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    • Compound mg was purified as an

    2023-02-25

    Compound 3 (29 mg) was purified as an amorphous, colourless powder. Molecular formula C20H28O4 was established by HRESIMS (Fig. S11) and corroborated with 1H and 13C NMR spectroscopic data (Table 1). IR spectrum showed bands for hydroxyl (3433 cm−1) and carbonyl (1686 cm−1) groups. 13C NMR spectrum exhibited twenty signals, being two methyl groups at δC 29.6 and 13.3 (C-18 and C-20, respectively), and two carboxyl groups at δC 180.4 and 177.7 (C-19 and C-17, respectively). In addition, the spectrum showed sixteen signals of non-oxigenated carbons. DEPT 135 spectrum showed that, from the twenty carbon atoms, eight were methylene, two were methyl, four were methyne and six were quaternary. 1H NMR spectrum showed a signal at δH 1.98–2.01. Although its shape and integral points to a methyl group (Fig. S3), HSQC indicated the overlap of signals of hydrogen atoms at C-6β, C-12 and C-13. HMBC spectrum showed correlations between the signal of H-1α and C-2, C-9, C-10 and C-20; H-9 and C-7, C-11, C-14 and C-20; H-14b and C-7, C-9, C-13 and C-15; and H-15α and C-7, C-8, C-9, C-16 and C-17. Some HMBC correlations for SIN-1 chloride 3 are shown in Fig. 3. NOESY spectrum showed correlations between H-1β with H-9; H-3β with H-5; H-6α with H-20; H-9 with H-1β, H-5 and H-15β; and between H-18 with H-3β, H-5 and H-6β. All correlations in HMBC and NOESY corroborated with the maintenance of the original skeleton. Structure of compound 3, trachyloban-17,19-dioic acid, was elucidated as a new trachylobane diterpene. Proposed structure of 3 was confirmed by extensive heteronuclear-2D- correlations experiments HMBC, HSQC and NOESY (Table S1, supporting information). Compound 4 (16 mg) was purified as an amorphous, colourless powder. Molecular formula C20H30O4 was established by HRESIMS (Fig. S19) associated with the 1H and 13C NMR spectroscopic data (Table 1). IR spectrum showed bands for hydroxyl (3420 cm−1) and carbonyl (1696 cm−1) groups. 13C NMR spectrum exhibited twenty signals of carbon atoms, being two of methyl groups at δC 29.7 and 16.4 (C-18 and C-20, respectively), and five signals of quaternary carbon atoms, including a carboxyl group at δC 180.4 (C-19). In addition, there were observed two signals of olefinic carbon atoms at δC 128.0 and 133.1 (C-11 and C-12, respectively), and two signals of carbinolic carbon atoms at δC 67.9 and 87.2 (C-17 and C-16, respectively). DEPT 135 spectrum showed eight methylene, two methylic, five are methyne and five quaternary carbon atoms. 1H NMR spectrum showed two signals of hydrogen atoms in oxygenated carbons, one at δH 4.01 (J = 10.6 Hz) and other at δH 4.17 (J = 10.6 Hz), both were assigned to H-17a and H-17b, respectively. Also there were observed two singlet of methyl hydrogen atoms at δH 1.38 (H-18) and 1.18 (H-20), and two double doublet (dd) at δH 5.64 and 6.21, which were assigned to H-11 and H-12 respectively. In compound 4 only two signals of methyl groups were present, one of them was hydroxylated (H-17). Through the HMBC correlations, compound 4 was determined to have a kaurane diterpene skeleton, due to a rearrangement that took place in compound 2. Some HMBC correlations for compound 4 are shown in Fig. 3. Compound 4 presents NOESY correlations between H-17 with H-11 and H-12, indicating that stereochemistry of C-17 must be β. The structure of compound 4, ent-16β,17-dihydroxykaur-11-en-19-oic acid, was elucidated as being a new compound. Proposed structure of 4 was confirmed by extensive heteronuclear-2D-correlation experiments HMBC, HSQC and NOESY (Table S2, supporting information). Compounds 1–4 were submitted to bioassay of acetylcholinesterase (AChE) inhibitory activity to evaluate possible improvement of activity after structural modifications performed by biotransformation. Currently, anti-AChE drugs are the main drugs used to control Alzheimer's disease [18]. Discovery of new compounds with activity against AChE may be the key to relief patients with this disease that affects more than 24 millions of people around the world [19]. Activity of the substrate 1 was lower than 50% at the highest concentration used in this assay and, therefore, IC50 was not determined for substrate 1. The results for the derivatives obtained by biotransformation of 1 showed that modification of trachyloban-19-oic acid skeleton improved the power of inhibiting AChE (IC50 values varying from 0.06 to 0.48 µM) (Table 2). Compound 3 (IC50 = 0.06 µM) presented the best result being about six times more active than galanthamine (IC50 = 0.38 µM), the positive control. The second best result was found for compound 2 (IC50 = 0.31 µM). Although compound 2 has been previously reported, this is the first report on its anti-AChE activity. The results suggest that oxidation of C-17 in the trachylobane diterpene skeleton leads to a significant increase in anti-AChE activity. Rearrangement of trachylobane to kaurane skeleton with C-17 oxidation (compound 4) also showed significant anti-AChE activity but it was smaller when compared to the activity of compounds 2 and 3. Our results suggests that compounds 2–4 may be good therapeutic candidates for development of drugs to treat patients with Alzheimer’s disease.