Isoquinoline Alkaloids from Berberis vulgaris as Potential Lead

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Isoquinoline Alkaloids from Berberis vulgaris as Potential Lead Compounds for the Treatment of Alzheimer’s Disease Anna Hostalkova,† Jana Marikova,‡ Lubomir Opletal,† Jan Korabecny,§,⊥ Daniela Hulcova,†,∥ Jiri Kunes,‡ Lucie Novakova,# Daniel I. Perez,▽ Daniel Jun,§,⊥ Tomas Kucera,§ Vincenza Andrisano,○ Tomas Siatka,∥ and Lucie Cahlikova*,†

J. Nat. Prod. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/31/19. For personal use only.

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ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic ‡ Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic § Department of Toxicoloxy and Military Pharmacy, Trebesska 1575, 500 05 Hradec Kralove, Czech Republic ⊥ Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic ∥ Department of Pharmacognosy, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic # Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic ▽ Centro de Investigaciones Biologicas-CSIC, Avenida Ramiro de Maeztu 9, 28040 Madrid, Spain ○ Department for Life Quality Studies, University of Bologna, Corso D’Augusto 237, 47921 Rimini, Italy S Supporting Information *

ABSTRACT: Three new alkaloids, bersavine (3), muraricine (4), and berbostrejdine (8), together with seven known isoquinoline alkaloids (1−2, 5−7, 9, and 10) were isolated from an alkaloidal extract of the root bark of Berberis vulgaris. The structures of the isolated compounds were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRMS, and optical rotation, and by comparison of the obtained data with those in the literature. The NMR data of berbamine (5), aromoline (6), and obamegine (7) were completely assigned employing 2D NMR experiments. Alkaloids isolated in sufficient amounts were evaluated for their in vitro acetylcholinesterase, butyrylcholinesterase (BuChE), prolyl oligopeptidase, and glycogen synthase kinase-3β inhibitory activities. Selected compounds were studied for their ability to permeate through the blood−brain barrier. Significant human BuChE (hBuChE) inhibitory activity was demonstrated by 6 (IC50 = 0.82 ± 0.10 μM). The in vitro data were further supported by computational analysis that showed the accommodation of 6 in the active site of hBuChE.

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cholinergic hypothesis, it was postulated that a deficit of the neurotransmitter acetylcholine (ACh) in the cortex aggravates cognitive performances.4 Accordingly, ACh levels can be maintained via the inhibition of acetylcholinesterase (AChE). Such an approach is an effective aid in the amelioration of the AD symptoms.5 Indeed, several drugs have been approved defined upon this hypothesis, like donepezil, galantamine, and rivastigmine.6 Similar to AChE, another cholinesterase, butyrylcholinesterase (BuChE; E.C. 3.1.1.8), can also terminate the action of ACh. Decreased AChE activity is typical in the later stages of AD in contrast with BuChE levels, which remain either normal or even elevated in the brain.7 Beyond ACh inactivation, cholinesterases play an important role in

lzheimer’s disease (AD), a neurodegenerative disorder, is the leading cause of dementia among the elderly. During the progression of this disease, the number of neurons is reduced significantly, which leads to gradual cognitive loss, inability to perform routine tasks, difficulty in learning, loss of language skills, and personality changes.1 The disease itself is becoming a slow pandemic, and it is expected that there will be more than 130 million sufferers by 2050.2,3 Given the multifaceted nature of AD, several hypotheses, including cholinergic, amyloid beta (Aβ), hyperphosphorylation of τprotein, calcium dyshomeostasis, and oxidative stress, have been put forward.2 From a pathological point of view, the most pronounced selective cholinergic neuronal loss is accompanied by the formation of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated τ-protein and extracellular senile plaques (SPs) formed by Aβ.1 Focused on this © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 19, 2018

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DOI: 10.1021/acs.jnatprod.8b00592 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

barberry”, which is a widespread species found from Central Europe to northwest Iran and that is also used as a food additive.18,19 Various parts of this plant have been used extensively in Iranian traditional medicine for their antibacterial, antipyretic, antipruritic, and antiarrhythmic properties.18,20 More than 20 isoquinoline alkaloids of various structural types have already been isolated from B. vulgaris. Previous phytochemical studies led to the isolation and characterization of alkaloids such as benzylisoquinolines, protoberberines, bisbenzylisoquinolines, quaternary protoberberines, and others.21−23 Prior phytochemical investigations of common barberry led to the isolation and identification of new isoquinoline dimers and secobisbenzylisoquinolines with interesting biological profiles.24,25 The major component and the most studied alkaloid from Berberis species is berberine, a quaternary alkaloid. Berberine has multiple therapeutic actions, and its potential use has been described in various disorders.26 Clinical trials have reported antihyperlipidemia properties, insulin-sensitizing action, beneficial effects on the cardiovascular system, and others, with a favorable safety profile. Berberine also improves cognitive impairment by promoting autophagic clearance and inhibiting the production of Aβ in the amyloid precursor protein (APP)/τ-protein/presenilin 1 mouse model of AD.27 On the contrary, some limitations have been reported given by its pharmacological interaction with other targets and low oral availability due to poor absorption through the gut wall.20 Therefore, tertiary uncharged alkaloids are the focus of this work because they usually display a better ability to pass through biological membranes.

their noncholinergic function; for example, AChE has been shown to act as a pathological chaperone inducing a conformational transition of Aβ.8 This fact makes AChE a more attractive target in AD.9 Besides AChE/BuChE inhibitors, the N-methyl-D-aspartate (NDMA) receptor antagonist memantine is currently used for AD treatment, reducing glutamate excitotoxicity.10 In recent years, the cytosolic serine peptidase, prolyl oligopeptidase (POP; E.C. 3.4.21.26), has gained importance as a target for the treatment of neuropsychiatric and neurodegenerative diseases, such as schizophrenia, bipolar affective disorder, Parkinson’s disease, and AD.11 POP has been studied as a potential therapeutic target for many years, and selective inhibitors were discovered in the 1980s. The common catalytic preference of POP is the cleavage of prolylcontaining peptides smaller than 3 kDa at the C-terminus of proline residues.12 POP is also implicated in the metabolism of inositol-1,4,5-triphosphate (IP3), a key molecule in the transduction cascade of neuropeptide signaling. The effect of reduced POP activity on IP3 concentration indicates an intracellular function of this peptidase, which may have an impact on cognitive enhancements due to POP inhibition.13 In general, it can be regarded as one of the active factors in the processes of learning and memory. In fact, some POP inhibitors have been found experimentally to be potentially efficacious antidementia drugs.11 Another neuropathological characteristic of AD is the presence of NFTs consisting of paired helical filaments, with the main component being hyperphosphorylated τ-protein. The phosphorylation of τ-proteins is primarily dependent on glycogen synthase kinase 3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5).14 A number of studies have emerged describing diverse molecules that inhibit GSK-3β, such as manzamine alkaloids,15 pyrazolopyrimidines,16 pyridyloxadiazoles,17 and others. Nowadays, the portfolio of drugs used for treatment of AD is narrow, and the search for new and effective therapy of the disease is urgently needed.6 One of the interesting sources of potential active compounds for the treatment of AD are plants of the Berberis genus, especially B. vulgaris L. (Berberidaceae), known as “common

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RESULTS AND DISCUSSION Continuing with the search for novel neuroprotective constituents from medicinal plants, an alkaloidal extract of the root bark of B. vulgaris was investigated. Extensive chromatographic purification led to the isolation of three new (3, 4,and 8) and known isolated alkaloids (1, 2, 5−7, 9, and 10). The known compounds were identified by comparison with published spectroscopic and physical data as 8-oxoberberine (1),28 berbidine (2),29,30 berbamine (5),31,32 aromoline (6),33−35 obamegine (7),31,33,34 berberine (9),36 B



Article

and palmatine (10) (Chart 1).30 The isolated alkaloids belong to the protoberberine, bisbenzylisoquinoline, isoquinolone− isoquinoline, and isoquinoline−benzylisoquinoline structural types. The new compound 3, named bersavine, was obtained as yellowish crystals. The HRMS of 3 showed a protonated molecular ion peak [M + H]+ at m/z 694.3853, corresponding to the molecular formula C42H51N3O6 (694.3851 calcd for C42H52N3O6+). The 1H and 13C NMR data were quite similar to those for compound 5, which led to the assumption that 3 is structurally related to 5. All proton and carbon signals of 3 were completely assigned employing 2D NMR experiments such as gCOSY, gHSQC, gHMBC, and NOESY. In these experiments, two tetrahydroisoquinoline fragments were determined. One of them was found to be 1,6,7,8tetrasubstituted with two methoxy groups in positions 6 and 7 and with substituents in positions 1 and 8. The second one is 1,6,7-trisubstituted with a methoxy group in position 6 and two substituents at positions 1 and 7. These fragments are interconnected by an ether bridge. The deshielding effect of the oxygen atom on carbons C-8 (δC 149.3) and C-7′ (δC 145.0) and the NOESY correlation between H-8′ and the methyl protons of C-7−OCH3 supported the constitution of an ether linker. In a gCOSY experiment, the methylene protons of H-9 were correlated to H-1 as well as H-9′ with H1′. In the gHMBC spectrum, correlations between C-9 and C10, C-14 were acquired, as well as correlations between C-9′ and C-10′, C-14′. These sp2 carbons belonged to two aromatic rings, which were substituted differently and joined by an ether connection, a second one in this molecule. The constitution of a 1,4-disubstituted benzene ring was established by gCOSY and gHMBC spectra (Figure 1). There was a need to specify a

second ether bridge had to be located between deshielded carbons C-11 (δC 149.6) and C-12′ (δC 156.3). The configuration was investigated by NOESY and by comparing its optical rotation [α]D25+101.2 (c 0.134; CHCl3) with that of compound 5 ([α]D25 + 43.6 (c 0.101; CHCl3)). The absolute configuration of 3 was assigned as the (1R,1′S)-isomer. Muraricine (4) was obtained as a greenish amorphous solid. The molecular formula was determined to be C30H36N2O5 from the protonated molecular ion peak [M + H]+ found at m/ z 505.2698 (505.2697 calcd for C30H37N2O5+) in the positiveion HRMS. The 1H NMR data of 4 revealed the presence of seven aromatic protons in four signals. Three of them were singlets (δH 4.86, H-8; 6.47, H-5′; 6.66, H-5). Another two signals were assigned to a para-disubstituted aromatic ring (δH 6.65−6.60, m, AA′BB′, H-10; 6.58−6.63, m, AA′BB′, H-11, and H-13). The 13C NMR spectrum of 4 exhibited 28 carbon resonances assignable via gHSQC data to three methoxy groups (δC 55.9, C-6′; 56.1, C-6; 60.0, C-7′), two N−CH3 groups (δC 41.2, N-2; 45.0, N-2′), three methylene groups (δC 24.1, C-4; 27.7, C-4′; 39.3, C-9), three nitrogenated methylene groups (δC 44.9, C-3; 52.0, C-3′; 52.5, C-1′), one methine carbon (δC 64.1, C-1), five sp2 methine carbon atoms (δC 108.1, C-5′; 111.8, C-5; 115.1, C-8; 117.9, C-11; 130.8, C-10), six oxygenated quaternary sp2 carbon atoms (δC 138.8, C-7′; 144.1, C-8′; 144.2, C-7; 147.5, C-6; 152.1, C-6′; 156.2, C-12), and five quaternary sp2 carbons (δC 118.2, C-8a′, 125.3, C-4a; 125.6, C-8a; 128.7, C-4a′; 128.9, C-9a). The 2D NMR experiments, including gCOSY, gHSQC, and gHMBC, confirmed two N-methylated tetrahydroisoquinoline fragments with a different substitution (Figure 2). The central

Figure 2. Key gHMBC, 1H−1H gCOSY, and NOESY correlations of 4.

tetrahydroisoquinoline fragment was substituted at C-1, C-6, and C-7 and at a nitrogen. Carbon atom C-6 was substituted with a methoxy group. The gCOSY and gHMBC spectra were used to establish the location of C-9 as a methylene bridge between C-1 and C-9a. The carbon 9a belonged to the paradisubstituted benzene ring, which has an oxygenated quaternary sp2 carbon atom (δC 156.2, C-12). Therefore, this electronegative substituent on C-12 was recognized as a phenolic hydroxy group. Thus the interconnection of the specified fragments remained to be resolved. The deshielding effect of the electronegative oxygen atom on carbon C-7 (δC 144.2) and C-8′ (δC 144.1) supported an ether bridge between these sp2 carbons. This connection was substantiated by the NOESY data, which exhibited correlations between H-8 and H-1′ and H-8 and H-7′−OCH3. The constitution of this ether

Figure 1. 1H−1H gCOSY, key gHMBC, and NOESY correlations of 3. The deuterium-induced isotope effect is described by red numbers.

1,2,3,5-tetrasubstituted aromatic ring. The methylene group H15 protons were correlated to the carbons C-12 and C-14 in the gHMBC experiment. Thus carbon C-15 should have been bound to C-13 of a 1,2,3,5-tetrasubsituted benzene ring. Consequently, carbon C-15 belonged to the (diethylamino)methyl group. The constitution of this group was obtained from gHMBC and gCOSY spectra (Figure 1). The connection of the 1,4-disubstituted and 1,2,3,5-tetrasubstituted benzene rings was proven by a deuterium-induced isotope effect. Employing this effect, the position of a phenolic group on C-12 (δC 146.8; Δδ(C‑12′) = −0.33) was determined. Therefore, the C



Article

one methoxy group (δC 55.9) correlated to the deshielded quaternary sp2 carbon C-12 (δC 148.8) and one phenolic group. The remaining substituent of these rings was established as an ether bridge connecting two deshielded quaternary sp2 carbons (δC 145.0; 144.2). There were two possibilities for the interconnection. The ether bridge was bound to carbon C-11 (δC 145.0) by one bond. The second bond should be linked either to carbon C-11′ (δC 144.2) or to carbon C-12′ (δC 145.9). This problem was solved using a deuterium-induced isotope effect, which proved the position of the phenolic hydroxy group at carbon C-12′ (Δδ(C‑12′) = −0.33). Therefore, it was apparent that the ether bridge joined the remaining carbons C-11 and C-11′. The relative configuration of 8 was not established because of the large distance of the chiral centers that did not allow the detection of the required nuclear Overhauser effect. Unfortunately, a crystalline form of compound 8 could not be prepared for X-ray crystallographic study. The NMR spectroscopic data previously given were also revised for compounds 5−7.31−35 For all of the compounds in this study (3−8), proton and carbon signals were completely assigned and revised employing 2D NMR experiments such as gCOSY, gHSQC, gHMBC, and NOESY; these data are summarized in Tables 1 and 2. All isoquinoline alkaloids isolated in sufficient amounts were assayed for their human erythrocyte acetylcholinesterase (hAChE), human serum butyrylcholinesterase (hBuChE), and POP inhibition activities. Active compounds were also studied for their GSK-3β inhibition potency and passive permeation through the blood−brain barrier (BBB). Galanthamine and huperzine A were used as positive controls in the hAChE/hBuChE assays and Z-pro-prolinal and berberine in the POP assay. The results are summarized in Table 3. In the hAChE assay, quaternary alkaloids 9 and 10 showed low micromolar inhibition potency, whereas the uncharged compounds 1, 3, and 8 showed less potent inhibition activity (Table 3). The quaternary nitrogen in 9 and 10 plays an important role in binding with the enzyme.39 On the contrary, these compounds might have limited permeation through the BBB.40 On the basis of this fact, this study was concentrated on the determination of the biological profile of tertiary isoquinoline alkaloids. However, all of the compounds in this investigation displayed only poor-to-moderate hAChE inhibition ability, mostly in the high micromolar region. It is noteworthy that 9 and 10 can be highlighted as the only inhibitors surpassing the inhibitory potency of galanthamine. Progressive decrease in AChE levels in AD patient brains is somewhat counterbalanced by elevated BuChE activity.41 Indeed, BuChE supersedes the role of AChE in the hydrolysis of ACh in the later stages of AD.7 From this point of view, hBuChE inhibition activity pinpointed 6 with an IC50 value of 0.82 ± 0.10 μM as the most potent and selective hBuChE inhibitor in the compound subset. The second top-ranked hBuChE inhibitor was denoted as 8 and was one order of magnitude less active than 6. The relevance of selective BuChE inhibitors is considered to be a very significant issue because specific BuChE inhibitors not only improve cognition but also reduce levels of APP, which is the source of Aβ that forms the major component of amyloid plaques.42 All of the other isoquinoline alkaloids in this study displayed nonselective cholinesterase profiles. Kinetic analysis of 6 was used for the determination of the inhibition pattern against hBuChE. The compound displayed a mixed mode of inhibition (p < 0.05),

bridge was unexceptional. This linked tetrahydroisoquinoline fragment was substituted by methoxy groups on C-6′ (δC 152.1) and C-7′ (δC 138.8). The methoxy group on C-7′ was determined by the gHMBC experiment. In the gCOSY experiment, correlations were observed between H-5′ and carbon C-7′ and the hydrogen of the methoxy group with C-7′. The methoxy group on C-6′ was determined using the NOESY spectrum. A correlation of the methoxy group on C-6′ and hydrogen H-5′ was observed. Detailed gHMBC, gCOSY, and NOESY correlations supporting the proposed structure are depicted in Figure 2. Compound 4 has one chiral carbon (C1). The absolute configuration of 4 was investigated by comparing its optical rotation [α]D25 −17.4 (c 0.115; CHCl3) with those of the similar published compounds (+)-armepavine37 and (−)-armepavine.38 Compound 4 was thus assigned as the (1R)-isomer. Another new compound, berbostrejdine (8), was obtained as a yellowish amorphous solid. The molecular formula was determined to be C37H42N2O7 from the protonated molecular ion peak [M + H]+ found at m/z 627.3054 (627.3065 calcd for C37H43N2O7+). At first sight, the 1H and 13C NMR spectra of compound 8 showed the signals of two very similar compounds. On the contrary, the measured mass suggested the occurrence of only one molecule. The 13C NMR data showed almost identical signals for the tetrahydroisoquinoline parts of the molecule that were confirmed employing gHSQC, gHMBC, COSY, and NOESY experiments (Figure 3).

Figure 3. 1H−1H gCOSY, and key gHMBC correlations of 8. The deuterium-induced isotope effect has been described by red numbers.

However, there were noticeable differences in the rest of the molecule. The methine proton H-1 showed correlations with the H-9 methylene protons in the gCOSY spectrum as well as proton H-1′ with the H-9′ protons. These H-9 and H-9′ methylene protons represented a link between the tetrahydroisoquinoline and aromatic fragments of the molecule. This assumption was supported by the gHMBC spectrum, from which the correlations between H-9, C-14, and C-10 were acquired, as well as the correlations between C-9′, C-10′, and C-14′. The gCOSY spectrum also confirmed that a doublet of doublets signal for H-14′ (δH 6.65, J = 8.2 Hz, J = 1.8 Hz) correlated to H-13′ and hence had to be a part of this 1,2,4trisubstituted benzene ring. The second 1,2,4-trisubstituted benzene ring was similarly confirmed. The main difference between the constitution of their two fragments was the arrangement on the trisubstituted benzene rings. There was D


Journal of Natural Products Table 1. 1H and

13

Article

C NMR Data for Bersavine (3), Berbamine (5), Aromoline (6), and Obamegine (7)

bersavine (3)a position

δC

1 3

63.4 45.5

4

24.9

4a 5 6 7 8 8a 9

130.5 107.0 153.5 138.4 149.3 121.6 38.8

9a 10 11 12 13 14 15 17 18 1′ 3′

134.0 116.5 149.6 146.8 123.2 124.5 57.2 47.6 11.5 64.4 46.1

4′

26.1

4a′ 5′ 6′ 7′ 8′ 8a′ 9′

129.9 112.5 151.3 145.0 121.1 128.8 37.3

9a′ 10′ 11′ 12′ 13′ 14′ N-2−CH3 C-6− OCH3 C-7− OCH3 N-2′−CH3 C-6′− OCH3

136.2 131.4 122.3 156.3 122.5 133.4 43.1 56.3

δH (J in Hz) 3.90 d (9.2) 3.28−3.22 m; 2.75−2.70 m 2.94−2.81 m; 2.54−2.45 m 6.40−6.37 m

3.01−2.84 m; 2.62−2.56 m 6.40−6.37 m

6.56 d (1.3) 3.83 d (8.7) 2.71 q (7.3) 1.16 t (7.3) 4.00−3.92 m 3.49−3.40 m; 2.94−2.81 m 3.01−2.84 m; 2.94−2.81 m 6.66 s

5.98 bs 3.28−3.22 m; 2.94−2.81 m 7.35 dd (8.2, 1.9) 7.08 dd (8.2, 2.2) 6.51 dd (8.4, 2.2) 6.40−6.37 m 2.18 s 3.72 s

berbamine (5)b δC 62.2 45.1 23.8 129.4 105.4 151.8 136.9 148.1 120.6 38.5 134.0 115.1 147.4 143.6 114.5 123.5 − − − 63.7 46.0 25.5 128.8 111.2 149.8 143.5 119.8 127.4 37.8 135.7 130.3 121.4 153.6 121.7 132.2 42.8 55.7

aromoline (6)b

δH (J in Hz)

δC

3.91−3.83 m 3.30−3.24 m; 2.88−2.76 m 2.88−2.76 m; 2.46−2.38 m

3.02 d (13.8); 2.63−2.58 m

6.82 d (8.1) 6.76 d (8.1) − − − 3.91−3.83 m 3.50−3.39 m; 2.88−2.76 m 2.96−2.88 m; 2.88−2.76 m

60.7 44.3

4.05 d (10.7) 3.29−3.24 m; 2.88−2.77 m

28.2

2.82−2.74 m; 2.46−2.40 m

23.0

2.88−2.77 m; 2.46−2.41 m

24.4 122.9 104.7 146.5 133.6 141.7 122.6 39.7

6.51 s

5.98 bs 3.30−3.24 m; 2.96−2.88 m

138.4 128.8 121.6 152.9 120.7 131.5 43.5 55.3

7.29 dd (8.1, 2.0) 7.10 dd (8.1, 2.1) 6.61 dd (8.1, 2.1) 6.49−6.39 m 2.26 s 3.75 s

δH (J in Hz)

3.58−3.55 m 2.46−2.40 m

130.6 116.9 146.8 143.6 114.5 124.5 − − − 60.7 45.0

6.49−6.39 m

δC

64.4 50.8

130.5 111.3 148.5 144.0 117.4 128.7 38.4

6.27 s

δH (J in Hz)

obamegine (7)b

61.0

3.09 s

60.5

3.11 s

−

42.6 55.9

2.55 s 3.56 s

42.8 55.5

2.58 s 3.58 s

41.7 56.1

6.35 s

6.66 s 3.02−2.96 m; 2.82−2.74 m

5.62 bs

6.78 d (8.1) 6.73 dd (8.1, 1.1) − − − 4.18 d (6.2) 3.25−3.17 m; 2.89 dd (12.5, 6.5) 3.07−3.02 m; 2.67 dd (16.8, 6.5) 6.30 s

3.28 d (12.8); 2.82−2.74 m

7.39 dd (8.3, 1.4) 6.92−6.88 m 6.41 dd (7.1, 1.6) 6.92−6.88 m 2.53 s 3.58 s − 2.52 s 3.80 s

124.2 107.3 147.0 136.1 143.8 121.8 38.9 132.6 114.5 148.3 143.4 115.1 122.8 − − − 65.0 45.9 25.2 130.5 112.3 149.4 143.5 121.4 129.7 38.3 135.3 130.3 122.5 154.3 122.7 132.1 42.5 56.0

6.35 s

2.97−2.92 m; 2.68 dd (15.2, 10.7) 6.22 d (1.3)

6.78 d (8.1) 6.67 dd (8.1, 1.3) − − − 3.70 dd (10.5, 4.4) 3.48−3.41 m; 2.88−2.77 m 2.97−2.92 m

6.73 s

6.05 s 3.34−3.29 m; 2.88−2.77 m

7.32 dd (8.3, 2.2) 7.07 dd (8.3, 2.5) 6.79 6.44 2.31 3.78

dd (8.3, 2.5) dd (8.3, 2.2) s s

− 42.9 56.1

− 2.52 s 3.89 s

a

Spectra were acquired in methanol-d4 at 500 MHz (1H) and 125.7 MHz (13C). bSpectra were acquired in chloroform-d1 at 500 MHz (1H) and 125.7 MHz (13C). J values are in parentheses and reported in hertz. Chemical shifts are given in ppm. Assignments were confirmed by COSY, NOESY, gHSQC, and gHMBC experiments.

increased, whereas Vmax was diminished at higher concentrations of 6. Corresponding values for Ki of 6.41 ± 1.77 μM and Ki′ of 39.7 ± 5.3 μM were determined. In recent studies, POP inhibitors have demonstrated a high potential to become effective antidementia drugs.43 POP inhibition can represent an additional supporting approach in AD treatment, and hence the POP inhibitory ability of the isolated compounds was tested. The most pronounced

consistent with the graphical representation of the corresponding Lineweaver−Burk plot in Figure 4. This means that 6 binds reversibly to both free hBuChE and the hBuChE-substrate complex, influencing the binding of the substrate in the active site of the enzyme and interacting with its peripheral anionic site. The intersection of lines is located above the x axis, which corresponds with higher affinity to the free hBuChE than to the enzyme−substrate complex (Ki < Ki′). Km was slightly E


Journal of Natural Products Table 2. 1H and

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C NMR Data for Muraricine (4) and Berbostrejdine (8)a muraricine (4)

berbostrejdine (8)

position

δC

δH (J in Hz)

δC

δH (J in Hz)

1 3 4 4a 5 6 7 8 8a 9 9a 10 11 12 13 14 1′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′ 9′ 9a′ 10′ 11′ 12′ 13′ 14′ N-2−CH3 N-2′−CH3 C-6−OCH3 C-6′−OCH3 C-7′−OCH3 C-12−OCH3

64.7 44.9 24.1 125.3 111.8 147.5 144.2 115.1 125.6 39.3 128.9 130.8 117.9 156.2 − − 52.5 52.0 27.7 128.7 108.1 152.1 138.8 144.1 118.2 − − − − − − − 41.2 45.0 56.1 55.9 60.0 −

3.61 dd (11.4, 3.1) 3.42−3.39 m; 3.05−2.99 m 3.05−2.99 m; 2.80 ddd (14.8, 5.7, 0.7)

64.4 46.7 24.6 124.3 110.8 145.8 143.8 114.4 128.5 40.3 132.3 121.2 145.0 148.8 112.4 125.4 64.4 46.7 24.5 124.3 110.7 145.7 143.7 114.2 128.6 40.7 131.1 119.8 144.2 145.9 116.0 125.3 41.9 42.0 55.7 55.7 − 55.9

3.74−3.68 m 3.17−3.05 m; 2.82−2.73 m 2.82−2.73 m; 2.60−2.53 m

6.66 s

4.86 s 3.37−3.29 m; 2.52 dd (11.4, 2.5) 6.61 d (7.8) 6.54 d (7.8) − − 3.78 d (15.0); 3.30 d (15.0) 3.17−3.12 m; 2.58−2.42 m 3.37−3.29 m; 2.85−2.69 m 6.47 s

− − − − − − − 2.60 2.61 3.89 3.80 3.39

s s s s s −

6.50 s

6.28 s 3.17−3.05 m; 2.82−2.73 m 6.74 bs

6.83−6.80 6.83−6.80 3.74−3.68 3.17−3.05 2.82−2.73

m m m m; 2.82−2.73 m m; 2.60−2.53 m

6.48 s

6.26 s 3.17−3.05 m; 2.82−2.73 m 6.70 bs

6.83−6.80 m 6.65 dd (8.2, 1.8) 2.47 s 2.45 s 3.80 s 3.80 s − 3.78 s

a

Spectra were acquired in chloroform-d1 at 500 MHz (1H) and 125.7 MHz (13C). J values are in parentheses and reported in hertz. Chemical shifts are given in ppm. Assignments were confirmed by COSY, NOESY, gHSQC, and gHMBC experiments.

through the lipid membrane of commercial drugs was determined together with 6 (Table 3). An assay validation was made comparing the reported permeability values of the commercial drugs with experimental data (Figure S2, Supporting Information). A good correlation between experimental and described values was obtained Pe(exptl) = 0.9079(bibl) − 0.696 (R2 = 0.974). From this equation and following the pattern established in the literature for BBB permeation prediction, compounds could be classified as CNS + when they presented a permeability of >3.88 × 10−6 cm s−1. On the basis of the obtained results, the permeability of 6 through the BBB remains rather speculative. However, the major limitation of the PAMPA-BBB assay is that it only takes into account passive permeation, thus omitting other mechanisms normally employed in a compound’s distribution, like active transport. From this point of view, the therapeutic potentiality of 6 is worthy of further investigation.

inhibitory activity was displayed by 3, with an IC50 value of 67 ± 6 μM, with this being a one-fold better POP inhibitor than berberine (IC50 = 142 ± 21 μM), which is recognized as a POP inhibitor (IC50 = 145 μM).44 Taking into account the balanced hBuChE and POP inhibitory abilities of 6, the compound was further submitted for GSK-3β inhibition potency at a concentration of 10 μM. Unfortunately, the assayed compound demonstrated only weak inhibition activity (Table 3). The crucial part in the process of AD therapeutics drug development is the ability of the drug to reach the CNS, thus crossing the BBB. Accordingly, the screening for BBB penetration in early drug discovery programs provides important information for compound selection.45 The parallel artificial membrane permeability assay (PAMPA) was selected as a useful technique to predict passive diffusion through biological membranes, as described by Di et al., employing a brain lipid porcine membrane.46 The in vitro permeability (Pe) F



Article

Table 3. hAChE, hBuChE, and POP Inhibitory Activities of the Alkaloids Isolated from the Root Bark of Berberis vulgaris compound 8-oxoberberine (1) berbidine (2) bersavine (3) muraricine (4) berbamine (5) aromoline (6) obamegine (7) berbostrejdine (8) berberine (9) palmatine (10) galanthaminee huperzine Ae Z-pro-prolinale

hAChE IC50 (μM)a

hBuChE IC50 (μM)a

SI for hAChEb

POP IC50 (μM)a

GSK-3β (% inhibition at 10 μM)c

PAMPA-BBB permeability (Pe; 10−6 cm s−1)d

>100

68 ± 6

1.1

>200

n.d.

n.d.

n.d. 68 ± 11 >100 >100 >100 97 ± 3 66 ± 8

n.d. >100 68 ± 6 >100 0.8 ± 0.1 >100 6.9 ± 1.0

− 2.0 0.2 0.4 0.008 1.5 0.1

n.d. 67 ± 6 >200 >200 189 ± 32 >200 n.d.

n.d. n.d. n.d. n.d. 10 ± 3 n.d. n.d.

n.d. n.d. n.d. n.d. 1.8 ± 0.1 CNS+/CNS− n.d. n.d.

0.7 ± 0.1 1.7 ± 0.2 1.7 ± 0.1 0.033 ± 0.001 n.d.

30.7 ± 3.5 >100 42.3 ± 1.3 >100 n.d.

44 86 25 >15.100 −

142 ± 21 >200 >200

n.d. n.d. n.d. n.d. n.d.

0.1 ± 0.1 CNS− n.d. n.d. n.d. n.d.

0.00275 ± 0.0022

Results are the mean of six replications. bDetermined as ratio hBuChE IC50 (μM)/hAChE IC50 (μM). cTested at 10 μM compound concentration. dCNS+: high BBB permeation predicted with Pe (10−6 cm·s−1) > 4.0; CNS−: low BBB permeation predicted with Pe (10−6 cm·s−1) < 2.0; CNS±: BBB permeation uncertain with Pe (10−6 cm·s−1) from 4.0 to 2.0; n.d.: not determined. eReference compound. a

responsible for the ligand binding. To achieve this aim, a molecular modeling simulation exploiting the crystal structure of hBuChE (PDB ID: 4BDS) was carried out.47 It is well established that AChE and BuChE share ∼54% amino acid sequence identity.48 Because of the less conservative active site of BuChE, where 6 out of 14 aromatic residues compared with AChE are replaced by aliphatic amino acid residues, the volume of the cavity reaches ∼200 Å3.49 Taking into account the bulkiness of 6, this is presumably the main reason reflecting the high affinity of this compound for hBuChE over hAChE. Indeed, the docking experiment between hBuChE and 6 revealed good ligand fitting at the bottom of the hBuChE gorge (Figure 5). These findings are also corroborated by the high binding energy of the hBuChE−6 complex (−14 kcal· mol−1). Compound 6 was seen to be lodged near all of the catalytic triad residues, thus compromising the access for follow-up cleavage of substrate by the catalytic machinery. In more detail, His438 (3.5 Å) enables parallel π−π interaction with the phenolic moiety of 6. This part of the molecule is also

Figure 4. Steady-state inhibition of hBuChE hydrolysis of the substrate butyrylthiocholine by compound 6 at different concentrations. Lineweaver−Burk plots of initial velocity at increasing substrate concentrations (2.5−20.0 mM) are presented. Lines were derived from a linear regression of the data points.

The high affinity and selectivity of 6 to hBuChE indicated the need for the elucidation of the structural determinants

Figure 5. Most favorable docking pose for 6 within the hBuChE active site (PDB ID: 4BDS). (A) Spatial conformation of ligand 6 displayed in blue, important amino acid residues responsible for ligand accommodation in purple, and catalytic triad in yellow. (B) 2D representation of 6 in hBuChE. Panel A was generated with PyMol 2.0.6.50 Panel B was created with Discovery Studio 2016 Client software (v 17.2.0.16349).51 G



Article

Fraction I (1.037 g) was subjected to preparative TLC (cyclohexane−ethyl acetate−diethylamine 50:40:10; 3×) yielding obamegine (7), which was subsequently recrystallized from EtOH (25 mg). Fraction J (8.439 g) was suspended in 2% HCl (50 mL), diluted with distilled H2O, and filtered. The filtrate was alkalized with Na2CO3 and successively partitioned with CHCl3 to give 4.5 g purified alkaloid mixture. This was further treated by preparative TLC to give a minor yellowish compound, berbostrejdine (8, 13 mg). Fraction A-4 (2.3 g) was subjected to preparative TLC (npropanol−formic acid−H2O 90:1:9) to give two yellowish compounds, berberine (9, 1.8 g) and palmatine (10, 50 mg). Bersavine (3). (11S,31R)-55-[(Diethylamino)methyl]-16,36,37-trimethoxy-12,32-dimethyl-11,12,13,14,31,32,33,34-octahydro-2,6-dioxa-1(7,1),3(8,1)-diisoquinolina-5(1,3),7(1,4)-dibenzenacyclooctaphan-54ol: Yellowish crystals; [α]D25 +101.2 (c 0.134, CHCl3); for 1H and 13C NMR data, see Table 1; ESIMS m/z (%) 694 (58) [M + H]+, 621 (100), 578 (12), 396 (6), 381 (6), 348 (25), 311 (6); HRMS m/z 694.3853 [M + H]+ (calcd for C42H52N3O6+, 694.3851). Muraricine (4). 4-({7-[(6,7-Dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-8-yl)oxy]-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl}methyl)phenol: Greenish amorphous solid; [α]D25 −17.4 (c 0.115, CHCl3); for 1H and 13C NMR data, see Table 1; ESIMS m/z (%) 505 (49) [M + H]+, 413 (12), 253 (100); HRMS m/z 505.2698 [M + H]+ (calcd for C30H37N2O5+, 505.2697). Berbostrejdine (8). 1-(4-Hydroxy-3-{5-[(7-hydroxy-6-methoxy-2methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl]-2methoxyphenoxy}benzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol: Yellowish amorphous solid; [α]D25 +25.2 (c 0.127, CHCl3); for 1H and 13C NMR data, see Table 2; ESIMS m/z (%) 627 (100) [M + H]+, 314 (59); HRMS m/z 627.3054 [M + H]+ (calcd for C37H43N2O7+, 627.3065). Preparation of Enzymes: hAChE and hBuChE. Enzymes were prepared from freshly drawn blood collected by venipuncture from healthy volunteers. A written informed consent was obtained, and the Ethics Committee of Charles University, Faculty of Pharmacy approved the study on January 2018. It conformed to Declaration of Helsinki. Whole blood was used in the screening experiments, analyzing the inhibition potency of alkaloids against human cholinesterases. To the collected blood sample was immediately added asodium citrate 3.4% (w/v) in an amount of 2 mL per 18 mL of blood, according to Steck and Kant, with slight modification.52 Plasma (hBuChE) was removed from the whole blood by centrifugation at 4000 rpm in a Boeco U-32R centrifuge fitted with a Hettich 1611 rotor. Red blood cells were transferred to 50 mL tubes and washed three times with 5 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride (centrifugation under same conditions). The washed erythrocytes were stirred with 5 mM phosphate buffer (pH 7.4) for 10 min to ensure lysis. The lysed cells were dispensed for subsequent measurement. The activity of the enzyme preparations was measured immediately after preparation and adjusted with 5 mM phosphate buffer (pH 7.4) to reach the activity of blank sample A = 0.08−0.15 for hAChE and A = 0.15−0.20 for hBuChE. hAChE and hBuChE Inhibition Assay. hAChE and hBuChE activities were determined using a modified method of Ellman, with acetylthiocholine iodide (ATChI) and butyrylthiocholine iodide (BuTChI) as substrates, respectively.53 In brief, 8.3 μL of either blood cell lysate or plasma dilutions (at least six different concentrations), 283 μL of 5 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), and 8.3 μL of the sample dilution in dimethyl sulfoxide (DMSO) (40, 10, 4, 1, 0.4, and 0 mM) were added to the semimicro cuvette. The reaction was initiated by the addition of 33.3 μL of 10 mM substrate (ATChI or BuTChI). The final proportion of DTNB and substrate was 1:1. The increase in absorbance (ΔA) at 436 nm for AChE and 412 nm for BuChE was measured for 1 min at 37 °C using a spectrophotometer (Synergy HT multi-detection microplate reader). Each measurement was repeated six times for every concentration of enzyme preparation. The % inhibition was calculated

implicated in a conventional hydrogen bond with Ser198 (2.8 Å). Lastly, Glu197 showed van der Waals forces to 6. Tyr332 enabled the formation of T-shaped π−π stacking with one of the 1,2,3,4-tetrahydroisoquinoline moieties (3.9 Å). The other one is anchored to Asp70 via both π−π and cation−π forces. All of these interactions make a complex web responsible for favorable ligand settling.

■

EXPERIMENTAL SECTION

General Experimental Procedures. All solvents were treated by using standard techniques before use. All reagents and catalysts were purchased from commercial sources (Sigma-Aldrich, Czech Republic) and used without purification. The NMR spectra were obtained in CDCl3 and CD3OD at ambient temperature on a VNMR S500 (Varian) spectrometer operating at 500 MHz for 1H and 125.7 MHz for 13C. Chemical shifts were recorded as δ values in parts per million (ppm) and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal (CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C; CD3OD: 3.30 ppm for 1H and 49.0 ppm for 13C). Coupling constants (J) are given in hertz. For unambiguous assignment of 1H and 13C signals, 2D NMR experiments, namely, gCOSY, gHSQC, gHMBC, and NOESY, were measured using standard parameter settings and standard pulse programs provided by the producer of the spectrometer. HRMS were obtained with a Waters Synapt G2-Si hybrid mass analyzer of a quadrupole time-of-flight (QTOF) type coupled to a Waters Acquity I-Class UHPLC system. Molecular ions were detected in positive mode. Compounds were observed under UV light (254 and 366 nm) and visualized by spraying with Dragendorff’s reagent. Plant Material. The root bark of B. vulgaris was obtained from the herbal dealer Kottas Pharma (Vienna, Austria). The root bark was harvested from 6−8 year old bushes. Taxonomic identification and verification of the plant material was performed by Prof. Lubomir Opletal, and a voucher specimen (AL-201) was deposited at the Department of Pharmaceutical Botany, Faculty of Pharmacy, Charles University. Extraction and Isolation. The root bark of B. vulgaris (30 kg) was dried, chopped, and extracted with 95% ethanol (EtOH) denaturated with methanol (MeOH) three times (3 × 30 min) under a reflux, and filtered. The filtrate was evaporated under a vacuum to obtain an EtOH extract (2 kg), which was suspended in 2% HCl (6 L), diluted with distilled H2O (6 L), and filtered. The filtrate was defatted with diethyl ether, alkalized with Na2CO3, and successively partitioned with CHCl3, yielding 128 g of residue. For purification, the residue was dissolved in CHCl3 (2.5 L) and passed through a column of Al2O3 (2 kg); four fractions (A-1−4) were obtained. Fraction A-1 (59.5 g) was further fractionated by CC (aluminum oxide; 230−400 mesh, 2.38 kg), eluting with petroleum ether (with boiling range 40−80 °C) gradually enriched with CHCl3 (85:15, 80:20, 70:30, 60:40, 50:50, 40:60, 20:80), and then CHCl3 with EtOH (99:1, 80:20). Fractions of 250 mL were collected and monitored by TLC. Eleven main fractions were obtained (A−J). The detailed plant extraction and column chromatography of a summary alkaloidal extract are described in previous report.24,25 Fraction C (1.591 g) was further crystallized from a CHCl3 and EtOH mixture to give 8-oxoberberine (1, 415 mg). Fraction E (0.208 g) was treated by preparative TLC (toluene− diethylamine 90:10; 2×) to give berbidine (2, 4.1 mg). Fraction H (25.7 g) was further fractionated by CC on Al2O3, as described in previous work.25 Preparative TLC of fraction H-I (1.028 g; toluene−CHCl3−diethylamine 45:45:10; 2×) gave bersavine (3, 470 mg) and muraricine (4, 24 mg). Part (1 g) of fraction H-II (12.6 g) was further treated by preparative TLC (cyclohexane−ethyl acetate−diethylamine 50:40:10; 3×) to obtain berbamine (5), which was recrystallized from a CHCl3 and EtOH mixture (230 mg). Fraction H-III (6.8 g) was subjected to preparative TLC (cyclohexane−ethyl acetate−diethylamine 50:40:10; 3×) to give aromoline (6) and crystallized from a CHCl3 and EtOH mixture (139 mg).

(

according to the following formula: %I = 100 − 100 × H

ΔABl ΔASa

),



Article

where ΔABl is the increase in absorbance of the blank sample and ΔASa is the increase in absorbance of the measured sample. Inhibition potency of the tested compounds was expressed as an IC50 value (concentration of inhibitor, which causes 50% cholinesterase inhibition). hBuChE Inhibition Mechanism. The procedure for the determination of the inhibition mechanism was similar to that for the determination of IC50, with a difference in that uninhibited and inhibited processes were observed using four different concentrations of BuTChI (2.5, 5, 10, and 20 mM). The dependence of absorbance (412 nm) versus time was recorded. Each measurement was performed in triplicate. The values of Vmax and Km of the Michaelis−Menten kinetics as well as the values of Ki and Ki′ were calculated by nonlinear regression from the substrate velocity curves. Linear regression was used for the calculation of Lineweaver−Burk plots. The type of enzyme inhibition was elucidated from the nonlinear regression analysis. Results for each type model of inhibition (competitive, noncompetitive, uncompetitive, and mixed) were compared by a sum-of-squares F-test. All calculations were performed using GraphPad Prism software version 6.07 for Windows (GraphPad Software, San Diego, CA). Prolyloligopeptidase Assay. POP (EC 3.4.21.26) was dissolved in phosphate-buffered saline (PBS; 0.01 M Na/K phosphate buffer, pH 7.4, containing 137 mM NaCl and 2.7 mM KCl); the specific activity of the enzyme was 0.2 U/mL. The assay was performed in standard polystyrene 96-well microplates with a flat and clear bottom. Stock solutions of tested compounds were prepared in DMSO (10 mM). Dilutions (10−3 to 10−7 M) were prepared from the stock solution with deionized H2O; the control was performed with the same DMSO concentration. POP substrate, (Z)-Gly-Pro-p-nitroanilide, was dissolved in 50% 1,4-dioxane (5 mM). For each reaction, PBS (170 μL), tested compound (5 μL), and POP (5 μL) were incubated for 5 min at 37 °C. Then, substrate (20 μL) was added, and the microplate was incubated for 30 min at 37 °C. The formation of p-nitroanilide, directly proportional to the POP activity, was measured spectrophotometrically at 405 nm using a microplate ELISA reader (Multimode microplate reader Synergy 2, BioTek Instruments, Winooski, VT). The inhibition potency of tested compounds was calculated by nonlinear regression analysis and was expressed as an IC50 value (concentration of inhibitor which causes 50% POP inhibition). All calculations were performed using GraphPad Prism software version 6.07 for Windows (GraphPad Software). CNS Penetration: in Vitro Parallel Artificial Membrane Permeability Assay − Blood Brain Barrier. The prediction of brain penetration was evaluated using a PAMPA. Ten commercially available drugs, PBS solution at pH 7.4, ethanol, and dodecane were purchased from Sigma, Acros Organics, Merck, Aldrich, or Fluka. The porcine polar brain lipid (PBL) was from Avanti Polar Lipids (Madrid, Spain). The donor plate was a 96-well filtrate plate (Multiscreen IP Sterile Plate PDVF membrane, pore size 0.45 μM), and the acceptor plate was an indented 96-well plate (Multiscreen), both from Millipore. Filter PDVF membrane units (diameter 30 mm, pore size 0.45 μm) from Symta (Madrid, Spain) were used to filter the samples. A 96-well plate UV reader (Thermo Scientific, Multiscan spectrum) was used for the UV measurements. Test compounds (2−6 mg of caffeine, enoxacine, hydrocortisone, desipramine, ofloxacine, piroxicam, and testosterone; 12−15 mg of promazine and verapamil; and 23 mg of atenolol) were dissolved in EtOH (1000 μL). One hundred μL of this compound stock solution was taken, and 1400 μL of EtOH and 3500 μL of PBS at pH 7.4 buffer were added to reach 30% EtOH concentration in the experiment. These solutions were filtered. The acceptor 96-well microplate was filled with 180 μL of PBS−EtOH (70:30). The donor 96-well plate was coated with 4 μL of porcine brain lipid in dodecane (20 mg mL−1), and after 5 min, 180 μL of each compound solution was added. One to two mg of each test compound to be determined for its ability to pass the brain barrier was dissolved in 1500 μL of EtOH and 3500 μL of PBS (pH 7.4) buffer, filtered, and then added to the donor 96-well plate. Then, the donor plate was carefully put on the acceptor plate to form a “sandwich”, which was left undisturbed for 2 h 45 min at 25 °C.

During this time, the compounds diffused from the donor plate through the brain lipid membrane into the acceptor plate. After incubation, the donor plate was removed. The UV plate reader determined the concentration of compounds and clinically used drugs in the acceptor and donor wells. Each sample was analyzed at three to five wavelengths, in three wells, and in two independent runs. Results are given as means (standard deviation (SD)), and the average of the two runs is reported. Nine quality-control compounds (previously mentioned) of known BBB permeability were included in each experiment to validate the analysis set. Molecular Modeling Studies. hBuChE was obtained from RCSB Protein Data Bank (PDB ID: 4BDS) (crystal structure of hBuChE).47,54 The receptor structure was prepared by DockPrep function of UCSF Chimera (version 1.4) and converted to pdbqt-files by AutodockTools (v. 1.5.6).55,56 Flexible residues selection was based on either previous experience with hBuChE or the spherical region around the binding cavity.57−59 Three-dimensional structures of ligands were built by Open Babel (v. 2.3.1), minimized by Avogadro (v 1.1.0), and converted to pdbqt-file format by AutodockTools.60 The docking calculations were made by Autodock Vina (v. 1.1.2) with the exhaustiveness of 8.61 The calculation was repeated 15 times for 6, and the best-scored result was selected for manual inspection. The visualization of enzyme−ligand interactions was prepared using The PyMOL Molecular Graphics System.50 2D diagrams were created with Dassault Systèmes BIOVIA Discovery Studio Visualizer.51

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00592. HRMS and 1D and 2D NMR data for bersavine (3), muraricine (4), and berbostrejdine (8) and permeability in the PAMPAf-BBB assay for compounds aromoline (6) and berberine (10) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +420 495 067 311. Fax: +420 495 067 162. E-mail: [email protected]. ORCID

Lucie Cahlikova: 0000-0002-1555-8870 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This project was supported by Charles University grants (SVV UK 260 412; 260 401; Progres/UK Q40 and Q42), by MH CZ - DRO (University Hospital Hradec Kralove, No. 00179906), by the grant of the Ministry of Defense of the Czech Republic − “Long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence”, and by EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/ 0000841) cofunded by ERDF.

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REFERENCES

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