Diterpene Alkaloids from the Roots of Aconitum moldavicum and Assessment of Nav 1.2 Sodium Channel Activity of Aconitum Alkaloids Botond Borcsa 1, László Fodor 2, Dezső Csupor 1, Peter Forgo 1, Attila Molnár V. 3, Judit Hohmann 1 1 Department of Pharmacognosy, University of Szeged, Szeged, Hungary 2 Gedeon Richter Plc., Budapest, Hungary 3 Department of Botany, University of Debrecen, Debrecen, Hungary
Abstract !
A new aconitane alkaloid, 1-O-demethylswatinine (1), was isolated from the root of Aconitum moldavicum together with the known compounds cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8). The structures were established by means of HRESIMS, 1D and 2D NMR spectroscopy, including 1H-1H COSY, NOESY, HSQC, and HMBC experiments, resulting in complete 1H‑NMR chemical shift assignments for 1–4. The effects of the isolated compounds 4–8, together with eighteen other Aconitum diterpene and norditerpene alkaloids with different skeletal types and substitution patterns, were studied on Nav 1.2 channels by the whole-cell patch clamp technique, using the QPatch-16 automated patch clamp system. Pyroaconitine, ajacine, septentriodine, and delectinine demonstrated significant Nav 1.2 channel inhibition (57–42 %) at 10 µM concentration; several other compounds (acovulparine, acotoxicine, hetisinone, 14-benzoylaconine-8-O-palmitate, aconitine, and lycoctonine) exerted moderate inhibitory activity (30–22 %), while the rest of the tested alkaloids were considered to be inactive. On the basis of these results and by exhaustive comparison of data of previously published computerized QSAR studies on diterpene alkaloids, certain conclusions on the structure-activity relationships of Aconitum alkaloids concerning Nav 1.2 channel inhibitory activity are proposed.
Key words Aconitum moldavicum · Ranunculaceae · aconitine · norditerpene alkaloids · Nav 1.2 sodium channel inhibitory activity Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica
Aconitum species native to Asia are herbs used as painkillers, anaesthetics, cardiotonics, and antirheumatic agents in traditional Oriental medicine [1, 2]. Characteristic compounds of these plants are the diterpenoid alkaloids [3]. Antinociceptive and anti-epileptic activity effects of diterpene alkaloids may be mediated by an action on voltage-gated Na+ channels. The analgesic effect of Aconitum preparations had been attributed earlier to aconitine (23), the main alkaloid of several species. Aconitine and numerous diterpene alkaloids have been demonstrated to have peripheral analgesic, antinociceptive properties in different test models [3]. Alkaloids that activate voltage-dependent Na+ channels are antinociceptive since they depolarize neurons permanently, and hence block the neuronal conduction
[4]. Na+ channel blockers possess antinociceptive activity by inhibiting neuronal activity [4, 5]. Inhibition of neuronal activity and anti-epileptiform activity of several diterpene alkaloids has been demonstrated by Ameri et al. on rat hippocampal slices in vitro. This anti-epileptiform activity of diterpene alkaloids is also in line with their blockade of the Na+ channels since Na+ channels are known to be involved in the genesis of abnormal activities in epilepsy. Na+ channel-blocking compounds, e.g., lappaconitine, frequency-dependently inhibit experimentally induced epileptiform activity by sparing the normal neuronal activity [3]. Voltage-gated sodium channels (VGSCs) in sensory neurons have been found to play a crucial role, among others, in several chronic painful neuropathies of different aethiology. Pharmacons that exhibit a use-dependent blockade of these channels, like anticonvulsants, antiarrhythmics, and local anaesthetics, have long been used, for example in the treatment of chronic neuropathic or inflammatory pain states, epilepsies, migraine, and neurodegeneration related to ischaemia [6, 7]. VGSCs (Nav 1.x) are members of the superfamily of ion channels that includes voltage-gated potassium and calcium channels, however, in contrast to these channel molecules, different sodium channel isoforms are relatively similar regarding their functional properties [8]. Nine isoforms of the alpha subunit have been discovered and characterised, each consisting of four homologous domains forming the voltage-gated aqueous pore. These subunits are > 75 % identical over the amino acid sequences comprising the transmembrane and extracellular domains. Despite of this high level of homogeneity, the distinct expression patterns and variable associations with accessory β-subunits enable a specialised functional role of the various isoforms [8]. Developing subtype selective pharmacons that only block the channel isoforms specifically involved in the disease to be treated is a widely used strategy in modern drug research, as reviewed by Tarnawa et al. who also discuss patent information on several subtype selective compound families being under development [6]. Theoretically, such compounds offer a relatively good side-effect profile, by leaving the channel subtypes involved in important physiological functions, such as the heart type Nav 1.5, untouched. Unfortunately, sodium channel blockers currently applied with different therapeutic indications discriminate poorly between Nav 1.x subtypes, and their tolerable therapeutic indices are rather the consequence of their use-dependent properties. Another important aspect is that compounds used therapeutically should be devoid of or have the least minimal activity against the hERG cardiac potassium channel being involved in cardiac toxicity [9, 10]. The present paper reports on a phytochemical investigation of a diterpene alkaloid-accumulating species, Aconitum moldavicum Hacq. (Ranunculaceae) (Carpathian or eastern monkshood), which is a 60–200 cm tall plant occurring in the eastern and central parts of Europe, extending to Romania and West Ukraine. One new [1-O-demethylswatinine (1)] and seven already known norditerpene alkaloids [cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and " Fig. 1) were isolated from the MeOH extract preajacine (8)] (l pared from the fresh roots of A. moldavicum by a combination of different chromatographic methods, including CC, VLC, CPC, and preparative TLC. The structures of the isolated compounds were elucidated by spectroscopic methods. Compound 1 was isolated as an amorphous solid with [α]28 D + 22 (c 0.1, CHCl3,). It was shown by HRESIMS to have the molecular formula of C24H39NO8 according to the quasimolecular ion peak Borcsa B et al. Diterpene Alkaloids from …
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Fig. 1 Chemical structures of the investigated diterpene alkaloids (1–27).
at m/z 470.2761 [M + H]+ (calcd. for C24H40O8 470.2754), which afforded fragment ions at m/z 452 [M + H – H2O]+ and 320 [M + H – CH3OH]+. The 1H and 13C JMOD (J-Modulated Spin-Echo Experiment) spectra of 1 indicated the presence of an N-ethyl group (δH 1.11 t, 2.97 m and 2.85 dq; δC 50.3 and 14.0). Singlet signals at δH 3.35, 3.43, and 3.45 (each 3H, s) and carbon signals at δC 56.3, 57.8, and 58.0 demontrated the presence of three methoxy groups. Further, the JMOD spectrum suggested that the skeleton consisted of 19 carbons, including six methylenes, eight meth" Table 1). From the HSQC ines, and five quaternary carbons (l spectrum, the chemical shifts of the protonated carbons were assigned, and the proton-proton connectivities were then studied. The 1H-1H COSY spectrum defined structural fragments with correlated protons: –CHR–CH2–CH2– (unit A, C-1–C-3) (δH 4.06 brs, 1.72 m, 1.68 m, 1.98 m and 1.47 m), –CH–CHR– (unit B, C-5–C-6) (δH 2.15 s, 4.03 s), –CH–CHR–CH–CH2– (unit C, C-9–C-14–C-13– C-12) (δH 2.83 d, 4.11 t, 2.58 dd, 2.34 d, and 1.95 dd), –CH2–CH– (unit D, C-15–C-16) (δH 2.63 dd, 1.80 dd, and 3.26 dd), two isolated methylenes [δH 3.40 d, 3.70 d (C-18), and 2.45 d, 2.49 d (CBorcsa B et al. Diterpene Alkaloids from …
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" Fig. 1). The long-range correla19)], and one N-ethyl group (l tions detected in the HMBC spectrum proved that structural elements A–D and the two methylene groups (C-18 and C-19), together with the quaternary carbons C-4, C-7, C-8, C-10, and C11, build up an aconitane diterpene substituted in C-1, 6, 7, 8, " Table 1, Fig. 2). The location of the me10, 14, 16, 18 positions (l thoxy groups were established via the HMBC experiment. The long-range correlations of C-6, C-14, and C-16 with the protons at δH 4.03 s, 4.11 t, and 3.26 dd demonstrated the presence of methoxy groups at C-6, C-14, and C-16, respectively. The four hydroxy groups in 1 were located of necessity on C-1, C-7, C-8, C10, and C-18. The stereochemistry and relative configuration of " Table 1). As 1 were studied by means of a NOESY experiment (l reference point, the β stereochemistry of H-5 was used, which is characteristic for norditerpene alkaloids. The Overhauser effects between H-5/H‑9, H-5/H-3β, H-3β/H‑1, H-9/H-14, H-9/8-OH, H14/13, H-13/H-12β, H-12β/H‑14, and 8-OH/H-6 were indicative of the β position of these protons and 8-OH group. On the other hand, NOE effects observed between H-6/H-19, H-17/H-16, H-
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Table 1 NMR data of compounds 1, 3, and 4 [500 MHz (1H), 125 MHz (13C), CDCl3, δ (ppm) (J = Hz)]. 1 1
H
13
C
69.8 34.6
HMBC (C→H)
1 2α 2β 3α 3β 4
4.06 brs 1.72 m 1.68 m 1.98 m 1.47 m –
38.2
5 6
2.15 s 4.03 s
40.8 91.0
7 8
– –
87.2 76.8
9 10 11 12α 12β 13 14 15α 15β 16
2.83 d (2.5) – – 2.34 d (15.1) 1.95 dd (15.1, 7.5) 2.58 dd (7.4, 4.6) 4.11 t (4.6) 2.63 dd (14.7, 8.5) 1.80 dd (14.7, 8.3) 3.26 dd (8.5, 8.3)
53.7 83.0 54.1 41.0
5, 6, 15α, 15β 14, 6, 8-OH, 9, 17, 15α, 15β 15α, 12α 9, 12α 5, 6, 17, 12α 16
38.2 82.9 34.6
9, 12α, 15α 14-OMe, 12α 8-OH, 9
82.0
17
2.74 d (1.7)
66.4
9, 12α, 12β, 15α, 15β, 16-OMe 5, 19a, 19b
18a 18b 19a 19b 20a 20b 21 1-OMe 6-OMe 14-OMe 16-OMe 1-OH 8-OH
3.70 d (10.5) 3.40 d (10.5) 2.49 d (11.5) 2.45 d (11.5) 2.97 dq (19.4, 7.3) 2.85 dq (19.4, 7.3) 1.11 t (7.3) – 3.43 s 3.45 s 3.35 s 7.50 s 3.99 s
66.9
3α, 19a, 19b
57.1
5, 17, 20
26.7
3α
18a, 18b, 19a, 19b 6, 5, 18a, 19a, 19b, 3α, 2β 6, 17, 18b, 19a, 19b 5, 6-OMe, 17
NOESY
3
4
1
1
H
H
2α, 3α, 3β, 12α 1 5, 9 1 1 –
3.74 brs 1.67 m 1.58 m 2.10 m 1.91 m –
3.58 t (7.9, 9.2) 2.15 m 2.15 m 1.60 m 1.50 m –
2β, 6, 9 5, 18a, 19a
1.84 m 1.92 m 1.62 m 2.10 m –
1.95 s 3.89 s – –
2.23 t (5.9) 1.87 m – 2.06 m 1.60 m 2.34 m 4.24 t (5.0) 2.42 dd (16.3, 9.3) 2.07 m 3.39 dd (9.2, 4.7)
2.88 d (4.7) – – 3.07 d (15.7) 1.70 m 2.49 dd (7.8, 4.6) 4.10 t (4.8, 4.5) 2.64 dd (15.3, 10.0) 1.70 m 3.17 t (8.5, 7.2)
2.82 s
2.84 d (2.1)
3.46 d (10.5) 3.29 d (10.5) 2.36 d (10.7) 2.08 d (10.8) 2.55 dq (12.4, 7.1) 2.46 dq (12.4, 7.1) 1.13 t (7.1) – – – 3.35 s 3.03 brs*
3.64 d (10.7) 3.37 d (10.9) 2.60 d (12.0) 2.32 d (12.0) 2.90 m 2.81 dq (19.2, 7.2) 1.05 t (7.2) 3.26 s 3.45 s 3.44 s 3.33 s – 4.08 s
– – 2β, 5, 14, 8-OH – – 1, 16, 17 13, 14 12b, 14 9, 12β, 13 16, 17 12α, 15α, 17 12α, 15α, 16, 20a, 21 6 6
50.3
19a
17
14.0 – 58.0 57.8 56.3 – –
20a, 20b – 6 14 16 – –
17 – 8-OH 8-OH 9, 6-OMe, 16-OMe
* not assigned signal
17/H-20, H-16/H-12α, H-16/H-15α exhibited the α-orientation of H-6, H-16 and corroborated that the N-containing bridge, between C-19 and C-17, including the N-ethyl group, is below the plane of ring A. All of the above evidence was used to propose the structure of this compound as 1-O-demethylswatinine (1). Compounds 3 and 4 were isolated as an amorphous solid and identified as columbianine (3) and swatinine (4). These compounds are known diterpenes of the Aconitum genus (found in A. laeve, A. ferox, and A. lamarckii) [11–13], but our 2D NMR studies provided the complete 1H assignments for columbianine (3) " Table 1). In addition, the and swatinine (4) for the first time (l known alkaloids cammaconine (2), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8) were also isolated from A. moldavicum and identified by comparing their spectral data with those reported in the literature and by TLC co-chromatography with an authentic sample [14–16]. The effects on Nav 1.2 channels of the isolated compounds 4–8 were analysed by the automated whole-cell patch clamp tech-
nique. In this study, other diterpene alkaloids (9–27) were also included, which were isolated from A. toxicum, A. anthora, " Table 2). This series of A. vulparia, and Consolida orientalis (l compounds represents diverse structural types, including bisnor(C18), nor- (C19), and diterpene (C20) alkaloids substituted with hydroxy, methoxy, keto, acetyl, and various aromatic ester groups. All the compounds are N-ethyl-substituted with the exception of acovulparine (15), which contains an azomethine group. Pyroaconitine (25) exerted the highest inhibitory activity (57%), thus measurements on the concentration dependence of " Fig. 3) have also been carried out, and the IC this activity (l 50 value for this compound was determined (7.3 ± 1.5 µM). Furthermore, ajacine (8), septentriodine (22), and delectinine (17) also demonstrated significant Nav 1.2 channel inhibition (44–42 %) at 10 µM; several other compounds (acovulparine (15), acotoxicine (14), hetisinone (11), 14-benzoylaconine-8-O-palmitate (24),
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Table 2 Inhibition of Nav 1.2 channels by compounds 4–27 at nominal concentration of 10 µM. Compound
Inhibition
sem
n
10 2 13 22 44 16
4 4 4 5 5 7
5 7 8 7 8 8
18 28 18 6 28 30 3 42 16 15 14 19
5 3 6 5 3 4 2 5 6 4 4 3
5 4 5 7 8 6 4 8 4 7 4 8
43 25 27 57 15 18
6 3 2 2 3 6
7 8 9 7 8 7
(%)
Fig. 2
Selected 1H-1H COSY (—) and HMBC (C→H) correlations for 1.
aconitine (23), and lycoctonine (7) exerted moderate inhibitory activity (30–22%), while the rest of the tested alkaloids were considered to be inactive. Concerning the relationship between the structural features and the Nav 1.2 inhibiting activity of the studied alkaloids, it seems relevant that the concomitant presence of a methoxy function at C-1, the presence of an oxygen-containing functionality at C-8, and the presence of a (substituted) aroyl function either at C-14 or at C-18 is necessary, with the parallel requirement that the number of oxygen functionalities in the molecule should be at least 4. Satisfaction of these stipulations can be seen in the case of all significantly (8, 17, 22, 25) and moderately active compounds (7, 14, 15, 23, 24). When C-1 methoxy is changed to hydroxyl, the inhibitory effects of the compounds decrease as it can be seen in the case of delectinine (17) (42 % inhibition) and takaosamine (16) (3% inhibition). The only, nonetheless very interesting, outlier compound is swatinine (4) (10% inhibition), which has a methoxy group at C-1, however, it is substituted with an unusual extra hydroxyl at C-10. Without this latter substituent, the substitution pattern of the compound is identical with that of acovulparine (15) (with the structural difference occurring on the N atom) (30 % inhibition). This surprising, and rather large activity difference, allows to hypothesise on the importance of hydroxy substitution at C-10 that may pose some kind of steric hindrance for the molecule when interacting with the channel receptor. However, it cannot be excluded that differences in the substitution of the N atom may also be of importance regarding this activity difference. Influence of the position of the aroyl functionality on the inhibitory activity is illustrated by the example of acotoxinine (20), which was found to be inactive, in spite of the fact that it has an aroyl functionality in the molecule, but it is positioned at C-8, not at C-14 or C-18, as it has been described above. These stipulations seem to be supported by a detailed QSAR analysis of 12 diterpene alkaloids, which revealed that the position of the aroyl/aroyloxy groups on C-4 or C-14 is the major determinant of the analgesic activity. Alkaloids with an aroyl or an aroyloxy group on C-14 exhibited an analgesic potency approximately 30 times higher than that of alkaloids with an aroyloxy group on C-4 in a model of acetic acid-induced writhing in rats [17]. Previously, it has also been proven that alkaloids with
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Swatinine (4) Gigactonine (5) Delcosine (6) Lycoctonine (7) Ajacine (8) 10-Hydroxy-8-O-methyltalatizamine (9) Isotalatizidine (10) Hetisinone (11) Aconosine (12) Dolaconine (13) Acotoxicine (14) Acovulparine (15) Takaosamine (16) Delectinine (17) Neolinine (18) Neoline (19) Acotoxinine (20) 14-Desacetyl-18-demethylpubescenine (21) Septentriodine (22) Aconitine (23) 14-Benzoylaconine-8-O-palmitate (24) Pyroaconitine (25) Songoramine (26) Songorine (27)
low affinity for the neurotoxin receptor site 2 of Na+ channels lack antinociceptive action [4]. As regards the three C20 diterpene alkaloids involved in this study, only one (hetisinone, 11) has been found to exert moderate activity, therefore no relevant deductions may be made concerning the structure-activity relationship of these compounds. It is noteworthy, though, to mention that songorine (27) was found practically inactive concerning its inhibitory action, which is in line with the previous results of Friese et al. when observing that this compound has failed to modulate Na+ channels [5]. However, in their study no exact subtype specification was provided on the examined Na+ channels. Further two aspects to be emphasised are that amongst the four most active compounds only one, delectinine (17), is not esterified (although satisfying all other presumably necessary conditions), and that both diester-type compounds [14-benzoylaconine-8-O-palmitate (24) and aconitine (23)] have been found to exert only moderate inhibitory activity on the studied particular channel subtype. In conclusion, it can be stated that distinctly minor substitutional changes seem to have rather firm impact on the inhibitory activity of diterpene alkaloids on Nav 1.2 channel, thus reinforcing the previous observations that minor structural differences may lead to completely different activities. Our results on Nav 1.2 channel inhibitory activity of selected diterpenoid alkaloids may open new perspectives in the research of channel subtype specific compounds, especially when considering that diterpene alkaloids comprise large and chemically variable biologically active compounds altogether having been poorly tested pharmacologically so far. The herein reported specific inhibitory activity of certain diterpene alkaloids on Nav 1.2 channel is intriguing, especially in light of the facts that in differ-
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Fig. 3 Concentration-dependent inhibition of pyroaconitine (25) on sodium current.
ent geographical regions of the world some diterpene alkaloid containing Delphinium and Aconitum species have long been used (both unprocessed and processed drugs) in the traditional medicine for the same analgetic and antiepileptic indications [1, 2] as those compounds being inhibitors of Nav 1.2, which have been reported to be currently under drug development [6]. Considering our herein results, especially in light of our previously reported results on the hERG K+ channel inhibitory activity of these diterpene alkaloids [18], it can be observed that certain compounds [14-benzoylaconine-8-O-palmitate (24), aconitine (23), gigactonine (5)] exerted noteworthy activity in both pharmacological tests, thus despite their promising Nav 1.2 inhibitory activity, these compounds should be considered as potentially harmful by causing adverse cardiovascular effects through their hERG effect, nevertheless, some of these compounds show some selectivity on hERG channels. Their potential cardiovascular safety risk should be evaluated based on their hERG IC50 value and in vivo efficient free plasma concentration. Among the studied active alkaloids, particularly those should be further pursued as model substances for pharmacological lead compounds as selective Nav 1.2 inhibitors, which exert minor or no hERG activity [18], i.e., ajacine (8) and delectinine (17). This is the first paper to report promising experimental results obtained in relation to a specific Nav 1.2 channel subtype inhibitory activity of diterpene alkaloids.
1-O-Demethylswatinine (1): amorphous solid; [α]28 D : + 22 (c 0.1, " Table 1. ESI‑MS‑MS: m/z CHCl3); 1H- and 13C‑NMR data: see l 470.2761 [M + H]+ (calcd. for C24H40NO8, m/z 470.2754). Bioassay: CHO cells stably expressing human Nav 1.2 sodium channels were investigated by the whole-cell patch clamp using the QPatch-16 automated patch clamp system. Cells were held at − 65 mV and activated by a train consisting of 20 steps to 0 mV for 8 ms at 10 Hz. Train protocols were applied at 10-s intervals. The current traces were recorded, and the amplitude of peak current evoked by the last pulse within a train was measured as test parameter. The inhibition was calculated from the peak currents in the presence and absence of the test compound. The control solution contained the same concentration of the vehicle (DMSO or HCl) as the solutions of the test compound. Solutions containing the test compounds were applied to the cells for 6–8 min. Aconitine (23) was purchased from Sigma-Aldrich. Semisynthetic preparations of benzoylaconine 8-O-palmitate (24) and pyroaconitine (25), alike the isolation of 10-hydroxy-8-O-methyltalatizamine (9), isotalatizidine (10), and hetisinone (11) from A. anthora; aconosine (12), dolaconine (13), acotoxicine (14), delectinine (17), neolinine (18), neoline (19), acotoxinine (20), songoramine (26), and songorine (27) from A. toxicum; acovulparine (15) and septentriodine (22) from A. vulparia; delcosine (6), gigactonine (5), takaosamine (16) and 14-desacetyl-18-demethylpubescenine (21) from Consolida orientalis were described ear" Fig. 1). The purities of the compounds were inlier in ref. [18] (l vestigated by means of 1H‑NMR spectroscopy [19], and all found to be > 95 %. Lamotrigine (purity > 98 %; Sigma-Aldrich) was used as positive control. Lamotrigine at 300 µM caused 89 ± 2% (n = 9) inhibition of sodium current.
Supporting information General experimental procedures, extraction and isolation, and NMR spectra of compound 1 are available as Supporting Information.
Acknowledgements !
The publication was supported by the European Union and cofunded by the European Social Fund (TÁMOP‑4.2.2.A‑11/1/ KONV-2012–0035). Financial support of the Hungarian Scientific Research Fund (OTKA K109846) is gratefully acknowledged.
Conflict of Interest !
The authors declare no conflict of interest.
References
Materials and Methods !
Plant material: A. moldavicum was collected near Eger in October 2006. The plant material was identified by Attila Molnár V. (Department of Botany, University of Debrecen, Hungary). A voucher specimen (No. 807) has been deposited in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary. For chromatographic purification, a combination of different chromatographic methods, including CC, VLC, CPC, and preparative TLC has been applied (for details see Supporting Information).
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received revised accepted
May 17, 2013 October 7, 2013 December 16, 2013
Bibliography DOI http://dx.doi.org/10.1055/s-0033-1360278 Published online January 22, 2014 Planta Med 2014; 80: 231–236 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Judit Hohmann Department of Pharmacognosy University of Szeged Eotvos u. 6 6720 Szeged Hungary Phone: + 36 62 54 64 53 Fax: + 36 6 25 45 7 04 [email protected]
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Abstract !
A new aconitane alkaloid, 1-O-demethylswatinine (1), was isolated from the root of Aconitum moldavicum together with the known compounds cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8). The structures were established by means of HRESIMS, 1D and 2D NMR spectroscopy, including 1H-1H COSY, NOESY, HSQC, and HMBC experiments, resulting in complete 1H‑NMR chemical shift assignments for 1–4. The effects of the isolated compounds 4–8, together with eighteen other Aconitum diterpene and norditerpene alkaloids with different skeletal types and substitution patterns, were studied on Nav 1.2 channels by the whole-cell patch clamp technique, using the QPatch-16 automated patch clamp system. Pyroaconitine, ajacine, septentriodine, and delectinine demonstrated significant Nav 1.2 channel inhibition (57–42 %) at 10 µM concentration; several other compounds (acovulparine, acotoxicine, hetisinone, 14-benzoylaconine-8-O-palmitate, aconitine, and lycoctonine) exerted moderate inhibitory activity (30–22 %), while the rest of the tested alkaloids were considered to be inactive. On the basis of these results and by exhaustive comparison of data of previously published computerized QSAR studies on diterpene alkaloids, certain conclusions on the structure-activity relationships of Aconitum alkaloids concerning Nav 1.2 channel inhibitory activity are proposed.
Key words Aconitum moldavicum · Ranunculaceae · aconitine · norditerpene alkaloids · Nav 1.2 sodium channel inhibitory activity Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica
Aconitum species native to Asia are herbs used as painkillers, anaesthetics, cardiotonics, and antirheumatic agents in traditional Oriental medicine [1, 2]. Characteristic compounds of these plants are the diterpenoid alkaloids [3]. Antinociceptive and anti-epileptic activity effects of diterpene alkaloids may be mediated by an action on voltage-gated Na+ channels. The analgesic effect of Aconitum preparations had been attributed earlier to aconitine (23), the main alkaloid of several species. Aconitine and numerous diterpene alkaloids have been demonstrated to have peripheral analgesic, antinociceptive properties in different test models [3]. Alkaloids that activate voltage-dependent Na+ channels are antinociceptive since they depolarize neurons permanently, and hence block the neuronal conduction
[4]. Na+ channel blockers possess antinociceptive activity by inhibiting neuronal activity [4, 5]. Inhibition of neuronal activity and anti-epileptiform activity of several diterpene alkaloids has been demonstrated by Ameri et al. on rat hippocampal slices in vitro. This anti-epileptiform activity of diterpene alkaloids is also in line with their blockade of the Na+ channels since Na+ channels are known to be involved in the genesis of abnormal activities in epilepsy. Na+ channel-blocking compounds, e.g., lappaconitine, frequency-dependently inhibit experimentally induced epileptiform activity by sparing the normal neuronal activity [3]. Voltage-gated sodium channels (VGSCs) in sensory neurons have been found to play a crucial role, among others, in several chronic painful neuropathies of different aethiology. Pharmacons that exhibit a use-dependent blockade of these channels, like anticonvulsants, antiarrhythmics, and local anaesthetics, have long been used, for example in the treatment of chronic neuropathic or inflammatory pain states, epilepsies, migraine, and neurodegeneration related to ischaemia [6, 7]. VGSCs (Nav 1.x) are members of the superfamily of ion channels that includes voltage-gated potassium and calcium channels, however, in contrast to these channel molecules, different sodium channel isoforms are relatively similar regarding their functional properties [8]. Nine isoforms of the alpha subunit have been discovered and characterised, each consisting of four homologous domains forming the voltage-gated aqueous pore. These subunits are > 75 % identical over the amino acid sequences comprising the transmembrane and extracellular domains. Despite of this high level of homogeneity, the distinct expression patterns and variable associations with accessory β-subunits enable a specialised functional role of the various isoforms [8]. Developing subtype selective pharmacons that only block the channel isoforms specifically involved in the disease to be treated is a widely used strategy in modern drug research, as reviewed by Tarnawa et al. who also discuss patent information on several subtype selective compound families being under development [6]. Theoretically, such compounds offer a relatively good side-effect profile, by leaving the channel subtypes involved in important physiological functions, such as the heart type Nav 1.5, untouched. Unfortunately, sodium channel blockers currently applied with different therapeutic indications discriminate poorly between Nav 1.x subtypes, and their tolerable therapeutic indices are rather the consequence of their use-dependent properties. Another important aspect is that compounds used therapeutically should be devoid of or have the least minimal activity against the hERG cardiac potassium channel being involved in cardiac toxicity [9, 10]. The present paper reports on a phytochemical investigation of a diterpene alkaloid-accumulating species, Aconitum moldavicum Hacq. (Ranunculaceae) (Carpathian or eastern monkshood), which is a 60–200 cm tall plant occurring in the eastern and central parts of Europe, extending to Romania and West Ukraine. One new [1-O-demethylswatinine (1)] and seven already known norditerpene alkaloids [cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and " Fig. 1) were isolated from the MeOH extract preajacine (8)] (l pared from the fresh roots of A. moldavicum by a combination of different chromatographic methods, including CC, VLC, CPC, and preparative TLC. The structures of the isolated compounds were elucidated by spectroscopic methods. Compound 1 was isolated as an amorphous solid with [α]28 D + 22 (c 0.1, CHCl3,). It was shown by HRESIMS to have the molecular formula of C24H39NO8 according to the quasimolecular ion peak Borcsa B et al. Diterpene Alkaloids from …
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Fig. 1 Chemical structures of the investigated diterpene alkaloids (1–27).
at m/z 470.2761 [M + H]+ (calcd. for C24H40O8 470.2754), which afforded fragment ions at m/z 452 [M + H – H2O]+ and 320 [M + H – CH3OH]+. The 1H and 13C JMOD (J-Modulated Spin-Echo Experiment) spectra of 1 indicated the presence of an N-ethyl group (δH 1.11 t, 2.97 m and 2.85 dq; δC 50.3 and 14.0). Singlet signals at δH 3.35, 3.43, and 3.45 (each 3H, s) and carbon signals at δC 56.3, 57.8, and 58.0 demontrated the presence of three methoxy groups. Further, the JMOD spectrum suggested that the skeleton consisted of 19 carbons, including six methylenes, eight meth" Table 1). From the HSQC ines, and five quaternary carbons (l spectrum, the chemical shifts of the protonated carbons were assigned, and the proton-proton connectivities were then studied. The 1H-1H COSY spectrum defined structural fragments with correlated protons: –CHR–CH2–CH2– (unit A, C-1–C-3) (δH 4.06 brs, 1.72 m, 1.68 m, 1.98 m and 1.47 m), –CH–CHR– (unit B, C-5–C-6) (δH 2.15 s, 4.03 s), –CH–CHR–CH–CH2– (unit C, C-9–C-14–C-13– C-12) (δH 2.83 d, 4.11 t, 2.58 dd, 2.34 d, and 1.95 dd), –CH2–CH– (unit D, C-15–C-16) (δH 2.63 dd, 1.80 dd, and 3.26 dd), two isolated methylenes [δH 3.40 d, 3.70 d (C-18), and 2.45 d, 2.49 d (CBorcsa B et al. Diterpene Alkaloids from …
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" Fig. 1). The long-range correla19)], and one N-ethyl group (l tions detected in the HMBC spectrum proved that structural elements A–D and the two methylene groups (C-18 and C-19), together with the quaternary carbons C-4, C-7, C-8, C-10, and C11, build up an aconitane diterpene substituted in C-1, 6, 7, 8, " Table 1, Fig. 2). The location of the me10, 14, 16, 18 positions (l thoxy groups were established via the HMBC experiment. The long-range correlations of C-6, C-14, and C-16 with the protons at δH 4.03 s, 4.11 t, and 3.26 dd demonstrated the presence of methoxy groups at C-6, C-14, and C-16, respectively. The four hydroxy groups in 1 were located of necessity on C-1, C-7, C-8, C10, and C-18. The stereochemistry and relative configuration of " Table 1). As 1 were studied by means of a NOESY experiment (l reference point, the β stereochemistry of H-5 was used, which is characteristic for norditerpene alkaloids. The Overhauser effects between H-5/H‑9, H-5/H-3β, H-3β/H‑1, H-9/H-14, H-9/8-OH, H14/13, H-13/H-12β, H-12β/H‑14, and 8-OH/H-6 were indicative of the β position of these protons and 8-OH group. On the other hand, NOE effects observed between H-6/H-19, H-17/H-16, H-
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Table 1 NMR data of compounds 1, 3, and 4 [500 MHz (1H), 125 MHz (13C), CDCl3, δ (ppm) (J = Hz)]. 1 1
H
13
C
69.8 34.6
HMBC (C→H)
1 2α 2β 3α 3β 4
4.06 brs 1.72 m 1.68 m 1.98 m 1.47 m –
38.2
5 6
2.15 s 4.03 s
40.8 91.0
7 8
– –
87.2 76.8
9 10 11 12α 12β 13 14 15α 15β 16
2.83 d (2.5) – – 2.34 d (15.1) 1.95 dd (15.1, 7.5) 2.58 dd (7.4, 4.6) 4.11 t (4.6) 2.63 dd (14.7, 8.5) 1.80 dd (14.7, 8.3) 3.26 dd (8.5, 8.3)
53.7 83.0 54.1 41.0
5, 6, 15α, 15β 14, 6, 8-OH, 9, 17, 15α, 15β 15α, 12α 9, 12α 5, 6, 17, 12α 16
38.2 82.9 34.6
9, 12α, 15α 14-OMe, 12α 8-OH, 9
82.0
17
2.74 d (1.7)
66.4
9, 12α, 12β, 15α, 15β, 16-OMe 5, 19a, 19b
18a 18b 19a 19b 20a 20b 21 1-OMe 6-OMe 14-OMe 16-OMe 1-OH 8-OH
3.70 d (10.5) 3.40 d (10.5) 2.49 d (11.5) 2.45 d (11.5) 2.97 dq (19.4, 7.3) 2.85 dq (19.4, 7.3) 1.11 t (7.3) – 3.43 s 3.45 s 3.35 s 7.50 s 3.99 s
66.9
3α, 19a, 19b
57.1
5, 17, 20
26.7
3α
18a, 18b, 19a, 19b 6, 5, 18a, 19a, 19b, 3α, 2β 6, 17, 18b, 19a, 19b 5, 6-OMe, 17
NOESY
3
4
1
1
H
H
2α, 3α, 3β, 12α 1 5, 9 1 1 –
3.74 brs 1.67 m 1.58 m 2.10 m 1.91 m –
3.58 t (7.9, 9.2) 2.15 m 2.15 m 1.60 m 1.50 m –
2β, 6, 9 5, 18a, 19a
1.84 m 1.92 m 1.62 m 2.10 m –
1.95 s 3.89 s – –
2.23 t (5.9) 1.87 m – 2.06 m 1.60 m 2.34 m 4.24 t (5.0) 2.42 dd (16.3, 9.3) 2.07 m 3.39 dd (9.2, 4.7)
2.88 d (4.7) – – 3.07 d (15.7) 1.70 m 2.49 dd (7.8, 4.6) 4.10 t (4.8, 4.5) 2.64 dd (15.3, 10.0) 1.70 m 3.17 t (8.5, 7.2)
2.82 s
2.84 d (2.1)
3.46 d (10.5) 3.29 d (10.5) 2.36 d (10.7) 2.08 d (10.8) 2.55 dq (12.4, 7.1) 2.46 dq (12.4, 7.1) 1.13 t (7.1) – – – 3.35 s 3.03 brs*
3.64 d (10.7) 3.37 d (10.9) 2.60 d (12.0) 2.32 d (12.0) 2.90 m 2.81 dq (19.2, 7.2) 1.05 t (7.2) 3.26 s 3.45 s 3.44 s 3.33 s – 4.08 s
– – 2β, 5, 14, 8-OH – – 1, 16, 17 13, 14 12b, 14 9, 12β, 13 16, 17 12α, 15α, 17 12α, 15α, 16, 20a, 21 6 6
50.3
19a
17
14.0 – 58.0 57.8 56.3 – –
20a, 20b – 6 14 16 – –
17 – 8-OH 8-OH 9, 6-OMe, 16-OMe
* not assigned signal
17/H-20, H-16/H-12α, H-16/H-15α exhibited the α-orientation of H-6, H-16 and corroborated that the N-containing bridge, between C-19 and C-17, including the N-ethyl group, is below the plane of ring A. All of the above evidence was used to propose the structure of this compound as 1-O-demethylswatinine (1). Compounds 3 and 4 were isolated as an amorphous solid and identified as columbianine (3) and swatinine (4). These compounds are known diterpenes of the Aconitum genus (found in A. laeve, A. ferox, and A. lamarckii) [11–13], but our 2D NMR studies provided the complete 1H assignments for columbianine (3) " Table 1). In addition, the and swatinine (4) for the first time (l known alkaloids cammaconine (2), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8) were also isolated from A. moldavicum and identified by comparing their spectral data with those reported in the literature and by TLC co-chromatography with an authentic sample [14–16]. The effects on Nav 1.2 channels of the isolated compounds 4–8 were analysed by the automated whole-cell patch clamp tech-
nique. In this study, other diterpene alkaloids (9–27) were also included, which were isolated from A. toxicum, A. anthora, " Table 2). This series of A. vulparia, and Consolida orientalis (l compounds represents diverse structural types, including bisnor(C18), nor- (C19), and diterpene (C20) alkaloids substituted with hydroxy, methoxy, keto, acetyl, and various aromatic ester groups. All the compounds are N-ethyl-substituted with the exception of acovulparine (15), which contains an azomethine group. Pyroaconitine (25) exerted the highest inhibitory activity (57%), thus measurements on the concentration dependence of " Fig. 3) have also been carried out, and the IC this activity (l 50 value for this compound was determined (7.3 ± 1.5 µM). Furthermore, ajacine (8), septentriodine (22), and delectinine (17) also demonstrated significant Nav 1.2 channel inhibition (44–42 %) at 10 µM; several other compounds (acovulparine (15), acotoxicine (14), hetisinone (11), 14-benzoylaconine-8-O-palmitate (24),
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Table 2 Inhibition of Nav 1.2 channels by compounds 4–27 at nominal concentration of 10 µM. Compound
Inhibition
sem
n
10 2 13 22 44 16
4 4 4 5 5 7
5 7 8 7 8 8
18 28 18 6 28 30 3 42 16 15 14 19
5 3 6 5 3 4 2 5 6 4 4 3
5 4 5 7 8 6 4 8 4 7 4 8
43 25 27 57 15 18
6 3 2 2 3 6
7 8 9 7 8 7
(%)
Fig. 2
Selected 1H-1H COSY (—) and HMBC (C→H) correlations for 1.
aconitine (23), and lycoctonine (7) exerted moderate inhibitory activity (30–22%), while the rest of the tested alkaloids were considered to be inactive. Concerning the relationship between the structural features and the Nav 1.2 inhibiting activity of the studied alkaloids, it seems relevant that the concomitant presence of a methoxy function at C-1, the presence of an oxygen-containing functionality at C-8, and the presence of a (substituted) aroyl function either at C-14 or at C-18 is necessary, with the parallel requirement that the number of oxygen functionalities in the molecule should be at least 4. Satisfaction of these stipulations can be seen in the case of all significantly (8, 17, 22, 25) and moderately active compounds (7, 14, 15, 23, 24). When C-1 methoxy is changed to hydroxyl, the inhibitory effects of the compounds decrease as it can be seen in the case of delectinine (17) (42 % inhibition) and takaosamine (16) (3% inhibition). The only, nonetheless very interesting, outlier compound is swatinine (4) (10% inhibition), which has a methoxy group at C-1, however, it is substituted with an unusual extra hydroxyl at C-10. Without this latter substituent, the substitution pattern of the compound is identical with that of acovulparine (15) (with the structural difference occurring on the N atom) (30 % inhibition). This surprising, and rather large activity difference, allows to hypothesise on the importance of hydroxy substitution at C-10 that may pose some kind of steric hindrance for the molecule when interacting with the channel receptor. However, it cannot be excluded that differences in the substitution of the N atom may also be of importance regarding this activity difference. Influence of the position of the aroyl functionality on the inhibitory activity is illustrated by the example of acotoxinine (20), which was found to be inactive, in spite of the fact that it has an aroyl functionality in the molecule, but it is positioned at C-8, not at C-14 or C-18, as it has been described above. These stipulations seem to be supported by a detailed QSAR analysis of 12 diterpene alkaloids, which revealed that the position of the aroyl/aroyloxy groups on C-4 or C-14 is the major determinant of the analgesic activity. Alkaloids with an aroyl or an aroyloxy group on C-14 exhibited an analgesic potency approximately 30 times higher than that of alkaloids with an aroyloxy group on C-4 in a model of acetic acid-induced writhing in rats [17]. Previously, it has also been proven that alkaloids with
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Swatinine (4) Gigactonine (5) Delcosine (6) Lycoctonine (7) Ajacine (8) 10-Hydroxy-8-O-methyltalatizamine (9) Isotalatizidine (10) Hetisinone (11) Aconosine (12) Dolaconine (13) Acotoxicine (14) Acovulparine (15) Takaosamine (16) Delectinine (17) Neolinine (18) Neoline (19) Acotoxinine (20) 14-Desacetyl-18-demethylpubescenine (21) Septentriodine (22) Aconitine (23) 14-Benzoylaconine-8-O-palmitate (24) Pyroaconitine (25) Songoramine (26) Songorine (27)
low affinity for the neurotoxin receptor site 2 of Na+ channels lack antinociceptive action [4]. As regards the three C20 diterpene alkaloids involved in this study, only one (hetisinone, 11) has been found to exert moderate activity, therefore no relevant deductions may be made concerning the structure-activity relationship of these compounds. It is noteworthy, though, to mention that songorine (27) was found practically inactive concerning its inhibitory action, which is in line with the previous results of Friese et al. when observing that this compound has failed to modulate Na+ channels [5]. However, in their study no exact subtype specification was provided on the examined Na+ channels. Further two aspects to be emphasised are that amongst the four most active compounds only one, delectinine (17), is not esterified (although satisfying all other presumably necessary conditions), and that both diester-type compounds [14-benzoylaconine-8-O-palmitate (24) and aconitine (23)] have been found to exert only moderate inhibitory activity on the studied particular channel subtype. In conclusion, it can be stated that distinctly minor substitutional changes seem to have rather firm impact on the inhibitory activity of diterpene alkaloids on Nav 1.2 channel, thus reinforcing the previous observations that minor structural differences may lead to completely different activities. Our results on Nav 1.2 channel inhibitory activity of selected diterpenoid alkaloids may open new perspectives in the research of channel subtype specific compounds, especially when considering that diterpene alkaloids comprise large and chemically variable biologically active compounds altogether having been poorly tested pharmacologically so far. The herein reported specific inhibitory activity of certain diterpene alkaloids on Nav 1.2 channel is intriguing, especially in light of the facts that in differ-
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Fig. 3 Concentration-dependent inhibition of pyroaconitine (25) on sodium current.
ent geographical regions of the world some diterpene alkaloid containing Delphinium and Aconitum species have long been used (both unprocessed and processed drugs) in the traditional medicine for the same analgetic and antiepileptic indications [1, 2] as those compounds being inhibitors of Nav 1.2, which have been reported to be currently under drug development [6]. Considering our herein results, especially in light of our previously reported results on the hERG K+ channel inhibitory activity of these diterpene alkaloids [18], it can be observed that certain compounds [14-benzoylaconine-8-O-palmitate (24), aconitine (23), gigactonine (5)] exerted noteworthy activity in both pharmacological tests, thus despite their promising Nav 1.2 inhibitory activity, these compounds should be considered as potentially harmful by causing adverse cardiovascular effects through their hERG effect, nevertheless, some of these compounds show some selectivity on hERG channels. Their potential cardiovascular safety risk should be evaluated based on their hERG IC50 value and in vivo efficient free plasma concentration. Among the studied active alkaloids, particularly those should be further pursued as model substances for pharmacological lead compounds as selective Nav 1.2 inhibitors, which exert minor or no hERG activity [18], i.e., ajacine (8) and delectinine (17). This is the first paper to report promising experimental results obtained in relation to a specific Nav 1.2 channel subtype inhibitory activity of diterpene alkaloids.
1-O-Demethylswatinine (1): amorphous solid; [α]28 D : + 22 (c 0.1, " Table 1. ESI‑MS‑MS: m/z CHCl3); 1H- and 13C‑NMR data: see l 470.2761 [M + H]+ (calcd. for C24H40NO8, m/z 470.2754). Bioassay: CHO cells stably expressing human Nav 1.2 sodium channels were investigated by the whole-cell patch clamp using the QPatch-16 automated patch clamp system. Cells were held at − 65 mV and activated by a train consisting of 20 steps to 0 mV for 8 ms at 10 Hz. Train protocols were applied at 10-s intervals. The current traces were recorded, and the amplitude of peak current evoked by the last pulse within a train was measured as test parameter. The inhibition was calculated from the peak currents in the presence and absence of the test compound. The control solution contained the same concentration of the vehicle (DMSO or HCl) as the solutions of the test compound. Solutions containing the test compounds were applied to the cells for 6–8 min. Aconitine (23) was purchased from Sigma-Aldrich. Semisynthetic preparations of benzoylaconine 8-O-palmitate (24) and pyroaconitine (25), alike the isolation of 10-hydroxy-8-O-methyltalatizamine (9), isotalatizidine (10), and hetisinone (11) from A. anthora; aconosine (12), dolaconine (13), acotoxicine (14), delectinine (17), neolinine (18), neoline (19), acotoxinine (20), songoramine (26), and songorine (27) from A. toxicum; acovulparine (15) and septentriodine (22) from A. vulparia; delcosine (6), gigactonine (5), takaosamine (16) and 14-desacetyl-18-demethylpubescenine (21) from Consolida orientalis were described ear" Fig. 1). The purities of the compounds were inlier in ref. [18] (l vestigated by means of 1H‑NMR spectroscopy [19], and all found to be > 95 %. Lamotrigine (purity > 98 %; Sigma-Aldrich) was used as positive control. Lamotrigine at 300 µM caused 89 ± 2% (n = 9) inhibition of sodium current.
Supporting information General experimental procedures, extraction and isolation, and NMR spectra of compound 1 are available as Supporting Information.
Acknowledgements !
The publication was supported by the European Union and cofunded by the European Social Fund (TÁMOP‑4.2.2.A‑11/1/ KONV-2012–0035). Financial support of the Hungarian Scientific Research Fund (OTKA K109846) is gratefully acknowledged.
Conflict of Interest !
The authors declare no conflict of interest.
References
Materials and Methods !
Plant material: A. moldavicum was collected near Eger in October 2006. The plant material was identified by Attila Molnár V. (Department of Botany, University of Debrecen, Hungary). A voucher specimen (No. 807) has been deposited in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary. For chromatographic purification, a combination of different chromatographic methods, including CC, VLC, CPC, and preparative TLC has been applied (for details see Supporting Information).
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received revised accepted
May 17, 2013 October 7, 2013 December 16, 2013
Bibliography DOI http://dx.doi.org/10.1055/s-0033-1360278 Published online January 22, 2014 Planta Med 2014; 80: 231–236 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Judit Hohmann Department of Pharmacognosy University of Szeged Eotvos u. 6 6720 Szeged Hungary Phone: + 36 62 54 64 53 Fax: + 36 6 25 45 7 04 [email protected]
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