Bioactive steroidal alkaloids from Buxus macowanii Oliv.

Steroids 95 (2015) 73–79

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Bioactive steroidal alkaloids from Buxus macowanii Oliv. Cheuk W. Lam a,b, Andrew Wakeman a, Abin James a, Athar Ata a,⇑, Robert M. Gengan c, Samir A. Ross d a

Department of Chemistry, Richardson College for the Environmental and Science Complex, The University of Winnipeg, 599 Portage Avenue, Winnipeg, MB R3B 2G3, Canada Department of Chemistry, The University of Manitoba, Winnipeg, MB R3T 2N2, Canada c Department of Chemistry, Durban University of Technology, Durban, South Africa d National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, University of Mississippi, MS 38677, USA b

a r t i c l e

i n f o

Article history: Received 21 July 2014 Received in revised form 18 November 2014 Accepted 3 December 2014 Available online 18 December 2014 Keywords: Buxus macowanii Acetylcholinesterase inhibitory activity Macowanioxazine Macowamine 31-Hydroxybuxatrienone 16a-Hydroxymacowanitriene

a b s t r a c t Chemical investigation of the crude methanolic extract of Buxus macowanii resulted in the isolation of five new steroidal alkaloids, 31-hydroxybuxatrienone (1), macowanioxazine (2), 16a-hydroxymacowanitriene (3), macowanitriene (4), macowamine (5), along with five known steroidal bases, Nb-demethylpapillotrienine (6), moenjodaramine (7), irehine (8), buxbodine B (9) and buxmicrophylline C (10). Structures of compounds 1–10 were elucidated with the aid of spectroscopic methods including 1D and 2D NMR techniques and mass spectrometry. Compounds 1, 3, and 4 belong to a rare class of Buxus alkaloids having D1,2 9(10 ? 19) abeo triene system. All isolates were evaluated for in vitro acetylcholinesterase (AChE) inhibitory activity and found to exhibit moderate to weak anti-AChE activity with IC50 values in the range of 10.8–98 lM. Compounds 1 and 6 were also moderately active in BACE1 inhibitory assay. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Plants of genus Buxus are rich source of steroidal alkaloids containing a unique steroidal–triterpenoidal pregnane-type structure with C-4 methyls, 9b, 10b-cycloartenol system and a degraded C-20 side chain [1]. Previous phytochemical studies on various plants of this genus, including Buxus papillosa, Buxus microphylla, Buxus hyrcana, Buxus natalensis and others, have resulted in the isolation of over 200 steroidal alkaloids [1–6]. A few of these alkaloids are reported to exhibit various biological activities including antimicrobial, anti-tuberculosis, and anti-acetylcholinesterase activities [1–6]. Buxus macowanii Oliv., commonly known as Cape Box, is a small growing evergreen tree which is native to Eastern Cape Forest of South Africa. This plant is used by local healers to treat wounds, and pains [7]. This plant has not been previously phytochemically investigated. Acetylcholine, a neurotransmitter, is present in the central nervous system, and carries out signal transduction between neurons [8]. Acetylcholinesterase (AChE), found in cholinergic synapses in central nervous system, deactivates acetylcholine by hydrolyzing it into acetic acid and choline. The excessive degradation of acetylcholine by AChE results in its deficiency, which is reported to be associated with Alzheimer’s disease (AD) [8,9]. The use of AChE ⇑ Corresponding author. Tel.: +1 (204) 786 9389; fax: +1 (204) 774 2401. E-mail address: [email protected] (A. Ata). http://dx.doi.org/10.1016/j.steroids.2014.12.002 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

inhibitors to enhance acetylcholine level in the brain is one of the most effective approaches to treat early symptoms of AD [8–11]. Four anti-AD drugs approved by the FDA namely, tacrine, donepezil, galantamine, and rivastigmine work by inhibiting the activity of AChE [12]. These inhibitors have limited effectiveness and several side effects [13,14]. Inhibition of AChE also serves as a strategy for the treatment of senile dementia, ataxia, myasthenia gravis and Parkinson’s disease [15]. Compounds with anti-AChE activity also help to prevent pro-aggregating activity of AChE leading to the deposition of b-amyloid plaques, another cause of AD [16,17]. In our continuing effort to discover new bioactive compounds from ethno-medicinally important plants [18–22], we collected the bark of B. macowanii from South Africa based on its reported ethno-medicinal properties. Its crude methanolic extract was active in our AChE inhibition assay with an IC50 value of 30 lg/mL. Our phytochemical studies on the methanolic extract of this plant afforded five new steroidal alkaloids, 31-hydroxybuxatrienone (1), macowanioxazine (2), 16a-hydroxymacowanitriene (3), macowanitriene (4), and macowamine (5), along with five known compounds, Nb-demethylpapillotrienine (6), moenjodaramine (7), irehine (8), buxbodine B (9) and buxmicrophylline C (10). Compounds 1–10 were identified by extensive NMR and mass spectral studies. Steroidal alkaloids 1–10 exhibited moderate to weak anti-AChE activity. As mentioned previously, AChE inhibitors also prevent the deposition of b-amyloid plaques in the brain. The

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C.W. Lam et al. / Steroids 95 (2015) 73–79

aspartic protease b-secretase (BACE1) is reported to be involved in the formation of b-amyloid oligomers and insoluble plaques in the brain of AD patient [23]. BACE1 Inhibitors can also serve as possible therapeutic strategy to cure AD [24]. Based on the involvement of this enzyme in the deposition of b-amyloid plaques in the brain, we also evaluated compounds 1–10 for their potential to inhibit the activity of BACE1. In this assay, compounds 1 and 6 were found to be moderately active. This paper describes the isolation and structure elucidation of steroidal alkaloids 1–10 as well as their anti-AChE and anti-BACE1 activities data.

Fraction F1 was subjected to preparative TLC (PTLC) using hexanes–acetone–diethylamine (95:5:2) to afford compounds 1 (3.1 mg) and 9 (4.7 mg). Compounds 3 and 8 were purified by performing PTLC on fraction F2 using hexanes–dichloromethane– diethylamine (80:20:2) as a mobile phase. Compounds 4, 7 and 10 were purified from fraction F3 by carrying out PTLC using hexane–acetone–diethylamine (90:10:1) as mobile phase. Fraction F4 was also subjected to PTLC using hexane–diethylamine (100:2) as mobile phase to afford compound 2, 5 and 6. 2.4. 31-Hydroxybuxatrienone (1)

2. Experimental section 2.1. General experimental procedures 1

H NMR experiments including 1D and 2D were recorded with a Bruker Avance-3 spectrometer at 400 MHz. 13C NMR spectra were acquired on the same instrument at 100 MHz. IR spectra were obtained with Varian 1000 FT-IR and UV spectra were acquired with a Shimadzu UV-2501PC spectrophotometer. Mass spectral studies were carried out on a Hewlett-Packard 5989B MS. The high-resolution mass spectra were recorded on Agilent mass spectrometer at the University of Mississippi. AChE inhibitory activity was measured spectrophotometrically using an ELISA microplate reader (VersaMax, Molecular Devices, USA). Optical rotations were measured on an Autopol-1V automatic polarimeter. Column chromatography was carried out on silica gel 60 (40–63 lm). 2.2. Plant material B. macowanii was collected from Umtamvuna Nature Reserve in South Africa in September 2007 by one of the authors (R.M.G.). The plant was identified by Mkhiphenj A Ngwenya, and a voucher specimen (NH132295-0) was deposited in the South Africa National Biodiversity Institute, Durban, South Africa.

White, amorphous solid (3.1 mg); m.p. 179–183 °C (crystallized in acetone) [a]20 D + 15.4 (c 0.3, CHCl3); UV (MeOH) kmax 332 and 234 nm; IR (KBr) mmax 3337, 2945 and 1653 cm1; 1H NMR (CDCl3, 400 MHz) (see Table 1); 13C NMR APT (CDCl3, 100 MHz) (see Table 1); HRTOFMS m/z 398.3004 [M++H] (calcd. for C26H39NO2, 397.2981). 2.5. Macowanioxazine (2) White, amorphous solid (14 mg); m.p. 183–185 °C (crystallized in acetone); [a]20 D  36.1 (c 0.21, CHCl3); UV (MeOH) kmax 246 and 236 nm; IR (KBr) mmax 3276, 2946 and 1099 cm1; 1H NMR (CDCl3, 400 MHz) (see Table 1); 13C NMR APT (CDCl3, 100 MHz) (see Table 1); HRTOFMS m/z 443.3566 [M++H] (calcd. for C28H46N2O2, 442.3559). 2.6. 16a-Hydroxymacowanitriene (3) White, amorphous solid (4.2 mg); m.p. 193–195 °C (crystallized in methanol); [a]20 D  28.7 (c 0.16, CH3OH); UV (MeOH) kmax 289 nm; IR (KBr) mmax 3554, 2927 and 1168 cm1; 1H NMR (CD3OD, 400 MHz) (see Table 2); 13C NMR APT (CD3OD, 100 MHz) (see Table 2); HRTOFMS m/z 441.3522 [M++H] (calcd. for C28H44N2O2, 440.3403). 2.7. Macowanitriene (4)

2.3. Extraction and isolation The bark of B. macowanii (9.6 kg) was grinded to powdered form and extracted with methanol at room temperature. The solvent was removed in vacuo to afford a gummy residue (1.4 kg). This gum was again dissolved in 80:20 (v/v) methanol–water. This aqueous methanolic extract was defatted with hexanes. The defatted extract was subjected to solvent–solvent partitioning with dichloromethane at pH 3.5, 7.0 and pH 9.5. The pH values 3.5 and 9.5 of the aqueous alcoholic extract were adjusted by using 10% solutions of HCl and NaOH, respectively. The dichloromethane extracts obtained at pH 3.5, 7.0 and 9.5 exhibited anti-AChE activity with IC50 values of 50, 160 and 22 lg/mL, respectively. In order to isolate anti-AChE compounds, the dichloromethane extract (112 g), obtained at pH 9.5, was loaded onto a silica gel column, and the column was eluted with hexane–dichloromethane (0– 100%) followed by dichloromethane–methanol (0–100%). It afforded several fractions which were pooled on the basis of the same Rf values on analytical TLCs to afford 15 fractions F1–F15. All of these fractions were evaluated for anti-AChE activity and fractions F1–F4 were found to be active in this assay. Fraction F1 was obtained on the elution of the silica gel column using dichloromethane–hexanes (55:45 v/v) as a mobile phase. Fraction F2 was obtained on elution of the silica gel column using two mobile phases: dichloromethane–hexane (95:5 v/v) and dichloromethane–methanol (99:1, v/v). Fraction F3 was obtained from the silica gel column on elution with dichloromethane–methanol (90:10, v/ v), and elution of the silica gel column with a mobile phase (dichloromethane–methanol, 85:15) afforded fraction F4.

Yellow, amorphous solid (27 mg); m.p. 185–187 °C (crystallized in toulene); [a]20 D  12.3 (c 0.21, CHCl3); UV (MeOH) kmax 290 nm; IR (KBr) mmax 2957, 1662 and 1215 cm1; 1H NMR (CDCl3, 400 MHz) (see Table 3); 13C NMR APT (CDCl3, 100 MHz) (see Table 3); HRTOFMS m/z 425.3450 [M++H] (calcd. for C28H44N2O, 424.3467). 2.8. Macowamine (5) White, amorphous solid (27 mg); m.p. 225–228 °C (crystallized in acetone); [a]20 D + 107 (c 0.09, CHCl3); UV (MeOH) kmax 218 nm; IR (KBr) mmax 3383, 2945 and 1667 cm1; 1H NMR (CDCl3, 400 MHz) (see Table 3); 13C NMR APT (CDCl3, 100 MHz) (see Table 3); HRTOFMS m/z 567.4105 [M++H] (calcd. for C35H54N2O4, 566.4084). 2.9. Nb-demethylpapillotrienine (6) White amorphous solid (11 mg); m.p. 180–1183 °C (crystallized 20 in toluene); [a]20 D 58 (c 0.47, CHCl3; lit. 31: [a]D + 62, CHCl3). 2.10. Moenjodaramine (7) White crystalline solid (8.1 mg); m.p. 175–178 °C (crystallized 20 in heptane); [a]20 D + 34 (c 0.33, CHCl3; lit. 32–33: [a]D + 33.3, CHCl3). 2.11. Irehine (8) Colorless crystalline material (6 mg); m.p. 20 [a]20 D + 38 (c 1.0, CHCl3; lit. 2: [a]D + 40, CHCl3).

148–151 °C;

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C.W. Lam et al. / Steroids 95 (2015) 73–79 Table 1 H and 13C NMR assignments for 31-hydroxybuxatrienone and macowanioxazine in CDCl3.

1

Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 30 31 32 33 Nb(CH3)2 Na–CH3

31-Hydroxybuxatrienone (1)

Macowanioxazine (2)

dC(APT)

dH (J in Hz)

dC(DEPT)

dH (J in Hz)b

153.4, CH 123.3, CH 204.8, C 50.7, C 43.2, CH 28.2, CH2 26.2, CH2 48.5, CH 133.4, C 139, C 137.0, CH 38.9, CH2 44.7, C 48.3, C 33.1, CH2 27.0, CH2 47.3, CH 15.9, CH3 143.8, CH 65.3, CH 12.4, CH3 15.8, CH3 63.9, CH2 17.0, CH3 – 36.0, CH3 –

6.96, d (9.9) 5.81, d (9.9) – – 2.98, m 1.49, m 1.81, m, 1.41, m 2.20, m – – 5.95, br, s 2.30, m, 2.17, m – – 1.64, m,1.43, m 2.06, m 2.03, m 0.84, s 6.49, s 3.27, br, s 1.33, d (5.8) 0.90, s 4.06, d (11.7), 3.52, d (11.7) 0.76, s – 2.90a, s, 2.70a, s –

39.3, CH2 25.9, CH2 71.1, CH 39.2, C 48.4, CH 28.7, CH2 25.3, CH2 49.3, CH 135.0, C 138.2, C 128.2, CH 38.4, CH2 43.7, C 46.5, C 44.8, CH2 69.4, CH 51.4, CH 16.7, CH3 129.7. CH 60.1, CH 10.8, CH3 13.9, CH3 77.9, CH2 17.5, CH3 88.4, CH2 36.4, CH3b 36.4, CH3b

2.25, m 1.89, m, 1.43, m 1.91, m – 1.97, m 1.87, m, 1.37, m 1.50, m, 1.21, m 2.15, m – – 5.50, br, s 2.11, m, 1.96, m – – 2.09, m, 1.60, m 4.65, m 1.94 m 0.94, s 5.95, s 3.49, m 1.20, d (6.5) 1.02, s 3.82, d (10.4), 3.25, d (10.4) 0.65, s 4.42, d (7.6), 3.57, d (7.6) 2.64, s 2.10, s

Multiplicity was determined by APT spectrum. a Signals collapsed into a singlet when the 1H NMR spectrum as recorded in acetone-d6. b Signals overlapped.

Table 2 H and 13C NMR assignments for 16a-hydrixymacowanitriene and macowanitriene.

1

Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 30 31 32 33 Nb(CH3)2 Na–CH3

16a-Hydrixymacowanitriene (3)a

Macowanitriene (4)b

dC(APT)

dH (J in Hz)

dC(DEPT)

dH (J in Hz)

124.0, CH 137.3, CH 71.6, CH 39.1, C 50.1, CH 28.4, CH2 29.2, CH2 50.1, CH 135.0, C 140.2, C 134.4, CH 39.6, CH2 45.4, Cc 45.4, Cc 34.4, CH2 71.6, CH 49.9, CH 16.2, CH3 137.8, CH 49.9, CH 11.2, CH3 13.6, CH3 78.3, CH2 17.9, CH3 89.0, CH2 36.7, CH3c 36.7, CH3c

5.70, 6.08, 2.84,  2.15, 1.30, 1.87, 2.15, – – 5.77, 2.29, – – 1.59, 3.64, 2.10, 0.82, 6.13, 2.10, 1.16, 0.97, 3.88, 0.80, 4.44, 2.22, 2.56.

123.9, CH 135.6, CH 69.9, CH 38.0, C 48.6, CH 27.0, CH2 27.1, CH2 48.4, CH 134.0, C 138.9, C 132.8, CH 38.6, CH2 44.0, Cc 44.4, Cc 33.3, CH2 27.7, CH2 48.6, CH 15.8, CH3 136.2, CH 48.4, CH 11.1, CH3 13.4, CH3 77.2 CH2 17.3, CH3 88.1, CH2 36.3, CH3c 36.3, CH3c

5.67, 6.04, 2.79,  2.09, 2.11, 2.10, 1.94, – – 5.72, 2.21, – – 1.52, 1.82, 2.09, 0.76, 6.08, 1.94, 1.03, 0.99, 3.89, 0.73, 4.47, 2.22, 2.42,

d (10.0) dd (10.0, 2.6) m m m, 1.78, m m, 1.35 m

br. s m, 2.13, m

m, 1.24, m m m s s m d (5.8) s d (10.6), 3.33, d (10.6) s d (8.1), 3.64, d (8.1) s s

Multiplicity of carbon signals of compounds 3 and 4 was determined by APT and DEPT spectra, respectively. a NMR data was acquired in CD3OD. b NMR spectra was recorded in CDCl3. c Signals overlapped.

d (10.1) dd (10.1, 2.6) m m m, 1.75, m m, 1.75, m m

br. s m, 2.06, m

m, 1.15, m m, 1.27, m m s s m d (6.1) s d (10.5), 3.27, d (10.5) s d (8.1), 3.67, d (8.1) s s

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C.W. Lam et al. / Steroids 95 (2015) 73–79

Table 3 H and 13C NMR assignments for macowamine in CDCl3.

1

Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 30 31 32 OCNCH3 NaCH3 Nb(CH3)2 OCH3 10 20 30 40 50 60

Macowamine (5) dC(APT)

dH (J in Hz)

22.1, CH2 27.7, CH2 69.2, CH 38.0, C 54.3, CH 20.9, CH2 30.8, CH2 39.8, CH 141.3, C 28.7, CH 120.2, CH 37.4, CH2 42.9, C 47.2, C 33.6, CH2 26.9, CH2 49.0, CH 14.6, CH3 43.4, CH2 61.8, CH 9.98, CH3 17.7, CH3 62.6, CH2 18.5, CH3 166.1, C 34.3, CH3 41.3, CH3 55.9, CH3 122.2, C 111.8, CH 146.3, C 150.3, C 114.1, CH 124.0, CH

1.96, m, 1.67, m 1.73, m, 1.45, m 2.26, m – 1.16, m 1.79, m, 1.53, m 2.30, m, 1.27, m 2.25, m – 1.19, m 5.44, br. s 2.16, m, 1.84, m – – 1.43, m, 1.37, m 1.90, m, 1.56, m 1.86, m 0.48, s 2.48, m, 2.26, m 2.48, m 0.835, d (6.5) 0.98, s 4.50, d, (11.3), 4.42, d (11.3) 0.80, s – 2.58, s 2.23, s 3.91, s – 7.47, d (1.7) – – 6.88, d (8.2) 7.49, dd (8.2, 1.7)

2.12. Buxbodine B (9) White amorphous solid (27 mg); m.p. 221–222 (crystallized in ethyl acetate); [a]20 D + 17 (c 0.19, CHCl3) (lit. 39). 2.13. Buxmicrophylline C (10) White needles (7 mg); m.p. 227–232 °C; [a]20 D + 60 (c 0.4, CHCl3; lit.40). 2.14. AChE inhibition assay AChE inhibitory activity of 1–10 was determined by our previously reported procedure [2,5,25]. Briefly, the assay was carried out at room temperature in 100 mM sodium phosphate buffer at pH 7.8. In a typical assay, 126 lL of buffer, 50 lL of 0.01 M DTNB [5,50 -dithiobis(2-nitrobenzoic acid)], 2 lL of enzyme, and 2 lL of solutions containing test compounds (1–10) were mixed and incubated for 30 min. The reaction was then initiated by the addition of 20 lL of 0.075 M acetylthiocholine. Hydrolysis of acetylthiocholine was monitored by the formation of 5-thio-2-nitrobenzoate anion at a wavelength of 406 nm. All assays were carried out in triplicate using a 96-well microplate reader. The percentage inhibition was calculated using the formula [(A0  A1/A0]  100, where A0 is the absorbance of the blank with no test compound and A1 is the absorbance value of each concentration of the test compounds. The IC50 values were calculated by plotting a concentration– response curve. The enzyme inhibition kinetic studies were carried out using the five different concentrations of substrate and inhibitors (1–10) using aforementioned bioassay procedure. The Lineweaver–Burk plot was used to determine the mode of inhibition.

2.15. BACE1 inhibition assay This assay was carried out according to the provided manual with slight modifications [26,27].

3. Results and discussion 3.1. Structure elucidation of compounds 1–10 Our first compound, 31-hydroxybuxatrienone (1), was isolated as a white amorphous solid. Its UV spectrum showed absorption maxima at 332 and 234 nm suggesting the presence of a D1,2 9(10 ? 19) abeo trienone chromophore. The IR spectrum displayed absorption bands at 3337 (OH), 2945 (CH), and 1653 (C@O) cm1. Compound 1 showed the [M+H]+ in the high-resolution time-offlight mass spectrum (HR-TOF-MS) at m/z 398.3004, appropriate for a molecular formula C26H39NO2, suggesting the presence of eight degrees of unsaturation. The 1H NMR spectrum (CDCl3, 400 MHz) of 1 exhibited three three-proton singlets at d 0.76, 0.84, and 0.90 due to C-32, C-18, and C-30 methyl protons, respectively. A doublet at d 1.33 (3H, J = 5.8 Hz) was assigned to the C-21 secondary methyl protons. The N, N-dimethyl protons appeared as two 3H singlets at d 2.70 and 2.90. These signals were collapsed into a 6H singlet (d 2.76) when the 1H NMR spectrum of 1 was recorded in acetone-d6. A set of two AB doublets, integrating for one protons each, centered at d 3.52 and 4.06 (J = 11.7 Hz) were due to the C-31 methylene protons. Their down-field chemical shift values suggested the presence of a geminal hydroxyl group. Four olefinic signals at d 5.81 (d, J = 9.9 Hz), 5.95 (br. s), 6.49 (s) and 6.96 (d, J = 9.9 Hz) were ascribed to the C-2, C-11, C-19 and C-1 methine protons, respectively. The COSY-45° spectrum 1 also confirmed the above mentioned 1H NMR chemical shift assignments as it showed vicinal couplings between H-1 (d 6.96) and H-2 (d 5.81). H-19 (d 6.49) showed cross-peaks with H-1 (d 6.96), H-2 (d 5.81) and H-11 (d 5.95). The C-11 methine proton also showed cross-peaks with the C-12 methylene protons (d 2.30 and 2.17). A combination of UV, 1H NMR and COSY spectral data confirmed the presence of the D1,2 9(10 ? 19) abeo triene system in 1. The 13C NMR spectrum (CDCl3, 100 MHz) showed the resonance of 25 carbons with one overlapping resonance due to N(CH3)2 carbons at d 36.0, indicating the presence of 26 carbons in 1. These resonances were differentiated as 5 methyl, 6 methylene, 8 methine, and 6 quaternary carbons with the aid of attached proton test (APT) experiment. Complete 1H and 13C NMR chemical shift assignments, as well as the 1H/13C one-bond shift correlations of 1, as determined from HSQC spectrum are shown in Table 1. The HMBC spectrum of 1 showed couplings of H2-31 (d 3.52 and 4.06) with C-3 (d 204.8) and C-4 (d 50.7). These HMBC interactions suggested the presence of a carbonyl group at C-3. Important HMBC interactions of 1 are shown in Fig. 1. The relative stereochemistry at all chiral centers was established with the aid of NOESY spectrum. The C-5 methine proton (d 2.98) showed an NOE with the C-32 methyl protons (d 0.76) which in turn showed cross-peaks with the C-17 methine proton (d 2.03). It has already been reported in the literature that H-5, H-17 and H3-32 have a-orientation in Buxus alkaloids [28–30]. Based on the cis relationships between H-5, H-17 and H3-32, as indicated by the NOESY spectrum, a-stereochemistry for H-5, H-17 and H3-32 was assigned. The C-30 methyl protons (d 0.90) showed cross-peaks with H-8 (d 2.20). The latter also showed an NOE with the C-18 methyl protons (d 0.84). As reported previously, H-8 and H3-18 have invariably b-orientation in Buxus alkaloids [28–30]. Based on these NOESY data, we assumed b-stereochemistry for H-8, H3-18, and H3-30. It has also been

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C.W. Lam et al. / Steroids 95 (2015) 73–79

H3 C 18

11

19 1

10

2 3

O

5

9

H3C30

12

13

H

20

17

14

Nb

H3C

CH3

16

H

15

32

H3C

Nb

CH3

CH2OH

CH3

1 N

2 R = OH 7R=H

H

CH3 H3C CH3

CH3

N

HO

3'

O

2'

4'

1'

5'

H

C

H

N H 3C CH3 H2C

6'

CH3

N O

2

CH3

N

OH

CH3

CH3 H3 C

N

CH3

CH3

N

CH3

O 9

N H

CH3

CH3

C CH3

H

CH3

N H3 C

N

O

CH3

4 N

OH

H O

H3 C

N

CH3

H3C

OH

H

3

H

8

CH3 H3C

O

HO

N 6

N

CH3

H

CH3

CH3

N

CH3

R

H H

H3C

H

CH3

CH2 OH 10

OH

H

5

4R=H

OH

H

H H3CO

Na HC H2C 3 CH2 O 3 R = OH

CH3

O

R

H3 C

H3 C

R

D

CH3

H

H

H

Na E H3 C CH2 O

CH3 H3C

H H3C

CH3

H 2C

OH CH 31 2 1

H3 C

C

H

A

CH3

7

6

Nb

CH3

B

8

H

4

CH3

N

CH3

CH3

21

H3CO HO

H O C N CH2OH 5

Fig. 1. Important HMBC interactions of compounds 1–5.

reported in the literature that C-4a methyl undergoes oxidation [1,28–30]. Based on these biogenetic consideration and NOESY spectral data, a- stereochemistry was proposed for the C-31 methylene group. These spectral data led us to propose structure 1 for this new alkaloid and named as 31-hydroxybuxatrienone. Our second compound, macowanioxazine (2), was isolated as a white amorphous solid. Its UV spectrum showed maximum absorptions at 236 and 246 nm suggesting the presence of a 9(10 ? 19) abeo diene system [28,29]. The IR spectrum displayed absorption bands at 3276 (OH), 2946 (CH), and 1099 (C–O) cm1. The HRTOF-MS showed the [M+H]+ ion at m/z 443.3566 that was in agreement with the molecular formula, C28H46N2O2. The 1H and 13C NMR data of 2 (Table 1) revealed that compound 2 was a Buxus alkaloid containing 9(10 ? 19) abeo diene system, a C-16/hydroxyl group and tetrahydrooxazine ring E. It was evidenced by the presence of four sp2 hybridized signals [C-9 (dC 135.0), C-11 (dC/H 128.2/5.50), C-10 (dC 138.2), and C-19 (dC/H 129.7/5.95)] and three down-field sp3 hybridized signals [C-16 (dC/H 69.4/4.65), C-31 (dC/H 77.9/3.82 and 3.25), and C-33 (dC/H 88.4/4.42 and 3.57)]. H-16 showed COSY interactions with the C-15 methylene (d 2.09 and 1.60) and C-17

methine (d 1.94) protons. The latter showed cross-peaks with the C-20 methine proton (d 3.49) which in turn exhibited vicinal couplings with the C-21 methyl protons (d 1.20). The HMBC spectrum of 2 showed couplings of H-33 (d 4.42 and 3.57) with C-3 (d 71.1) and C-31 (d 77.9) while H-31a exhibited long-range heteronuclear couplings with C-3 (d 71.1), C-4 (d 39.2) and C-30 (d 13.9). These HMBC interactions supported the presence of a tetrahydrooxazine ring E in 2. Important HMBC interactions of 2 are shown in Fig. 1. The NOESY spectrum indicated that chiral centers (C-4, C-5, C-8, C-13, C-14 and C-20) in compound 2 have the same stereochemistry as we have assigned for compound 1. H-16 (d 4.65) showed an NOE with C-18 methyl protons (d 0.94) suggesting the a-orientation of C-16/OH. H-3 (d 1.91) exhibited an NOE with H-5 (d 1.97) indicating a-orientation of H-3. A combination of UV, IR, MS and NMR spectral data helped us to establish structure 2 for this new alkaloid. The third compound, 16a-hydroxymacowanitriene (3), was isolated as a white amorphous solid and exhibited the molecular ion [M++H] peak at m/z 441.3522 that provided the molecular formula C28H44N2O2. Its UV spectrum showed maximum absorption at

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289 nm suggesting the presence of a D1,2 9(10 ? 19) abeo triene system [31]. The IR spectrum of 3 was identical to that of previously discussed compound 2 suggesting the presence of same functional groups. The NMR data (Table 2) of 3 was also similar to those of previously discussed compound 2 with the exception of olefinic resonances due to C-1 (dC/H 124.0/5.70) and C-2 (dC/H 137.3/6.08). The COSY-45° spectrum of 3 showed vicinal couplings between H-1 and H-2. These signals also showed vinylic couplings with H-19 (d 6.13) and H-11 (d 5.77) in the TOCSY spectrum. H-2 (d 6.08) exhibited vicinal coupling with the C-3 methine proton (d 2.84). Important HMBC interactions are shown in Fig. 1. The NOESY spectrum of 3 suggested the chiral centers (C-3, C-4, C-5, C-8, C-13, C-14, C-16, C-17, and C-20) had the same relative configurations as those of previously discussed compound 2. Based on these spectral data, structure of 3 was assigned to this new alkaloid and named as 16a-hydroxymacowanitriene. The fourth compound, macowanitriene (4), was isolated as a yellow amorphous solid. Its UV and IR spectra were similar to those of compound 3, with one exception that IR spectrum of 4 did not exhibit an absorption bands due to a hydroxyl group. The HR-TOF-MS showed a molecular ion peak [M++H] at m/z 425.3450 which provided the molecular formula, C28H44N2O. The NMR spectral data of 4 (Table 2) was similar to those of compound 3 with one exception that NMR spectra of 4 showed the absence of downfield signals due to the hydroxyl-bearing C-16. Instead, an up-field sp3 hybridized signal due to C-16 (dC/H 27.7/1.82 and 1.27) was observed. Important HMBC interactions of 4 are shown in Fig. 1. These spectral data led us to propose structure 4 for this new alkaloid and named as macowanitriene. Our fifth compound, macowamine (5), was isolated as a white amorphous solid. Its UV spectrum showed maximum absorption at 218 nm suggesting the presence of a benzamide chromophore [1–3,28–30]. The IR spectrum displayed absorption bands at 3383 (OH), 2945 (CH), and 1667 (amide C@O) cm1. The HR-TOF-MS showed a molecular ion peak at [M++H] at m/z 567.4105 which provided its molecular formula, C35H54N2O4. The NMR spectral data (Table 3) showed that compound 5 was also a Buxus alkaloid consisted of C-3/40 -hydroxy-30 -methoxybenzamide moiety, C-31/ hydroxyl group and 9(10 ? 19) abeo system with a double bond D9-11. The NMR spectral data featured three methine aromatic signals [C-20 (dC/H 111.8/7.47), C-50 (dC/H 114.1/6.88), C-60 (dC/H 124.0/ 7.49)], three quaternary aromatic signals [C-10 (dC 122.2), C-30 (dC 146.3) C-40 (dC 150.3)], two olefinic resonances [C-11 (dC/H 120.2/ 5.44) and C-9 (dC 141.3)], and one down-field sp3 hybridized signal due to C-31 (dC/H 62.6/4.50 and 4.42). The 1H and 13C NMR spectra also featured Na-CH3 signal at dC/H 34.3/2.58. The HMBC spectrum of 5 showed the cross-peaks of Na-methyl protons (d 2.58) with C-3 (d 69.2) and amide carbonyl carbon (d 166.1). The C-3/O-methyl protons (d 3.91) exhibited coupling with C-30 (d 146.3). H-20 (d 7.47) and H-60 (d 7.49) also showed HMBC interactions with amide carbonyl carbon (d 166.1). These HMBC spectral data indicated the substitution of a 40 -hydroxy-30 -methoxybenzamide group at C-3. Important HMBC interactions of 5 are shown in Fig. 1. The methoxy protons (d 3.91) showed an NOE with the C-20 methine proton (d 7.47) suggesting its substitution at C-30 . The NOESY spectrum of 5 indicated the same stereochemistry at all chiral centers as those of previously discussed compounds (1–4) and other reported Buxus alkaloid [1–5,28–30]. H-10 (d 1.19) showed NOE with H-8 (d 2.25) and H3-30 (d 0.98) suggesting its b-orientation. These spectral studies led to assign structure 5 to this new Buxus alkaloid and named as macowanine. The known alkaloid were characterized as Nb-demethylpapillotrienine (6), moenjodaramine (7), irehine (8), buxbodine B (9) and buxmicrophylline C (10) which were identified by comparing their 1 H, 13C NMR, UV, IR, and mass spectra with those of reported in the literature [31–38]. Compound 6 was previously isolated from the

Table 4 Anti-AChE activity result of compounds 1–10. Compounds

AChE (IC50 lM)a

1 2 3 4 5 6 7 8 9 10 Galanthamine

17.0 ± 1 32.5 ± 3 11.4 ± 2 10.8 ± 2 45 ± 4 19.0 ± 2 27 ± 1 98 ± 1.8 50 ± 1 20 ± 1 0.8 ± 0.2

a IC50 represents the concentration required to inhibit 50% of the enzyme activity. Galanthamine was used as a positive control. ‘‘±’’ represents the standard error of mean of these enzyme inhibition assays.

root of B. papillosa [31] while compound 7 was purified from B. papillosa [32–34,37], Buxus hildebrandtii [35], Buxus rugulosa [36] and B. hyrcana [3,38], and compounds 8, 9 and 10 were previously reported from B. hyrcana [2], Buxus bodinieri [39] and B. microphylla [40], respectively. 3.2. Enzyme inhibitory activities Compounds 1–10 were evaluated for their anti-AChE potentials, and AChE inhibition data are shown in Table 4. Compounds 3 and 4 exhibited higher anti-AChE activity compared to the rest of the isolated compounds (1–2, 5–10). The AChE inhibition kinetic studies on compounds 1–10 indicated that compounds 3 and 4 were competitive inhibitors while compounds 1–2 and 5–10 were noncompetitive inhibitors. Compounds 1–10 were also evaluated for their potential to inhibit the BACE1 activity and only compounds 1 and 6 were found to be active in this assay with IC50 values of 15.0 and 25.0 lM, respectively. Isolates 2–5 and 7–10 were inactive in this bioassay at a concentration of 250 lg/mL. 4. Conclusion In summary, our phytochemical studies on B. macowanii afforded five novel steroidal alkaloids (1–5) along with five known steroidal bases (6–10). Compounds 1, 3 and 4 belong to the rarely occurring class of Buxus alkaloids containing D1,2 9(10 ? 19) abeo triene system as only four compounds of this series have so far been reported in the literature [31,37,38]. Furthermore, compounds 3 and 4 consisted of tetrahydrooxazine ring E incorporated in their structures. To the best of our knowledge, this is the first report on compounds containing D1,2 9(10 ? 19) abeo triene system and tetrahydrooxazine ring E, from genus Buxus. The presence of these compounds in B. macowanii might be used as chemotaxonomic markers for the identification of this plant. Additionally, compounds 3 and 4 have shown strong anti-AChE activity compared to compounds 1–2 and 5–10. Further structure–activity relationship studies on compounds 3 and 4 are warranted in order to improve their bioactivity and to determine the active pharmacophore, responsible for the expression of this bioactivity. Acknowledgments Funding for this research was provided by the Manitoba Health Research Council, Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Winnipeg. A.A. is thankful to the Department of Chemistry, University of Manitoba, for granting him adjunct status.

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