxonomist at the Faculty of Science and Technology, University of Nairobi. The voucher specimens were deposited at both the University of Nairobi Herbarium (UoN_JM 2022_002) and at the Institute of Pharmaceutical Biology and Phytochemistry, University of Münster (IPBP 916-TS_JM_2022_002). The plant material was air-dried under shade at room temperature to constant weight and then ground into fine powder using a mill.

3.2. Extraction of Buxus Obtusifolia Leaves

In a Soxhlet apparatus, the powdered plant material (680 g) was exhaustively extracted in three equal parts with 1.5 L dichloromethane (CH2Cl2) for each part for 36 h until the supernatant was clear. The extracts were combined and evaporated in vacuo at 40 °C to obtain 35.3 g of crude extract, translating to 5.2% yield. In order to separate the alkaloids from the crude extract, an acid-base extraction was performed. For each batch of extraction, 5 g of the extract was once again dissolved in 100 mL of CH2Cl2. Using a separatory funnel, this was extracted seven times using 30 mL of diluted hydrochloric acid (aq., 1 M). The organic CH2Cl2 phases were combined and evaporated using a rotary evaporator at 40 °C to yield a total of 10.2 g of the neutral fraction. The aqueous phases were alkalized to ≈ pH 10 with sodium hydroxide solution (aq., 2 M) and exhaustively extracted with 400 mL (6 times) CH2Cl2 to yield, after evaporation, a total of 15.4 g of the alkaloid fraction (2.3% yield) which was then kept in a refrigerator at 4 °C for subsequent work.

3.3. Isolation of Alkaloids from Buxus Obtusifolia Leaf Extract

Using a CPC-250 (Gilson, Limburg, Germany) chromatography system, a portion of (5 g of the alkaloid fraction was separated using the centrifugal partition chromatography (CPC) technique that was previously utilized for B. sempervirens in our working group [11], with minimal modifications. This was accomplished using a biphasic solvent system with iso-hexane/ethyl acetate (8/2; v/v) as the upper phase and CH3OH/H2O/propan-2-ol (8/2/1; v/v/v) as the lower phase. The eluent system was equilibrated in a separatory funnel overnight and sonicated before the experiment. The alkaloid fraction (5 g in 8 portions of 0.5–1 g) was dissolved in 8 mL (6 mL upper phase + 2 mL lower phase) and filtered using a 0.45-micron syringe filter. In ascending mode (1200 rpm, 2 mL/min), portions of 4 mL were collected into test tubes. After termination of the elution mode (624 mL) the lower phase was also fractionated and collected into test tubes by stopping the rotation and increasing the flow rate (6 mL/min) at the same time. The collected fractions were monitored on pre-coated silica gel 60 F254 thin layer chromatography (TLC) plates, (Merck KGaA, Darmstadt, Germany) using a mobile phase of butan-1-ol:H2O:CH3COOH (10:3:1.5) (v/v/v) followed by spraying with Dragendorff’s reagent (bismuth subnitrate (0.85 g):H2O (40 mL): CH3COOH (10 mL):potassium iodide solution (40%; 20 mL)). Based on the TLC and LC/MS profiles, the eluates were combined in 16 subfractions (1–16). Of these, 12 were obtained from the upper phase while the lower phase afforded 4 subfractions (compare Scheme 1).
CPC subfractions 3 (351.0 mg), 4 + 5 (79.6 mg), 6–8 (138.1 mg), 9–10 (195.3 mg), 11 (70.4 mg) and 15 (447.5 mg) were separated by prep-HPLC on a RP-18 phase (VP 250/21 Nucleodur C-18 HTec with a VP 10/16 Nucleodur C-18 HTec pre-column, Macherey-Nagel, Düren, Germany) using binary gradients of H2O (+0.1% TFA; A) and ACN (+0.1% TFA; B). The following gradient conditions were used in all separations: 5–15% of B (0.1–15 min), 15–25% of B (15–30 min), 25–40% of B (30–45 min), 40–50% of B (45–50 min), 50–100% of B (50–55 min), and 100% of B (55– 60 min) at a flow rate of 10 mL/min and a column temperature of 40 °C. The separation of CPC subfraction 3 yielded compound 1 (17.3 mg, tR 21.5 min), 2 (1.3 mg, tR 26.8 min), 6 (1.8 mg, tR 19.9 min) and 13 (4.9 mg, tR 24.6 min). prep-HPLC of CPC subfractions 4 and 5 resulted in isolation of compound 12 (5.3 mg, tR 29.5 min) while compounds 7 (1.9 mg, tR 16.8), 3 (2.1 mg, tR 18.7 min), and 9a + 9b (1.2 mg, tR 32.5) were obtained from CPC subfractions 6-8. Compounds 4 (24.0 mg, tR 24.2), 10 (18.3 mg, tR 26.7), 11 (1.2 mg, tR 20.4), and 14 (3.1 mg, tR 27.3), were distributed in CPC subfractions 9 and 10 in varying concentrations while compound 8a + 8b (1.4 mg, tR 44.6) were isolated from CPC subfraction 11. The separation of CPC subfraction 15 resulted in isolation of compounds 5 (2.1 mg, tR 34.2), 15 (4.6 mg, tR 23.1), 16 (2.6 mg, tR 48.5), 17 (2.2 mg, tR 38.6), 18 (9.0 mg, tR 54.3), 19 (7.2 mg, tR 56.6), 20 (3.5 mg, tR 47.2), 21 (5.9 mg, tR 51.8), 22 (2.0 mg, tR 53.7), and 23 (1.5 mg, tR 55.1) 24 (2.8 mg, tR 32.3).

3.4. Spectrometric and Spectroscopic Analysis

The crude extract, alkaloid fraction, CPC subfractions and the isolated compounds were analysed using a Bruker Daltonics micrOTOFQII time-of-flight mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) fitted with an Apollo electrospray ionization source in positive mode at 3 Hz over a mass range of m/z 50–1500 using the following instrument settings: nebulizer gas (N2), 5 bar; dry gas (N2), 9 L/min, drying temperature, 200 °C; capillary voltage, 4500 V; end plate offset -500 V; transfer time 100 µs; collision gas (N2); collision energy 20 eV. MS2 spectra were acquired using a collision energy of 40 eV and an isolation width of 5 m/z units. Internal calibration (Quadratic + High Precision Calibration (HPC) mode) was performed for each analysis using the mass spectrum of a 10 mM solution of sodium formate solution prepared in propan-2-ol/water/formic acid/1M NaOH (50:50:0.2:1, v/v/v/v), and introduced into the source during LC re-equilibration via a divert valve equipped with a 20 µL sample loop. The compounds were separated on a C-18 column (Dionex Acclaim RSLC 120, Thermo Fisher Scientific, Waltham, MA, USA) and detected using a Dionex Ultimate DAD-3000 RS (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 200–400 nm. Using a binary gradient of H2O (+0.1% formic acid; A) and Acetonitrile (+0.1% formic acid; B) at a flow rate of 0.4 mL/min and column temperature of 40 °C, the following method previously developed for Buxus alkaloids [11] was used: 0–1.88 min: linear from 15% B to 30% B; 1.88–7.88 min: linear from 30% B to 33% B; 7.88–9.9 min: linear from 33% B to 50% B; 9.9–9.93 min: linear from 50% B to 100% B; 9.93–15.88: isocratic 100% B; 15.88–15.98 min: linear from 100% B to 15% B; 15.98–20.0 min: isocratic 15% B. The following sample concentrations were used: 10 mg/mL (crude extract), 1 mg/mL (alkaloid-enriched fraction and CPC subfractions), and 0.1 mg/mL (pure compounds). Each sample was injected at a volume of 2 μL. Data were analysed using the Bruker DataAnalysis 4.1 software.
1H and 13C (1D-NMR) and 1H/1H-COSY, 1H/1H-NOESY, 1H/13C-HSQC, and 1H/13C-HMBC (2D-NMR) spectra were recorded on an Agilent DD2 600 MHz spectrometer (Agilent, Santa Clara, CA, USA) at 26 °C in deuterated methanol (CD3OD). The recorded spectra were analysed with MestReNova version 15.0.0–34764 software and were referenced to the CD3OD solvent signals (1H: 3.310 ppm; and 13C: 49.000 ppm).
Circular dichroism (CD) spectra of compounds 5, 17, 18, 19, 21, 22 and 24 were measured with a Jasco J-815 CD spectrometer (Jasco, Gros-Umstadt, Germany). The compounds were dissolved in CH3OH (0.1 mg/mL) and measurement using a 0.1 cm Suprasil Quartz cuvette (Hellma, Mullheim, Germany).
The three-dimensional molecular models were created using the Molecular Operating Environment (MOE; version 2018.0101) software. The molecular models were energy minimized with the MMFF94x force field, and then a Low-Mode Molecular Dynamics (LowModeMD) conformational search was performed using default settings of MOE. The conformers with the lowest energy were selected and energy minimized using the semi-empirical Austin Model 1 (AM1) Hamiltonian (MOPAC module of MOEs).
The simulated ECD curve for compound 24 (Supplementary Materials, Figure S210) was obtained by Time-Dependent Density Functional Theory (TD-DFT) computations. Computational details are included in the caption of the Figure.

3.5. Spectral Data of the Isolated New Compounds

Cycloprotobuxoline-C N20-oxide (2): Yellow gum; +ESI-QqTOF-MS (m/z): 433.3773 [M + H]+ (calcd. for C27H49N2O2+: 433.3789), 217.1949 [M + 2H]2+ (calcd. for C27H50N2O22+: 217.1931). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S1–S10.
16α-Hydroxycycloprotobuxoline-C (3): White gum; +ESI-QqTOF-MS (m/z): 433.3840 [M + H]+ (calcd. for C27H49N2O2+: 433.3789), 217.2010 [M + 2H]2+ (calcd. for C27H50N2O22+: 217.1931). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S11–S21.
29-Trimethoxybenzoyloxy cycloprotobuxoline-C (5): Colorless gum; +ESI-QqTOF-MS (m/z): 627.4483 [M + H]+ (calcd. for C37H59N2O6+: 627.4368), 314.2334 [M + 2H]2+ (calcd. for C37H60N2O62++: 314.220). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S22–S34.
N3-Demethylcycloprotobuxoline-C (6): Yellow gum; +ESI-QqTOF-MS (m/z): 403.3722 [M + H]+ (calcd. for C26H47N2O+: 403.3683), 202.1918 [M + 2H]2+ (calcd. for C26H48N2O2+: 202.1878). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S35–S44.
16α-Hydroxy-N3-demethylcycloprotobuxoline-C (7): White gum; +ESI-QqTOF-MS (m/z): 419.3691 [M + H]+ (calcd. for C26H47N2O2+: 419.3632), 210.1910 [M + 2H]2+ (calcd. for C26H48N2O22+: 210.1853). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S45–S55.
Cycloprotobuxoline-D N3-trans- (8a) and cycloprotobuxoline-D N3-cis (8b) -formamide: White gum; +ESI-QqTOF-MS (m/z): 431.3678 (8a)/431.3671 (8b) [M + H]+ (calcd. for C27H47N2O2+: 431.3671), 216.1896 (8a)/216.1897 (8b) [M + 2H]2+ (calcd. for C27H48N2O22+: 216.1853). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S56–S66.
16a-Hydroxycycloprotobuxoline-C N3-trans-formamide (9a) and 16a-hydroxycycloprotobuxoline-C N3-cis-formamide (9b): White gum; +ESI-QqTOF-MS (m/z): 461.3808 (9a)/461.3775 (9b) [M + H]+ (calcd. for C28H49N2O3+: 461.3738), 231.1955 (9a)/231.1940 (9b) [M + 2H]2+ (calcd. for C28H50N2O32+: 231.1905). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 3 and Table 4, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S67–S80.
N3-Demethyl cyclonataminol (11): White gum; +ESI-QqTOF-MS (m/z): 431.3721 [M + H]+ (calcd. for C27H47N2O2+: 431.3632), 216.1937 [M + 2H]2+ (calcd. for C27H48N2O22+: 216.1853). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S81–S92.
Deoxycyclovirobuxeine-B (12): Yellow gum; +ESI-QqTOF-MS (m/z): 399.3785 [M + H]+ (calcd. for C27H47N2+: 399.3734), 200.1954 [M + 2H]2+ (calcd. for C27H48N22+: 200.1903). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S93–S105.
N20-Demethyl deoxycyclobuxoxazine A (15): Colorless gum; +ESI-QqTOF-MS (m/z): 415.3736 [M + H]+ (calcd. for C27H47N2O+: 415.3683), 208.1943 [M + 2H]2+ (calcd. for C27H48N2O2+: 208.1878). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S106–S118.
Obtusibuxeine A (16): Yellow gum; +ESI-QqTOF-MS (m/z): 374.3115 [M + H]+ (calcd. for C24H40NO2+: 374.3054). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S119–S132.
O10-Obtusifuranamine-A (17): Yellow gum; +ESI-QqTOF-MS (m/z): 593.3679 [M + H]+ (calcd. for C35H49N2O6+: 593.3585), 297.1921 [M + 2H]2+ (calcd. for C35H50N2O62+: 297.1829). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S133–S144.
O10-Obtusifuranamine-B (18): Yellow gum; +ESI-QqTOF-MS (m/z): 655.3817 [M + H]+ (calcd. for C40H51N2O6+: 655.3742), 328.1977 [M + 2H]2+, (calcd. for C40H52N2O62+: 328.1907). For 1H- and 13C-NMR data (600/150MHz, CD3OD), Table 5 and Table 6, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S145–S157.
16-Deoxy-O10-obtusifuranamine-B (19): Colorless gum; +ESI-QqTOF-MS (m/z): 639.3602 [M + H]+ (calcd. for C40H51N2O5+: 639.3792), 320.1993 [M + 2H]2+ (calcd. for C40H52N2O52+: 320.1933). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S158–S170.
Obtusiepoxamine-A (21): Yellow gum; +ESI-QqTOF-MS (m/z): 577.3728 [M + H]+ (calcd. for C35H49N2O5+: 577.3636), 289.1940 [M + 2H]2+ (calcd. for C35H50N2O52+: 289.1855). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S171–S183.
Obtusidienolamine-A (22): Colorless gum; Yellow gum; +ESI-QqTOF-MS (m/z): 579.3887 [M + H]+ (calcd. for C35H51N2O5+: 579.3792), 290.2016 [M + 2H]2+ (calcd. for C35H52N2O52+: 290.1933). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (CD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S184–S197.
Deoxyobtusidienolamine-A (23): Colorless gum; +ESI-QqTOF-MS (m/z): 563.3801 [M + H]+ (calcd. for C35H51N2O4+: 563.3843), 282.1999 [M + 2H]2+ (calcd. for C35H52N2O42+: 282.1958). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 5 and Table 6, respectively. For spectral data (LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S198–S209.
Obtusiaminocyclin (24): Yellow gum; +ESI-QqTOF-MS (m/z): 368.2660 [M + H]+ (calcd. for C24H34NO2+: 368.2584). For 1H- and 13C-NMR data (600/150MHz, CD3OD), see Table 7. For spectral data (ECD, UV, LC/MS, 1H-NMR, 13C-NMR, and 2D-NMR (HSQC, COSY, HMBC, and NOESY), see Figures S210–224.

3.6. In Vitro Bioassays

In vitro biological activity against Trypanosoma brucei rhodesiense (bloodstream trypomastigotes, STIB 900 strain), Plasmodium falciparum (intraerythrocytic form, NF54 strain) and cytotoxicity assay against rat skeletal myoblasts (L6 cell line) were carried out at the Swiss Tropical and Public Health Institute (Swiss TPH, Allschwil, Switzerland) in accordance with the established standard procedures, in exactly the same manner and with the same cell lines as described recently [14].

4. Conclusions

In the present study, a detailed phytochemical investigation of the dichloromethane extract from the leaves of Buxus obtusifolia was conducted for the first time. A total of 24 aminosteroid alkaloids were successfully isolated and characterized, confirming the plant’s richness as a source of antiprotozoal compounds. Among the isolated natural products, 18 were previously undescribed, including two compounds with novel carbocyclic skeletons (16 and 24), which could all be elucidated by their NMR and LC/MS data. Of particular note is compound 24, which possesses a unique hexacyclic skeleton featuring a cyclic amine alongside an unprecedented position of the cyclopropane moiety involving carbons C-9, C-19 and C-11. This structural arrangement represents a significant novelty amongst the isolated compounds. Interestingly, approximately 68% of the 9-19-cyclo-5α-pregnanes identified in our study (compounds 1–11) possess a hydroxy group at C-2, which is a rare structural feature among Buxus alkaloids. This characteristic may therefore serve as a potential chemotaxonomic marker for B. obtusifolia.
The alkaloid fraction, several CPC subfractions and a number of isolated alkaloids displayed prominent antiprotozoal activities, with compounds 12 and 5 showing the highest activities (IC50 values of <1.0 µmol/L) against Trypanosoma brucei rhodesiense and Plasmodium falciparum, respectively. This work further complements the ongoing studies of our group on the structural diversity of antiprotozoal aminosteroid alkaloids and contributes valuable molecular candidates that may serve as promising lead candidates for the development of therapies against Malaria and sleeping sickness. Three-dimensional quantitative structure–activity relationship (3D–QSAR) analyses of all aminosteroids obtained from B. obtusifolia in comparison with those previously reported from P. terminalis, B. sempervirens and H. africana by our laboratory are currently underway. Additionally, detailed mechanistic or molecular target studies will be interesting subjects for future studies. In order to investigate the dynamics of alkaloid accumulation in B. obtusifolia, particularly to determine the optimal harvesting period for isolating the bioactive alkaloids, a study on seasonal and climate-related variation is currently in progress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules30234558/s1: Spectral data of compounds 2, 3, 5–9, 11, 12, 15–19, and 21–24 (Figures S1–S224).

Author Contributions

Conceptualization, T.J.S.; investigation, J.W.M., M.K. and M.C.; resources, T.J.S.; L.K.O. and P.M.; data curation, J.W.M., M.K. and M.C.; writing—original draft preparation, J.W.M. and T.J.S.; writing—review and editing, L.K.O.; N.M.K.; P.M., M.K. and M.C.; supervision, T.J.S., L.K.O. and P.M.; project administration, T.J.S.; funding acquisition, T.J.S., and P.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received external funding in the form of a doctoral fellowship for Mr. Justus Wambua Mukavi from the Kenyan government, through National Research Fund-Kenya, in cooperation with the German Academic Exchange Service (NRF-DAAD). External funding was also received from Apothekerstiftung Westfalen-Lippe under project title: “Struktur-Wirkungsbeziehungen für die antiprotozoale Aktivität von Aminosteroiden und Amino-nortriterpenen aus Buxaceae und Apocynaceae”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original data of this study is detailed in the article and Supplementary Materials. The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

J.W.M. is most grateful to the Kenyan government, through National Research Fund—Kenya, in cooperation with the German Academic Exchange Service (NRF-DAAD) for a doctoral fellowship to Justus Mukavi at the University of Münster, Germany. The authors very cordially thank Apothekerstiftung Westfalen-Lippe for the financial support of this study. The authors thank the Kenya forest service (KFS) for providing the plant material and Patrick Mutiso of the University of Nairobi (Kenya) for the plant identification. Thanks are due to J. Köhler (Münster) for NMR measurements, J. Sendker (Münster) for the support with the LC/MS measurements and S. Keller-Maerki and R. Rocchetti (Allschwil) for assistance with the parasite and cytotoxicity assays. This work is part of the activities of the Research Network Natural Products against Neglected Diseases (ResNet NPND, see www.resnetnpnd.org (accessed on 20 November 2025)).

Conflicts of Interest

The authors declare no conflicts of interest.

Author Statement

Some of the results described in this article were previously published as conference abstracts [44,45]. This work is part of the doctoral thesis of Justus W. Mukavi.

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Molecules 30 04558 sch001
Scheme 1. Isolation scheme for aminosteroid alkaloids from Buxus obtusifolia.
Scheme 1. Isolation scheme for aminosteroid alkaloids from Buxus obtusifolia.
Molecules 30 04558 sch001
Molecules 30 04558 g001
Figure 1. Chemical structures of alkaloids isolated from Buxus obtusifolia. New compounds are marked by an asterisk (*).
Figure 1. Chemical structures of alkaloids isolated from Buxus obtusifolia. New compounds are marked by an asterisk (*).
Molecules 30 04558 g001
Molecules 30 04558 g002
Figure 2. UHPLC/+ESI-QqTOF-MS chromatogram of 8a and 8b (A); +ESI-QqTOF-MS/MS spectrum of 8a (B) and 8b (C); and detail of the 1H-NMR spectrum (showing 1H–1H coupling constants between H-2 and H-3) of 8a and 8b (D).
Figure 2. UHPLC/+ESI-QqTOF-MS chromatogram of 8a and 8b (A); +ESI-QqTOF-MS/MS spectrum of 8a (B) and 8b (C); and detail of the 1H-NMR spectrum (showing 1H–1H coupling constants between H-2 and H-3) of 8a and 8b (D).
Molecules 30 04558 g002
Molecules 30 04558 g003
Figure 3. Molecular models of the two C-7 epimers of 17 showing the dihedral angles (between H-5 and H-6 and between H-6 and H-7) and distances between the proton H-7 and the protons at C-15 and C-28, respectively. Based on the excellent agreement of the right model with the NMR spectra, compound 17 was assigned structure B.
Figure 3. Molecular models of the two C-7 epimers of 17 showing the dihedral angles (between H-5 and H-6 and between H-6 and H-7) and distances between the proton H-7 and the protons at C-15 and C-28, respectively. Based on the excellent agreement of the right model with the NMR spectra, compound 17 was assigned structure B.
Molecules 30 04558 g003
Molecules 30 04558 g004
Figure 4. Molecular models of the two stereoisomers of 21 (with the epoxide in α- and β-orientation) showing distances from H-6 to both H-8 and H-19b. Based on the excellent agreement of the right model with the NMR spectra, compound 21 was assigned structure B.
Figure 4. Molecular models of the two stereoisomers of 21 (with the epoxide in α- and β-orientation) showing distances from H-6 to both H-8 and H-19b. Based on the excellent agreement of the right model with the NMR spectra, compound 21 was assigned structure B.
Molecules 30 04558 g004
Molecules 30 04558 g005
Figure 5. Key COSY (bold lines) and HMBC (blue arrows) correlations of 24 (top); Molecular models of the two stereoisomers of 24 (with the amino bridge in α- and β-orientation) showing distances between H3-29 and H-2a, and between H3-30 and H-7b. Based on the excellent agreement of the right model with the NMR spectra, compound 24 was assigned structure B.
Figure 5. Key COSY (bold lines) and HMBC (blue arrows) correlations of 24 (top); Molecular models of the two stereoisomers of 24 (with the amino bridge in α- and β-orientation) showing distances between H3-29 and H-2a, and between H3-30 and H-7b. Based on the excellent agreement of the right model with the NMR spectra, compound 24 was assigned structure B.
Molecules 30 04558 g005
Table 1. Preliminary in vitro antiprotozoal and cytotoxic activity of the crude extracts from the leaves of Buxus obtusifolia, and the acid-base extraction fractions. Data (in µg/mL) are the means of two separate IC50 determinations ± the absolute deviations from the mean.
Table 1. Preliminary in vitro antiprotozoal and cytotoxic activity of the crude extracts from the leaves of Buxus obtusifolia, and the acid-base extraction fractions. Data (in µg/mL) are the means of two separate IC50 determinations ± the absolute deviations from the mean.
Test SampleTbrPfCytotox. L6SI (Tbr)SI (Pf)
Small-scale extracts
Twigs (CH3OH)6.3 ± 0.042.8 ± 0.2554 ± 2.48.519
Twigs (CH2Cl2)12 ± 0.403.1 ± 0.3357 ± 4.74.918
Leaves (CH3OH)13 ± 0.252.3 ± 0.3351 ± 2.94.122
Leaves (CH2Cl2)16 ± 0.451.1 ± 0.1126 ± 1.41.624
Fractions of the large-scale CH2Cl2 extract
Alkaloid fraction8.1 ± 3.10.69 ± 0.1442 ± 4.24.553
Lipophilic fraction14 ± 0.902.2 ± 0.1050 ± 0.73.523
Hydrophilic residue>100>50>100--
Positive controls0.004 ± 0.000 a0.002 ± 0.000 b0.007 ± 0.002 c--
Positive controls: a melarsoprol (Tbr), b chloroquine (Pf), c podophyllotoxin (Cytotoxic L6); selectivity indices (SI) = IC50 (Cytotox. L6)/IC50 (parasite).
Table 2. Antiprotozoal activity of centrifugal partition chromatography (CPC) subfractions and positive controls. Data (in µg/mL) are the means of two separate IC50 determinations ± the absolute deviations from the mean.
Table 2. Antiprotozoal activity of centrifugal partition chromatography (CPC) subfractions and positive controls. Data (in µg/mL) are the means of two separate IC50 determinations ± the absolute deviations from the mean.
Test SampleTbrPfCytotox.L6SI TbrSI Pf
Fr. 0122 ± 0.64.2 ± 0.667 ± 14316
Fr. 0242 ± 5.37.6 ± 1.458 a18
Fr. 030.8 ± 0.01.9 ± 0.526 ± 9.33414
Fr. 040.8 ± 0.01.6 ± 0.616 ± 2.52110
Fr. 054.4 ± 0.21.0 ± 0.215 ± 3.0315
Fr. 062.4 ± 0.31.1 ± 0.214 ± 3.2613
Fr. 072.9 ± 0.21.0 ± 0.115 ± 2.1515
Fr. 084.2 ± 0.21.1 ± 0.214 ± 2.4313
Fr. 092.9 ± 0.51.1 ± 0.212 ± 3.2411
Fr. 10a4.2 ± 0.31.2 ± 0.116 ± 1.3414
Fr. 10b3.6 ± 0.61.7 ± 0.514 ± 4.148
Fr. 113.4 ± 0.01.8 ± 0.412 ± 3.249
Fr. 123.3 ± 0.51.7 ± 0.418 ± 4.4510
Fr. 132.9 ± 0.21.8 ± 0.616 ± 3.669
Fr. 145.2 ± 0.41.1 ± 0.218 ± 0.4416
Fr. 155.2 ± 0.20.9 ± 0.117 ± 0.5318
Fr. 1617 ± 0.51.1 ± 0.528 ± 12225
Positive controls0.004 ± 0.000 b0.002 ± 0.000 c0.007 ± 0.003 d--
a Deviation not calculable as the result of one replicate was higher than the maximal concentration tested (>100 µg/mL). Positive controls: b melarsoprol (Tbr), c chloroquine (Pf), d podophyllotoxin (Cytotox.L6); selectivity indices (SI) = IC50 (Cytotox. L6)/IC50 (parasite).
Table 3. 1H-NMR data of compounds 1, 2, 3, 5, 6, 7, 8a, 8b, 9a, and 9b (600 MHz, CD3OD).
Table 3. 1H-NMR data of compounds 1, 2, 3, 5, 6, 7, 8a, 8b, 9a, and 9b (600 MHz, CD3OD).
δH [ppm] mult., J [Hz]
Pos.1235678a8b9a9b
11.67, d, (2H, 7.9)1.64, d, (2H, 8.0)1.63, m, (2H)1.68, m
1.63, m		1.64, m (2H)1.63, m (2H)1.72, m
1.63, m		1.72, m
1.63, m		1.71, dd, (12.5, 5.0)
1.63, m		1.71, dd, (12.5, 5.0)
1.63, m
23.94, dt, (10.5, 7.9)3.91, m3.90, ddd, (10.6, 8.9, 6.8)4.04, dt, (10.7, 5.0)3.75, dt, (10.5, 10.5, 5.3)3.74, dt, (10.5, 10.4, 5.5)4.08, dt, (10.6, 10.6, 5.0)4.02, dt, (10.7, 10.6, 5.1)4.08, dt, (10.6, 10.6, 5.0)4.01, dt, (10.6, 10.6, 5.0)
32.68, d, (10.5)2.65, d, (10.5)2.65, d, (10.6)2.88, d, (10.3)2.72, m2.72, d, (10.4)3.04, d, (10.5)3.94, d, (10.9)3.04, d, (10.5)3.94, d, (10.9)
4----------
51.59, m1.57, d, (12.4, 4.3)1.56, dd, (12.3, 4.0)1.98, m1.56, m1.56, m1.53, m1.53, m1.54, m1.54, m
61.72, m,
0.90 dq, (12.6, 2.5)		1.69, m
0.90, m		1.70, m
0.86, dd, (12.7, 2.3)		1.81, m
1.03, m		1.70, m
0.88, dd, (12.7, 2.4)		1.70, m
0.87, dd, (12.6, 2.2)		1.62, m
0.88, m		1.62, m
0.88, m		1.64, m
0.86, m		1.64, m
0.86, m
71.46, m
1.23, m		1.45, m
1.19, m		1.23, m
1.40, m		1.99, m
1.58, m		1.43, m
1.20, m		1.38, m
1.22, m		1.41, m
1.20, m		1.41, m
1.20, m		1.37, m
1.20, m		1.37, m
1.20, m
81.65, m1.60, m1.56, dd, (12.3, 4.0)1.68, m1.63, m1.56, m1.61, m1.61, m1.54, m1.54, m
9----------
10----------
112.16, m
1.27, m		2.16, m
1.24, m		2.16, m
1.23, m		2.14, m
1.27, m		2.14, m,
1.25, m		2.17, m
1.23, m		2.14, m
1.26, m		2.14, m
1.26, m		2.18, m
1.25, m		2.18, m
1.25, m
121.82, dd, (13.8,10.9)
1.70, m		1.86, m
1.76, m		1.84, brt, (12.2, 12.2)
1.62, m		1.80, m
1.69, m		1.79, t, (12.3, 12.3)
1.68, m		1.84, brt, (12.3, 12.3)
1.62, m		1.73, m, (2H)1.73, m, (2H)1.85, m
1.63, m		1.85, m
1.63, m
13----------
14----------
151.56, m, (2H)1.49, m, (2H)2.03, dd, (14.0, 9.4)
1.46, dd, (14.0, 2.2)		1.55, m, (2H)1.54, m, (2H)2.03, m
1.46, dd, (13.9, 2.3)		1.52, m, (2H)1.52, m, (2H)2.04, dd, (13.8, 9.5)
1.47, dd, (12.9, 2.3)		2.04, dd, (13.8, 9.5)
1.47, dd, (12.9, 2.3)
162.02, m,
1.61, m		2.18, m, (2H)4.30, ddd, (9.6, 7.5, 2.2)1.51, m
1.21, m		1.98, m
1.57, m		4.30, ddd, (9.7, 7.5, 2.2)2.02, m
1.54, m		2.02, m
1.54, m		4.30, ddd, (9.5, 7.6, 2.2)4.30, ddd, (9.5, 7.6, 2.2)
172.20, m2.33, q, (9.5, 9.5, 0.5)2.22, dd, (11.1, 7.6)2.17, m2.18, m2.22, dd, (11.1, 7.4)2.06, m2.06, m2.22, d, (11.1, 7.4)2.22, d, (11.1, 7.4)
181.10, s, (3H)1.10, s, (3H)1.08, s, (3H)1.09, s, (3H)1.08, s, (3H)1.09, s, (3H)1.08, s, (3H)1.08, s, (3H)1.09, s, (3H)1.09, s, (3H)
190.72, d, (4.5)
0.56, d, (4.5)		0.68, d, (4.4)
0.54, d, (4.4)		0.69, d, (4.5)
0.52, d, (4.5)		0.78, d, (4.5)
0.61, d, (4.5)		0.69, d, (4.5)
0.53, d, (4.5)		0.68, d, (4.4)
0.52, d, (4.4)		0.68, d, (4.5)
0.55, d, (4.5)		0.66, d, (4.4)
0.53, d, (4.4)		0.67, d, (4.4)
0.55, d, (4.4)		0.65, d, (4.4)
0.52, d, (4.4)
203.43, dq, (11.1, 6.6)3.81, dq, (10.5, 6.9)3.58, dq, (11.0, 6.6, 6.5, 6.5)3.41, dd, (11.2, 6.6)3.40, m3.58, m3.23, m3.23, m3.58, dq, (11.5, 6.7, 6.6, 6.6)3.58, dq, (11.5, 6.7, 6.6, 6.6)
211.32, d, (3H, 6.6)1.53, d, (3H, 6.7)1.31, d, (3H, 6.6)1.30, d, (6.6)1.30, d, (3H, 6.6)1.31, d, (3H, 6.7)1.32, d, (3H, 6.5)1.32, d, (3H, 6.5)1.32, d, (3H, 6.7)1.32, d, (3H, 6.7)
281.06, s, (3H)1.05, s, (3H)1.21, s, (3H)1.02, s, (3H)1.03, s, (3H)1.21, s, (3H)1.02, s, (3H)1.02, s, (3H)1.22, s, (3H)1.22, s, (3H)
291.14, s, (3H)1.11, s, (3H)1.11, s, (3H)4.62, d, (13.0)
4.17, d, (13.0)		1.08, s, (3H)1.08, s, (3H)0.90, s, (3H)0.90, s, (3H)0.90, s, (3H)0.90, s, (3H)
300.97, s, (3H)0.95, s, (3H)0.94, s, (3H)1.04, s, (3H)0.93, s, (3H)0.92, s, (3H)0.94, s, (3H)0.94, s, (3H)0.94, s, (3H)0.94, s, (3H)
312.94, s, (3H)2.92, s, (3H)2.92, s, (3H)2.97, s, (3H) 2.90, s, (3H)3.08, s, (3H)2.89, s, (3H)3.07, s, (3H)
33/342.92, s, (3H)3.50, s, (3H)2.96, s, (3H)2.90, s, (3H)2.90, s, (3H)2.96, s, (3H)2.67, s, (3H)2.67, s, (3H)2.96, s, (3H)2.96, s, (3H)
2.75, s, (3H)3.38, s, (3H)2.81, s, (3H)2.72, s, (3H)2.73, s, (3H)2.81, s, (3H) 2.81, s, (3H)2.81, s, (3H)
1′ - 8.01, s8.21, s8.01, s8.21, s
2′ -
3′/7′ 7.85, s, (2H)
4′/6′ -
5′ -
4′/6′-OCH3 3.91, s, (6H)
5′-OCH3 3.85, s, (3H)
Table 4. 13C-NMR data of compounds 1, 2, 3, 5, 6, 7, 8a, 8b, 9a, and 9b (150 MHz, CD3OD). Multiplicities according to 1H/13C-HSQC.
Table 4. 13C-NMR data of compounds 1, 2, 3, 5, 6, 7, 8a, 8b, 9a, and 9b (150 MHz, CD3OD). Multiplicities according to 1H/13C-HSQC.
δC [ppm]
Pos.1235678a8b9a9b
142.9, CH242.9, CH242.8, CH242.9, CH242.5, CH242.3, CH243.5, CH243.6, CH243.4, CH243.5, CH2
269.7, CH69.7, CH69.7, CH69.7, CH68.7, CH68.7, CH65.9, CH66.0, CH65.9, CH66.0, CH
376.8, CH76.8, CH76.7, CH70.7, CH67.2, CH67.2, CH74.2, CH66.6, CH74.2, CH66.5, CH
440.8, qC40.7, qC40.7, qC44.7, qC39.8, qC39.8, qC42.6, qC42.6, qC42.6, qC42.6, qC
548.9*, CH48.9*, CH48.9*, CH43.7*, CH48.9*, CH48.9*, CH49.6*, CH49.6*, CH49.4*, CH49.4*, CH
621.9, CH222.0, CH221.9, CH221.8, CH222.0, CH222.0, CH222.4, CH222.2, CH222.3, CH222.2, CH2
726.8, CH226.8, CH226.9, CH226.5, CH226.9, CH227.0, CH226.9, CH226.9, CH227.0, CH227.0, CH2
849.1*, CH49.1*, CH49.0*, CH48.8*, CH49.2*, CH49.0*, CH49.3*, CH49.3*, CH49.1*, CH49.1*, CH
920.6, qC20.6, qC20.0, qC20.5, qC20.6, qC20.1, qC20.5, qC20.5, qC20.0, qC20.0, qC
1025.8, qC25.6, qC25.9, qC25.0, qC25.9, qC26.1, qC26.1, qC26.2, qC26.2, qC26.3, qC
1127.2, CH227.3, CH227.2, CH227.1, CH227.2, CH227.2, CH227.2, CH227.2, CH227.2, CH227.2, CH2
1233.2, CH233.9, CH232.5, CH233.1, CH233.2, CH232.5, CH233.3, CH233.3, CH232.5, CH232.5, CH2
1347.2, qC49.9*, qC47.3, qC47.1, qC47.2, qC47.3, qC46.9, qC46.9, qC47.3, qC47.3, qC
1450.1, qC49.1*, qC48.8*, qC50.1, qC50.1, qC48.8*, qC50.1, qC50.1, qC48.8*, qC48.8*, qC
1536.5, CH237.2, CH247.9, CH236.4, CH236.5, CH248.0, CH236.5, CH236.5, CH248.0, CH248.0, CH2
1626.6, CH230.0, CH277.1, CH26.8, CH226.5, CH277.1, CH27.0, CH227.0, CH277.2, CH77.2, CH
1749.7, CH49.6, CH56.1, CH49.6, CH49.7, CH56.1, CH51.1, CH51.1, CH56.1, CH56.1, CH
1818.8, CH318.4, CH319.6, CH318.7, CH318.8, CH319.6, CH318.7, CH318.7, CH319.6, CH319.6, CH3
1930.3, CH230.0, CH230.7, CH230.1, CH230.3, CH230.7, CH230.5, CH230.5, CH230.9, CH230.9, CH2
2067.6, CH82.8, CH68.1, CH67.6, CH67.6, CH68.1, CH60.5, CH60.5, CH68.2, CH68.2, CH
2111.5, CH316.0, CH311.4, CH311.5, CH311.5, CH311.4, CH315.5, CH315.5, CH311.4, CH311.4, CH3
2819.7, CH320.0, CH321.2, CH319.7, CH319.8, CH321.3, CH319.8, CH319.8, CH321.3, CH321.3, CH3
2925.5, CH325.4, CH325.4, CH364.9, CH226.0, CH326.0, CH326.6, CH326.6, CH326.6, CH326.6, CH3
3015.9, CH315.9, CH315.9, CH312.5, CH316.0, CH316.0, CH318.7, CH318.7, CH318.7, CH318.7, CH3
3137.8, CH337.8, CH337.8, CH337.5, CH3 29.4, CH333.7, CH329.4, CH333.7, CH3
33/3443.6, CH357.1, CH343.7, CH343.5, CH343.5, CH343.7, CH329.9, CH329.9, CH343.7, CH343.7, CH3
35.9, CH351.8, CH336.6, CH335.9, CH335.9, CH336.6, CH3 36.6, CH336.6, CH3
1′ 167.1, qC 167.9, qC167.7, qC167.9, qC167.8, qC
2′ 125.7, qC
3′/7′ 108.2, CH
4′/6′ 154.7, qC
5′ 144.3, qC
4′/6′-OCH3 56.8, CH3
5′-OCH3 61.2, CH3
* The δC values were assigned from the HSQC or HMBC spectra due to overlap with the solvent signal.
Table 5. 1H-NMR data of compounds 11, 12, 15, 16, 17, 18, 19, 21, 22 and 23 (600 MHz, CD3OD).
Table 5. 1H-NMR data of compounds 11, 12, 15, 16, 17, 18, 19, 21, 22 and 23 (600 MHz, CD3OD).
δH [ppm] mult., J [Hz]
Pos.11121516171819212223
11.81, m
1.73, m		1.74, m (2H)1.74, m
1.53, m		1.66, m
1.50, m		5.90, dd, (9.5, 1.1)5.96, dd, (9.4, 1.2)5.98, dd, (9.5, 1.1)6.01, dd, (10.1, 3.0)5.64, m5.64, m
23.92, dt, (10.7, 10.7, 4.8)2.11, m
1.91, m		2.14, m
1.71, m		2.14, m
1.44, m		5.86, dd, (9.5, 3.7)5.91 dd, (9.5, 3.6)5.92, dd, (9.4, 3.7)5.51, dd, (10.1, 2.1)2.34, m
2.18, m		2.34, m
2.20, m
32.73, d, (10.7)3.23, d, (12.8, 3.8)3.21, m3.14, m4.71, dd, (3.7, 2.0)4.69, dd, (3.7 2.0)4.70, dd, (3.8 2.0)4.83, t, (2.6)4.03, m4.03, t, (6.0)
4---1.97, m
1.18 d, (11.8)		------
52.14, m2.11, m1.63, dd, (12.5, 4.4)1.69, m2.32, d, (11.3)2.53, d, (11.4)2.54, d, (11.4)2.26, m2.62, m2.62, m
65.71, d, (10.7)5.70, dd, (10.7, 1.4)1.35, m
0.97, dd, (12.4, 2.7)		1.50, m
0.88 dd, (13.3, 2.9)		5.05, dd, (11.3, 1.7)5.31, dd, (11.3, 1.7)5.32, dd, (11.4, 1.7)4.99, dd, (9.6, 2.1)4.94, dd, (10.0, 2.8)4.96 dd, (10.0, 2.8)
75.57, ddd, (10.7, 6.2, 3.2)5.60, br dt, (10.7, 6.1, 3.1)1.40, m
1.17, dd, (12.4, 2.9)		1.38, m
1.24, m		4.02, d, (1.7)4.13, d, (1.6)4.19, d, (1.7)3.89, d, (2.1)4.05, m4.09, d, (2.7)
82.67, m2.69, d, (2.4)1.67, m1.77 dd, (11.0, 5.9)2.13, m2.22, m2.28, m2.67, m2.12, m2.19, m
9----------
10----------
111.95, m
1.50, m		1.95, m,
1.52, m		2.09, m
1.27, dd, (4.9, 3.6)		2.09, m
1.43, m		5.52, m5.56, m5.57, m5.32, m5.35, m5.37, m
121.86, m
1.46, m		1.73, m
1.53, m		1.69, m (2H)2.05, m,
1.70, m		2.26, m
1.94, m		2.27, m
1.97, m		2.21, m
2.04, m		2.27, m
1.89, m		2.24, m
1.89, m		2.16, m
1.96, m
13----------
14----------
152.16, m
1.31, m		1.64, m
1.33, m		1.50, m1.82, m
1.51, m		2.19, m
1.49, dd, (13.7, 2.2)		2.24, m
1.52, dd, (13.7, 2.2)		1.77, m
1.61, m		2.30, m
1.44 dd, (14.0, 2.2)		2.04
1.51		1.58, m, (2H)
164.36, ddd, (8.7, 6.9, 1.6)2.01, m
1.60, m		2.01, m
1.54, m		4.39, m4.33, m4.36, ddd, (9.6, 7.5, 2.2)2.00, m
1.61, m		4.37, m4.33, m2.00, m
1.60, m
172.17, m2.01, m2.07, m3.10, d, 6.32.24, m2.26, m2.21, m2.21, m2.25, m2.20, m
181.03, s, (3H)1.02, s (3H)1.06, s (3H)0.91, s (3H)0.81, s (3H)0.86, s (3H)0.87, s (3H)0.88, s (3H)0.77, s (3H)0.77, s (3H)
190.84, d, (4.4)
0.04, d, (4.4)		0.80, d, (4.3)
0.02, d, (4.3)		0.74, d, (4.5)
0.54, d, (4.5)		0.54, d, (4.3)
0.18, d, (4.3)		3.09, m
2.59, d, (14.3)		3.16, m
2.64, d, (14.4)		3.16, m
2.66, m		2.79, m
2.22, m		3.42, m
2.93, d, (13.8)		3.41, m
2.95, m
203.60, m3.27, m3.23, m-3.59, m3.61, m3.44, m3.61, m3.59, m3.41, m
211.32, d, (3H, 6.7)1.32, d (3H, 6.5)1.32, d (3H, 6.5)2.16, s (3H)1.33, d, (3H, 6.6)1.34, d, (3H, 6.6)1.32, d, (3H, 6.5)1.33, d, (3H, 6.7)1.32, d, (3H, 6.6)1.30, d, (3H, 6.5)
281.06, s, (3H)0.82, s (3H)1.01, s (3H)1.14, s (3H)1.19, s (3H)1.20, s (3H)1.02, s (3H)1.09, s (3H)1.18, s (3H)1.01, s (3H)
291.20, s, (3H)1.25, s (3H)3.90, d, (11.1)
3.56, d, (11.1)		 3.70, d, (8.6)
3.60, d, (8.6)		3.74, d, (8.6)
3.62, d, (8.6)		3.74, d, (8.5)
3.62, d, (8.5)		1.09, s (3H)1.08, s (3H)1.08, s (3H)
300.93, s, (3H)1.00, s (3H)1.23, s (3H) 1.18, s (3H)1.15, s (3H)1.15, s (3H)1.08, s (3H)1.00, s (3H)1.01, s (3H)
31/322.94, s, (3H)2.98, s (3H)2.72, s (3H)2.69, s (3H)
2.81, s (3H)5.03, d, (8.6)
4.40, d, (8.6)
33/342.96, s, (3H)2.67, s (3H)2.67, s (3H) 2.96, s (3H)2.96, s (3H)2.90, s (3H)2.97, s (3H)2.96, s (3H)2.90, s (3H)
2.80, s, (3H) 2.81, s (3H)2.81, s (3H)2.73, s (3H)2.81, s (3H)2.81, s (3H)2.73, s (3H)
16- OCH3 3.18, s (3H)
1′ ---
2′ 1.95, s (3H)--2.10, s (3H)2.08, s (3H)2.08, s (3H)
3′/7′ 7.92, m7.92, m
4′/6′ 7.39, m7.38, m
5′ 7.55, m7.54, m
1″ ---
2″ ---
3″/7″ 7.83, m7.71, m7.71, m7.83, m7.76, m7.76, m
4″/6″ 7.47, m7.43, m7.42, m7.47, m7.46, m7.46, m
5″ 7.55, m7.53, m7.53, m7.54, m7.54, m7.53, m
Table 6. 13C-NMR data of compounds 11, 12, 15, 16, 17, 18, 19, 21, 22 and 23 (600 MHz, CD3OD).
Table 6. 13C-NMR data of compounds 11, 12, 15, 16, 17, 18, 19, 21, 22 and 23 (600 MHz, CD3OD).
δC [ppm]
Pos.11121516171819212223
140.5, CH230.5, CH233.1, CH231.3, CH2133.8, CH133.8, CH133.9, CH133.2, CH125.6, CH125.6, CH
269.3, CH21.6, CH222.6, CH229.7, CH2130.5, CH130.6, CH130.5, CH132.3, CH30.0, CH230.0, CH2
376.1, CH77.1, CH73.0, CH59.4, CH55.5, CH55.4, CH55.4, CH58.9, CH54.9, CH54.9, CH
440.5, qC40.8, qC40.8, qC36.5, CH247.6, qC47.7, qC47.7, qC40.1, qC38.7, qC38.7, qC
548.2, CH49.1*, CH45.5, CH38.5, CH50.9, CH51.0, CH51.0, CH59.5, CH50.3, CH50.4, CH
6126.4, CH126.2, CH21.2, CH228.4, CH279.5, CH80.1, CH80.1, CH83.0, CH81.0, CH81.0, CH
7130.9, CH131.2, CH26.1, CH225.7, CH272.6, CH72.9, CH72.9, CH83.1, CH72.4, CH72.5, CH
844.4, CH44.6, CH48.3, CH47.1, CH47.6, CH48.0, CH48.3, CH51.3, CH46.8, CH47.2, CH
921.2, qC22.3, qC21.0, qC24.1, qC133.9, qC133.9, qC133.8, qC132.9, qC138.5, qC138.5, qC
1027.9, qC29.2, qC25.9, qC31.1, qC81.1, qC81.3, qC81.3, qC81.1, qC136.8, qC136.8, qC
1125.7, CH226.3, CH226.7, CH227.6, CH2126.1, CH126.2, CH126.5, CH122.3, CH120.0, CH120.3, CH
1232.8, CH233.3, CH233.2, CH232.3, CH237.4, CH237.4, CH238.0, CH237.6, CH237.0, CH237.5, CH2
1347.6, qC46.9, qC46.9, qC48.5qC46.2, qC46.2, qC46.2, qC46.1, qC46.3, qC46.3, qC
1451.1, qC51.9, qC50.0, qC49.7, qC49.2*, qC49.2*, qC50.2, qC47.8, qC48.5, qC49.6, qC
1544.9, CH233.6, CH236.3, CH243.0, CH245.7, CH245.7, CH234.4, CH244.6, CH245.9, CH234.6, CH2
1676.9, CH26.7, CH226.9, CH282.7, CH76.6, CH76.6, CH26.0, CH277.2, CH76.7, CH26.0, CH2
1755.8, CH49.9, CH51.0, CH69.2, CH54.6, CH54.7, CH48.2, CH54.8, CH54.6, CH48.2, CH
1815.8, CH314.8, CH318.5, CH320.6, CH316.9, CH316.9, CH316.1, CH316.7, CH316.4, CH315.6, CH3
1918.8, CH218.5, CH230.5, CH226.5, CH245.2, CH245.2, CH245.1, CH240.4, CH246.5, CH246.4, CH2
2068.1, CH60.4, CH60.4, CH211.5, qC68.1, CH68.1, CH67.5, CH68.1, CH68.0, CH367.5, CH3
2111.8, CH315.9, CH315.5, CH331.5, CH311.4, CH311.4, CH311.6, CH311.4, CH311.4, CH311.6, CH3
2819.0, CH317.7, CH319.6, CH320.3, CH320.2, CH320.2, CH318.7, CH319.5, CH320.0, CH318.5, CH3
2925.4, CH325.0, CH378.1, CH2 79.1, CH279.1, CH279.1, CH228.2, CH328.9, CH328.9, CH3
3016.3, CH316.2, CH312.4, CH3 21.1, CH321.4, CH321.4, CH318.9, CH322.1, CH322.1, CH3
31/3237.8, CH347.0, CH386.4, CH230.6, CH3
40.7, CH335.6, CH3
33/3443.7, CH329.9, CH329.9, CH3 43.7, CH343.7, CH343.5, CH343.7, CH343.7, CH343.5, CH3
36.6, CH3 36.7, CH336.7, CH335.9, CH336.7, CH336.6, CH335.9, CH3
16- OCH3 57.5, CH3
1′ 172.9, qC167.9, qC167.9, qC173.3, qC172.6, qC172.6, qC
2′ 21.3, CH3131.0, qC131.0, qC20.8, CH321.8, CH321.8, CH3
3′/7′ 130.8, qC130.8, qC
4′/6′ 129.5, CH129.5, CH
5′ 134.6, CH134.5, CH
1″ 170.3, qC170.5, qC170.5, qC170.7, qC170.6, qC170.6, qC
2″ 135.1, qC135.1, qC135.1, qC135.8, qC136.5, qC136.5, qC
3″/7″ 128.6, CH128.6, CH128.6, CH128.5, CH128.3, CH128.3, CH
4″/6″ 129.5, CH129.4, CH129.4, CH129.6, CH129.6, CH129.6, CH
5″ 132.9, CH132.7, CH132.7, CH132.7, CH132.5, CH132.5, CH
* The δC values were assigned from the HSQC or HMBC spectra due to overlay with the solvent signal.
Table 7. NMR data of obtusiaminocyclin (24) in CD3OD. All assignments were confirmed by 1H/13C-HSQC, COSY, HMBC and NOESY correlations.
Table 7. NMR data of obtusiaminocyclin (24) in CD3OD. All assignments were confirmed by 1H/13C-HSQC, COSY, HMBC and NOESY correlations.
Pos.1H-NMR13C-NMR
δ (ppm)Mult.J [Hz]δ (ppm)
11.73
1.52		ddd
td		13.2, 9.6, 4.8
13.0, 4.4		30.0
22.21
1.90		ddd
m		14.1, 9.7,4.322.3
33.81d5.168.4
4---46.6
52.51br s (1H) 62.1
6---211.3
73.16
2.42		dd
m		16.0, 8.642.6
82.74dd8.4, 1.440.4
9---18.1
10---81.4
111.58dt (1H)10.9, 6.622.6
122.42
2.36		m
d
14.0		30.4
13---47.6
14---44.8
152.30
1.95		dd
d		17.0, 1.4
16.2		47.0
16---206.6
17---148.5
181.14s (3H) 23.9
191.30
1.18		t
dd		6.6
10.9, 6.3		16.9
206.51q7.5131.0
211.85d (3H)7.513.2
280.85s (3H) 20.6
291.34s (3H) 26.7
301.05s (3H) 23.8
Table 8. Antiprotozoal activity and cytotoxicity of compounds isolated from Buxux obtusifolia leaves. Data are the means of two separate IC50 determinations with absolute deviations from the mean. Compounds were tested as the monoa- or bis b-trifluoroacetates.
Table 8. Antiprotozoal activity and cytotoxicity of compounds isolated from Buxux obtusifolia leaves. Data are the means of two separate IC50 determinations with absolute deviations from the mean. Compounds were tested as the monoa- or bis b-trifluoroacetates.
CompoundTbr [µmol/L]Pf [µmol/L]Cytotox.L6 [µmol/L]SI TbrSI Pf
1 a6.7 ± 3.328 ± 0.7>100--
2 a3.8 ± 0.19.5 ± 0.996 ± 5.02510
3 a7.3 ± 0.116 ± 2.1107 ± 34157
4 a2.3 ± 0.31.1 ± 0.016 ± 1.9715
5 a1.6 ± 0.70.5 ± 0.05.9 ± 0.0412
6 a0.9 ± 0.23.0 ± 0.150 ± 115617
7 a48 ± 8.249 ± 3.978 *22
8a + 8b c41 ± 0.630 ± 1.852 ± 2212
9a + 9b c39 ± 2.026 ± 0.468 ± 0.723
10 a37 ± 1820 ± 1.351 ± 2.813
11 a14 ± 7.019 ± 1.150 ± 1.643
12 a0.8 ± 0.35.4 ± 1.282 ± 4.110315
13 a11 ± 0.38.4 ± 0.977 ± 4.879
14 a16 ± 0.511 ± 2.257 ± 1.245
15 a6.6 ± 3.55.8 ± 0.424 ± 1.544
16 b22 ± 1.713 ± 1.445 ± 5.123
17 b44 ± 1611 ± 2.250 ± 0.515
18 b4.9 ± 0.117 ± 1.527 ± 1.752
19 b2.0 ± 0.218 ± 0.816 ± 1.181
20 b23 ± 4.78.7 ± 2.843 ± 2.025
21 b2.2 ± 0.210 ± 1.134 ± 0.5163
22 b6.5 ± 0.324 ± 2.748 ± 2.272
23 b6.5 ± 0.430 ± 6.539 ± 5.971
24 b26 ± 1.26.4 ± 0.251 ± 1.628
Positive controls0.013 ± 0.0010.012 ± 0.0000.014 ± 0.004--
* Deviation not calculable as the result of one replicate was higher than the maximal concentration tested (>100 µg/mL). a mono-trifluroracetate; b bis-trifluoroacetate; c alkaloids isolated as mixture. positive controls: melarsoprol (Tbr), chloroquine (Pf), podophyllotoxin (Cytotox.L6); selectivity indices (SI) = cytotoxic IC50/IC50 of the target parasite.
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Mukavi, J.W.; Cal, M.; Kaiser, M.; Mäser, P.; Kimani, N.M.; Omosa, L.K.; Schmidt, T.J. Antiprotozoal Aminosteroid Alkaloids from Buxus obtusifolia (Mildbr.) Hutch. Molecules 2025, 30, 4558. https://doi.org/10.3390/molecules30234558

AMA Style

Mukavi JW, Cal M, Kaiser M, Mäser P, Kimani NM, Omosa LK, Schmidt TJ. Antiprotozoal Aminosteroid Alkaloids from Buxus obtusifolia (Mildbr.) Hutch. Molecules. 2025; 30(23):4558. https://doi.org/10.3390/molecules30234558

Chicago/Turabian Style

Mukavi, Justus Wambua, Monica Cal, Marcel Kaiser, Pascal Mäser, Njogu M. Kimani, Leonidah Kerubo Omosa, and Thomas J. Schmidt. 2025. "Antiprotozoal Aminosteroid Alkaloids from Buxus obtusifolia (Mildbr.) Hutch." Molecules 30, no. 23: 4558. https://doi.org/10.3390/molecules30234558

APA Style

Mukavi, J. W., Cal, M., Kaiser, M., Mäser, P., Kimani, N. M., Omosa, L. K., & Schmidt, T. J. (2025). Antiprotozoal Aminosteroid Alkaloids from Buxus obtusifolia (Mildbr.) Hutch. Molecules, 30(23), 4558. https://doi.org/10.3390/molecules30234558

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