C. glauca was selected on the basis of its utilization in traditional medicine. Its leaves were harvested in Bangang-Wabane village, Cameroon, in May 2016. The sample was authenticated by Mr. Francois NANA, a botanist of the National Herbarium of Cameroon (Yaounde, Cameroon), where a voucher specimen (55064/HNC) was deposited.

The air-dried and powdered leaves of C. glauca (1.2 kg) were extracted with methanol (3 × 10 L) for 72 h at room temperature to yield a crude extract (90 g) after evaporation of solvent. Part of this extract (85 g) was subjected to column chromatography (8 cm × 60 cm) over silica gel (300 - 400 mesh) eluted with gradients of n-Hex/EtOAc (90:10, 70:30, 50:50, 30:70) then EtOAc/MeOH (100:0, 90:10, 70:30, 50:50, 0:100) and 68 fractions of 300 mL each were collected. These fractions were combined on the basis of their TLC (Thin Layer Chromatography) profiles into five major fractions A-E: A (9 g, 1 - 14), B (14 g, 15 - 28), C (16 g, 29 - 43), D (12 g, 44 - 53) and E (23 g, 54 - 68). Fraction A with a fatty appearance was not further investigated. Fraction B (14 g) was separated on a silica gel (100 - 200 mesh) column chromatography using a gradient of n-Hex/Me₂CO (100:0 to 70:30) to yield caloncobalactone C (8, 12 mg). Fraction C (16 g) was separated on a silica gel (100 - 200 mesh) column eluted with gradients of n- Hex/EtOAc (90:10 to 0:100) then EtOAc/MeOH (95:5 to 0:100) and subfractions C.I to C.IV were obtained. Successive column chromatography of subfraction C.I (2.5 g) over silica gel (100 - 200 mesh) and Sephadex LH-20 eluted respectively with gradients of n-Hex/Me₂CO (90:10 to 100:0) and isocratic CH₂Cl₂/MeOH (1:1) solvents mixtures afforded glaucartanoic acid B (4, 9.5 mg). Repeated purifications of sub fraction C.II (4 g) on silica gel (100 - 200 mesh) column chromatography eluted with CH₂Cl₂/Me₂CO (100:0 to 50:50) afforded caloncobic acid A (6, 14 mg). Subfraction C.III (3.5 g) was subjected successively to a silica gel (100 - 200 mesh) column chromatography eluted with CH₂Cl₂/Me₂CO (100:0 to 70:30) and passage over Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1) to give glaucalactone (3, 19 mg). Sub fraction C.IV (1.5 g) precipitated in n-Hex/Me₂CO (80:20) and the deriving powder was purified over a Sephadex LH-20 column chromatography eluted with CH₂Cl₂/MeOH, (1:1) to afford glaucalactone B (9, 8.4 mg). Fraction D (12 g) was chromatographed on a silica gel (100 - 200 mesh) column eluted with gradients of n-Hex/EtOAc (90:10 to 0:100) then EtOAc/MeOH (95:5 to 0:100) to give three subfractions: D.I, D.II and D.III. Subfraction D.II (7 g) was subjected to a silica gel (100 - 200 mesh) column chromatography eluted with n-Hex/Me₂CO (from 90:10 to 0:100) and afforded sub fractions D.II.1 - D.II.3. Successive chromatography of subfraction D.II.2 (2.3 g) over a silica gel column eluted with n-Hex/Me₂CO (70:30 to 100:0) and Sephadex LH-20 eluted with CH₂Cl₂/MeOH (1:1) yielded glaucatarnoic acid A (1, 170 mg) and 3β,21β-dihydroxy-30-nor-(D:A)-friedoolean-20(29)-en-27-oic acid (5, 30 mg). Subfraction D.II.3 (1.8 g) was purified similarly to D.II.2 to furnish caloncobalactone (2, 14.3 mg). Fraction E (23 g) precipitated in EtOAc. Purification of the filtered powder over a silica gel column chromatography eluted with EtOAc/MeOH (95:5) followed by gel permeation over a Sephadex LH-20 column using CH₂Cl₂/MeOH, (1:1) gave caloncobic acid C (7, 8.3 mg).

Column chromatography was performed on silica gel G (100 - 200 and 300 - 400 mesh, Qingdao Haiyang Chemical Co., Ltd., People’s Republic of China) and Sephadex LH-20 (40 - 70 μm, Amersham Pharmacia Biotech AB, Sweden). TLC was carried out on precoated silica gel plates (Qingdao Haiyang Chemical Co.), and spots were visualized by heating the plates at 90˚C after they were dipped into a 10% ethanolic H₂SO₄ solution. Solvents were distilled prior to use.

Optical rotations were measured with a JASCO P-1020 digital polarimeter. The NMR spectra were recorded on Bruker AV-400 or DRX-500 NMR spectrometers. Chemical shifts (δ) are expressed in ppm with reference to TMS, and coupling constants (J) are given in Hz. ESIMS and HRESIMS were carried out on an API Qstar time-of-flight spectrometer.

The MeOH extract from the leaves of C. glauca was subjected to column chromatography (CC) over silica gel to afford nine known compounds including glaucartanoic acid A (1: C₃₀H₄₈O₅; m/z 488) [11], caloncobalactone B (2: C₃₀H₄₈O₆; m/z 504) [12], glaucalactone (3: C₂₉H₄₄O₄; m/z 456) [12], glaucartanoic acid B (4: C₃₀H₄₆O₅; m/z 486) [11], 3β,21β-dihydroxy-30-nor-(D:A)-friedoolean-20(29)-en-27-oic acid (5: C₂₉H₄₆O₄; m/z 458) [8], caloncobic acid A (6: C₃₀H₄₆O₄; m/z 470) [12], caloncobic acid C (7: C₃₀H₄₈O₆; m/z 504) [13], caloncobalactone C (8: C₃₃H₅₂O₅; m/z 528) [13] and glaucalactone B (9: C₂₉H₄₆O₄; m/z 458) [13]. Chemical derivatizations including acetylation and methylation of compound 1 and acetylation of compound 5 respectively, afforded five previously unreported semisynthetic derivatives (1a-1b and 5a). The structures of these compounds (Figure 1) were elucidated by the means of 1D and 2D NMR spectroscopy, MS and comparison of these data with those reported in the literature.

Compound 1a, obtained from the acetylation reaction of 1 (Scheme 1), was isolated as a white powder in acetone. The molecular formula C₃₄H₅₂O₇ was deduced from its HR-ESI-MS which displayed the pseudo-molecular ion peak at m/z 595.3612 [M + Na]⁺. This mass was 84 mass units higher than that of 1, indicating the addition of two acetyl units as compared to 1. The positive ion mode ESI-MS of 1a also confirmed the presence of two acetoxy groups in the molecule by displaying the ion peaks at m/z 513 [M − OAc]⁺ and 453 [M − H − 2 OAc]⁺.

The NMR data of 1a revealed additional signals compared to those of glaucartanoic acid A (1) at δ_H 2.08 and 1.94 (each 3H, s) in the ¹H-NMR spectrum (Table 1) and at δ_C 172.5, 172.1, 22.2 and 20.9 in the ¹³C-NMR spectrum (Table 2) assignable to two acetoxy groups. The downfield shift of the H-24 proton signal from δ_H 3.21 (in compound 1) to δ_H 5.27 (in 1a) and of C-25 carbon signal from δ_C 73.0 (in compound 1) to δ_C 84.2 (in 1a) indicated the locations of the acetoxy groups at C-24 and C-25 respectively. Compound 1a was thus elucidated as 24(S), 25-diacetoxy-3-oxocycloartan-30-oic acid and trivially named diacetylglaucartanoic acid A.

Compound 1b, obtained from the acetylation reaction of 1 (Scheme 1), was isolated as a white powder in acetone. Its molecular formula was determined as C₃₂H₅₀O₆ from the pseudo-molecular ion peak observed in its HR-ESI-MS at m/z 553.3505 [M + Na]⁺. This mass was 42 mass units higher than that of 1, corresponding to the addition of one acetyl unit. The presence of a remaining free hydroxyl group in 1b was further confirmed by the base peak observed at m/z 513 [M − H₂O + H]⁺ in its positive ion mode ESI-MS. Its ¹H and ¹³C NMR data (Table 1 and Table 2) compared to those of compound 1 revealed additional signals at δ_H 2.08 (3H, s) and δ_C 172.9 and 21.1 assignable to the acetoxy group. The downfield shift of the H-24 proton signal from δ_H 3.21 (in 1) to δ_H 4.78 (in 1b) together with the deshielding of the C-24 carbon signal at δ_C 80.8 (instead of δ_C 78.6 in 1) indicated the location of the acetoxy group at C-24. This was further confirmed by the HMBC correlation of H-24 with the carbonyl carbon at δ_C 172.9. Compound 1b was thus elucidated as a new semi-synthetic derivative named 24-acetylglaucartanoic acid A.

Compound 1c was obtained from the methylation reaction of 1 (Scheme 2), and precipitated as a white powder in acetone. Its HR-ESI-MS displayed a pseudo-molecular ion peak at m/z 525.3573 [M + Na]⁺ compatible with the molecular formula C₃₁H₅₀O₅, indicating an additional methyl group as compared to compound 1. Indeed, the only difference between the NMR data of compound 1c (Table 1 and Table 2) and those of compound 1 was the appearance of signals attributable to a methoxyl group in the ¹H and ¹³C NMR spectra of 1c at δ_H/δ_C 3.64/51.5. The cross-peak observed in its HMBC spectrum between the methoxyl protons and the carbonyl carbon at δ_C 178.4 (C-30) revealed the presence of a methyl ester group. Compound 1c was thus elucidated as 24(S), 25-dihydroxy-3-oxocycloartan-30-carboxylic acid methyl ester and trivially named glaucartanoic acid A methyl ester.

Compound 1d, obtained from the methylation reaction of 1 (Scheme 2), was isolated as a white powder in acetone. Its HR-ESI-MS displayed a pseudo-molecular ion peak at m/z 539.3820 [M + Na]⁺ compatible with the molecular formula C₃₂H₅₂O₅, indicating one more methoxyl than compound 1c. The NMR data of 1d (Table 1 and Table 2) confirmed the presence of two methoxyl groups at δ_H/δ_C 3.64/51.5 and 3.49/61.4 respectively. Detailed comparison of its 1D NMR spectra with those of compound 1 revealed the shifting of signals at 24-position at δ_H/δ_C 2.88/90.7 instead of δ_H/δ_C 3.21/78.6 in 1 indicating the location of one of the methoxyl groups at C-24. This was confirmed by the HMBC spectrum where a long-range connectivity was observed between H-24 (δ_H 2.88) and the methoxyl carbon at δ_C 61.4 (24-OMe). The HMBC correlation also observed between the second methoxyl signal at δ_H 3.64 and the carbonyl carbon at δ_C 178.4 (C-30) proved the existence of a methyl ester functionality at C-30. Compound 1d was thus characterized as 24(S)-methoxy-25-hydroxy-3-oxocycloartan-30-carboxylic acid methyl ester and was trivially named 24-methoxyglaucartanoic acid A methyl ester.

Compound 5a was obtained from the acetylation reaction of 5 (Scheme 3), and precipitated as a white powder in acetone. Its molecular formula was established as C₃₃H₅₀O₆ from the HR-ESI-MS displaying the pseudo-molecular ion peak at m/z 565.3503 [M + Na]⁺. This molecular formula in accordance with the pseudo-molecular mass furthermore 84 mass units higher than that of 5, revealed the addition of two acetyl units in the reaction product. The NMR data of 5a (Table 1 and Table 2) exhibited additional signals of two methyl groups at δ_H/δ_C 2.02 (3H, s)/21.5 and 1.99 (3H, s)/21.8 and for two ester carbonyls at δ_C 172.4 and 171.8 as compared to 5. Notable differences between the NMR data of compounds 5 and 5a also involved the downfield shift of signals at C-3 position from δ_H/δ_C 3.65/72.7 in compound 5 to δ_H/δ_C 4.83/75.7 in compound 5a, and at position C-21 from δ_H/δ_C 4.19/71.5 (in 5) to δ_H/δ_C 5.30/74.9 (in 5a). This implied the location of acetoxy groups at C-3 and C-21 positions in compound 5a which was further elucidated as 3β,21β-diacetoxy-30-nor-(D:A)-friedoolean-20(29)-en-27-oic acid.

The MeOH extract, isolated compounds and some new semisynthetic derivatives were evaluated for their antimicrobial activities against 9 microorganisms including four Gram-positive (Staphylococcus aureus ATCC25923, methicillin sensitive S. aureus MSSA1, methicillin resistant S. aureus MRSA3, methicillin resistant S. aureus MRSA4), two Gram-negative (Shigella flexneri SDINT, Pseudomonas aeruginosa) bacteria and three fungal strains (Candida albicans ATCC10231, Candida tropicalis PK233 and Cryptococcus neoformans H99) (Table 3). The MeOH extract displayed antimicrobial activity towards 9/9 (100%) of tested bacterial and fungal strains with good activity (MIC < 100 μg/mL) [24] against Staphylococcus aureus and Shigella flexneri SDINT ATCC25923. Among the phytochemicals, compounds 3 and 5 were the most active showing moderate activity (10 < MIC ≤ 100 μg/mL) [24] against 5/9 (55.55%) and 6/9 (66.66%) tested microbial species, respectively. As shown in Table 3, ciprofloxacin and nystatin used as standard drugs were more potent against yeasts, Gram-positive and Gram-negative bacteria than all the tested samples. A microbicidal effect with MMC/MIC ratio ≤ 4 was noted for most of the samples (MeOH extract, 2, 3, 5, 7, 8, 1a, 1b, 1d and 5a) indicating their lethal effect. In general, Gram-negative bacteria were found to be more sensitive to the tested samples when compared with Gram-positive bacteria whereas the bacterial species were resistant as compared to fungal strains. These variations may be due to genetic differences between the microorganisms. The known antimicrobial mechanisms associated to the group of chemicals to which the isolated compounds belong may explain the antimicrobial potency of the MeOH extract. Membrane disruption has been suggested as one of the likely mechanisms of action [25] [26]. This might also explain the antimicrobial activities of isolated triterpenoids and semisynthetic derivatives [25] [26]. Although the fungal strains were more sensitive to the tested compounds than the bacteria strains used in this study, the antibacterial activities of the acetylated (1a and 1b) and the methylated (1d) derivatives of cycloartane 1 were improved compared to that of the original substrate mostly on Gram-negative strains. Meanwhile the O-acetylation at both the C-3 and C-21 positions of friedelane 5 leading to compound 5a induced a decrease in the antibacterial activity on both Gram-positive and Gram-negative strains, but an

SA ATCC25923: Staphylococcus aureus ATCC25923; MSSA1: methicillin sensitive S. aureus MSSA1; MRSA3: methicillin resistant S. aureus MRSA3; MRSA4: methicillin resistant S. aureus MRSA4; SF: Shigella flexneri; PA: Pseudomonas aeruginosa; CA ATCC10231: Candida albicans ATCC10231; CT PK233: Candida tropicalis PK233; CN H99: Cryptococcus neoformans H99; MIC: minimum inhibitory concentration; MMC: Minimum microbicidal concentration; /: not determined; *: Ciprofloxacin and nystatin were used as reference drugs for bacteria and yeasts respectively.

increase in the antifungal activity. The lower antibacterial activity mainly on MRSA3 and MRSA4 strains observed for compound 3 with respect to its analogue 5 could arise from lactonization of the C-27 carboxylic group in compound 3. The difference in the antimicrobial activity of compounds having the same basic skeleton highlights the contribution of esterified carboxylic acid group and acetyl substituents in influencing the activity of this series of compounds.