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Mechanism of action of antiepileptic ceramide from Red Sea soft coral Sarcophyton auritum
Accepted Manuscript Mechanism of Action of Antiepileptic Ceramide from Red Sea Soft Coral Sarcophytonauritum Nermeen A. Eltahawy, Amany K. Ibrahim, Mohamed M. Radwan, Sawsan Zayton, Mohamed Gomaa, Mahmoud A. ElSohly, Hashim A. Hassanean, Safwat A. Ahmed PII: DOI: Reference:
S0960-894X(15)00874-4 http://dx.doi.org/10.1016/j.bmcl.2015.08.039 BMCL 23033
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
2 May 2015 7 August 2015 14 August 2015
Please cite this article as: Eltahawy, N.A., Ibrahim, A.K., Radwan, M.M., Zayton, S., Gomaa, M., ElSohly, M.A., Hassanean, H.A., Ahmed, S.A., Mechanism of Action of Antiepileptic Ceramide from Red Sea Soft Coral Sarcophytonauritum, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.08.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bioorganic & Medicinal Chemistry Letters
Mechanism of Action of Antiepileptic Ceramide from Red Sea Soft Coral Sarcophytonauritum Nermeen A. Eltahawya, Amany K. Ibrahima, Mohamed M. Radwanb, c,SawsanZaytond, Mohamed Gomaa e, Mahmoud A. ElSohlyb,f, Hashim A. Hassaneana, Safwat A. Ahmeda, * a
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA. c Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt. d Department of Pharmacology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. e Department of Medicinal chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. f Department of Pharmaceutics and Drug Delivery, School of Pharmacy, The University of Mississippi, University, MS 38677. b
*
To whom correspondence should be addressed. Dr. Safwat Ahmed, Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, Phone: 01092638387, Fax: (+20)064-3230741, e-mail: [email protected].
A RT I C L E I N F O
A BS T RA C T ~ U I O /
Article history: Received Revised Accepted Available online
Chemical investigation of the Red Sea soft coral Sarcophytonauritum led to the isolation and structure elucidation of a new ceramide N-((2S,3R,4E,6E)-1,3-dihydroxyhenicosa-4,6-dien-2yl)tridecanamide (1). Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. The anticonvulsant activity of the isolated ceramide was measured in vivo using the pentylenetetrazole (PTZ)-induced seizure model, where it successfully antagonized the lethality of pentylenetetrazole in mice. In addition, the isolated ceramide showed good anxiolytic activity when used in the light-dark transition box and the elevated plus maze compared to diazepam. The molecular modeling studies for the antiepileptic and antianxiety mechanism of the isolated ceramide suggested a CNS depressing activity possibly through GABA and serotonin receptors modulation. The pharmacological activity of the ceramide involved agonistic activity on GABA-A receptors but not 5HT3 receptors.
Keywords: Sarcophytonauritum ceramide Anticonvulsant Anxiolytic GABA receptors 5HT3 receptors
The marine environment is an exceptional reservoir of bioactive natural products, many of which exhibit structural/chemical features not found in terrestrial natural products1. In recent years, many bioactive compounds have been isolated from various marine organisms like tunicates, sponges, soft corals, sea hares, nudibranchs, bryozoans, sea slugs and marine organisms.2 Soft corals or Alcyonacea are an order of corals which do not produce calcium carbonate skeletons. Soft corals contain minute, spiny skeletal elements called sclerites, useful in species identification. Sclerites give these corals some degree of support and give their flesh a spiky, grainy texture that deters predators.3An abundance of unique secondary metabolites including sesquiterpenoids, diterpenoids, steroids, ceramides and other chemical compounds have been isolated and identified from various species of soft corals.4 Sarcophyton is a widespread genus of corals extending from Eastern Africa and the Red sea in the west to Polynesia in the east. About 14 species of the 36 known Sarcophyton species are found in the Red Sea.5
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There are few studies about chemical constituents of the marine soft coral Sarcophytonauritum,from which cytotoxic cembranoids have been isolated.6 Many marine invertebrates are rich sources of ceramides that differ in structure and biological properties from those of terrestrial organisms. Unusual ceramides have been isolated from sponges7-9, coelenterata10-12, crabs13, sea stars14, and ascidia15. As bioactive lipids, ceramides have been implicated in a variety of physiological functions including apoptosis, cell growth arrest and cell senescence.16 Ceramides are precursors of complex sphingolipids (SLs), which are important for normal functioning of both the developing and mature brain. Altered SL levels have been associated with many neurodegenerative disorders, including epilepsy and Alzeheimer disease.17-19 In our continuing search for new compounds isolated from the soft corals inhabited in various locations of the Red Sea20, we were able to isolate a new ceramide, compound 1 (Figure 1), from the Red Sea soft coralSarcophytonauritum. Compound 1 was screened for anxiolytic activity using the lightdark transition box and elevated plus maze. It was also screened forin vivo anticonvulsant activity using the pentylenetetrazole
(PTZ)-induced seizure model. Molecular modeling studies were also performed to further substantiate the proposed mechanism of action of this ceramide.It suggested a CNS depressing activity possibly through a GABA and serotonin receptors modulation. Moreover, the study includes assessment of the mechanism of anxiolytic activity for the isolated ceramide.
Figure 1: Compound 1
The soft coral (5 kg, wet weight) was freeze dried and then exhaustively extracted with MeOH/CH2Cl2 (1:1) for 5 days. The extract was concentrated till drying (150 g) under vacuum, then fractionated on vacuum liquid chromatography (VLC) using silica gel, (mesh 230–400 nm,) and step gradient elution with a non-polar solvent (n-Hexane) with increasing the polarity using CH2Cl2 then MeOH to give nine fractions(1-9). Fraction 5 (25% MeOH in CH2Cl2) was chromatographed using silica gel 230 x 400 mesh (40-63 µm) to give the ceramide ,eluted with 60% CH2Cl2 in hexane, and it was subjected to final purification on Sephadex LH-20 column using MeOH: CH2Cl2 (1:1) as an eluent, where compound 1 was obtained in pure form (5mg). Compound 1 was obtained as white powder, [α]D22= +3.1(c=0.33, CHCl3); IR (film) 2942, 1743, 1465, 1377 cm-1. The HRESIMS exhibited an [M+H]+ ion at m/z536.5043 (calcd536.5043), indicating the molecular formula of C34H66NO3 and three degrees of unsaturation as supported by NMR data. The 13 C-NMR spectrum, (table 1), showed 31 carbon resonances. Characteristic resonances of a 2-amino-1,3-diol unit of the hydrocarbon chain were observed at δC57.3 (C-2), 62.6 (C-1) and 73.7 (C-3). Also, there are four observed resonances for olifinic carbons at δC130.4 (C-4), 133.2 (C-5), 132.1 (C-6) and 131.6 (C7). In addition, there is a resonance at δC14.5 assigned for the two terminal methyl groups (C-21 and C-13') and at δC174.0 assigned for the amide carbonyl (C-1'). The 1H-NMR spectrum, (table 1), showed resonances of an amide proton doublet at δH 8.37 (d, J= 9.2 Hz) and protons of a long methylene chain at δH1.28, indicating a sphingolipid (SL) skeleton. Also characteristic resonances of a 2-amino-1,3-diol unit of the hydrocarbon chain were observed at δ H 4.77 (m),[4.31 (dd, J= 8.0, 4.8 Hz), 4.47 (dd, J= 8.0, 4.8 Hz)] and 4.86 (m) assigned for H-2, H2-1 and H-3 respectively and resonances for four olefinic protons at δH5.51 (m) assigned for (H-4 and H-7) , 6.01 (m) assigned for H-5 and 6.11 (m) for H-6.In addition, resonancescorresponding to aliphatic hydrocarbons at δH 0.88 (t, J= 6.8 Hz) assigned for H-21 and H-13', 1.28 (overlapped H, m), 1.39 (m) assigned for H-3' and 2.48 (t, J=7.6 Hz) assigned for H2'. The position of the double bonds were confirmed through the 1H-1H COSY spectrum, as there were correlations between H-3/H-4, H-4/H-5, H-5/H-6, H-6/H-7 and H-7/H2-8, as well as the HMBC spectrum which showed correlations from H3/C-5 (3JCH), H-4/C-2 (3JCH), H-6/C-4 (3JCH), H2-8/C-6 (3JCH) leading to the assignment of the C-4/C-5 and C-6/C-7 double bonds (Figure 2). The position and geometry of the double bonds were confirmed by 1H-1H COSY analysis and coupling constant data. The J4,5 value of (15.0 Hz) indicated the trans geometry of these double bonds. On the other hand, it is known that the geometry of the double bond in a long chain alkene can be determined from the 13C-NMR chemical shift of the methylene carbon atom next to the olefinic carbon atom. The carbon signal is observed near to
27 in the (Z) type and near to 32 in the (E) type.20 Therefore, compound 1was assigned as a 4E,6Esphingadiene type ceramide. GC-MS analysis of the fatty acid methyl ester of compound 1 was carried out after hydrolysis. The GC-MS analysis exhibited a peak at molecular ion of m/z (228) corresponding to a C13 fatty acid methyl ester which indicated the presence of only one terminal fatty acid tridecanoic (C13:0) acid. The configuration of compound 1 moieties was assigned through comparison of 13C NMR with analogs as reported in the literature. The chemical shifts of C-1 (δc 62.5), C2 (δc 54.6) and C-3 (δc 74.5) in CDCl3 were very similar to those of the neurotrophic ceramide, (4E,6E,2S,3R)-2-N-eicosanoyl-4,6tetradecasphingadienine21. Further confirmation of the absolute configuration of 1 was determined via the Mosher ester analysis protocol22.This evidence indicated the absolute configurations at C-2 and C-3 to be 2S and 3R, respectively.
Figure 2: Selected 1H-1H COSY (─), HMBC correlations (→) of
compound 1 Table 1.1H and 13C NMR spectral Data for compound 1 (400 MHz, δ in ppm, J in Hz, C5D5N) *
compound 1
No. 1 2 3 4 5 6 7 8 9-16
δH (No. of H, mult.,JHz) 2H; 4.31 (dd, 8.0, 4,8), 4.47 (dd, 8.0, 4,8) 1H, 4.77 (m) 1H, 4.86 (m) 1H, 5.51 (m) 1H, 6.01 (m) 1H, 6.11 (m) 1H, 5.51 (m) 2H, 2.03(m) 2H, 1.28 (m)
δC 62.6 57.3 73.7 130.4 133.2 132.1 131.6 32.6 30.5
17 2H, 1.28 (m) 30.0 18 2H, 1.28 (m) 37.4 19 2H, 1.28 (m) 30.2 20 2H, 1.28 (m) 23.4 21 3H, 0.88 (t, 6,8) 14.5 1' 174.0 2' 2H, 2.48 (t, 7.6) 33.4 3' 3H, 1.39 (m) 26.9 4'-10' 2H, 1.28 (m) 30.5 11' 2H, 1.28 (m) 32.6 12' 2H, 1.28 (m) 23.4 13' 3H, 0.88 (t, 6.8) 14.5 NH 8.37 (d, 9.2) * Chemical shifts (δ) are in ppm. Coupling constants (J) are in Hz
GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. To date, the GABA(A) receptors have not been crystallized. However, X-ray structure of a pentameric ligand gated ion channel from Erwiniachrysanthemi (ELIC) in complex with GABA and flurazepam has recently been deposited in the PDB (PDB code 2YOE).23This prokaryotic homolog ELIC
Compound 1 GABA modulator fitted well in the GABA allosteric site and established several hydrophobic and electrostatic interactions. Residues involved in ligand binding at the allosteric site include VAL73, PRO74, ASN60, ASN89, ILE39, LEU76, ALA75, THR87, GLY88, PRO85, SER84, PHE78. TYR102 may also play a role through potential hydrogen bonding withcompound 1 secondary OH (Figure 4).
schizophrenia and drug abuse.25, 26 Antagonists of the 5-HT3R, such as granisetron and ondansetron, are in clinical use as antiemetics to suppress nausea and vomiting induced by general anaesthetics, chemotherapeutics and radiotherapy. 25, 26 Docking of compound 1 in 5HT-1B receptors (PDB code 4IAQ) did not show initial promising results. The 5-HT3receptor differs markedly in structure and mechanism from the other 5-HT receptor subtypes, which are all G-protein-coupled. Therefore, we decided to study the possible binding pattern of compound 1 to 5HT-3 receptor. The 5-HT3 receptor is a member of the super family of ligand-gated ion channels, and consists of 5 subunits arranged around a central ion conducting pore, which is permeable to sodium, potassium, and calcium ions. Binding of the neurotransmitter 5-hydroxytryptamine to the 5-HT3 receptor opens the channel, which, in turn, leads to an excitatory response in neurons. To date the crystal structure of 5HT-3 has not been resolved. The crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor (PDB code 2YME) was used in our molecular modeling study to assess the possible mechanism of the CNS inhibitory activity of compound 1 as a potential HT-3 inhibitor.27, 28 The results showed that compound 1 docked well in the active site and established hydrophobic interactions with W53, W145, Y193, Y186, D162. A potential hydrogen bonding is also noted between compound 1 carbonyl oxygen and the C188 SH (Figure 6).
Figure 4: Compound 1 GABA modulator binding to GABA allosteric site;distances in Ǻ are indicated by green lines for potential hydrogen bonding.
Figure 6: Compound 1 binding to engineered 5-HTBP;distances in Ǻ are indicated by dotted lines for potential hydrogen bonding.
is also activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors and therefore represents a good target for GABA(A) molecular modeling studies. The docking studies revealed that the docking pattern of compound 1 is very close to that of the co-crystallized benzodiazepine (Flurazepam) suggesting a possible similar mode of action (Figure 3).
Figure 3:Compound 1 GABA modulator (yellow) and Flurazepam (red) bound to GABA allosteric site. Co-crystallised GABA occupying the GABA active sites on the opposite side of the receptors.
The electrostatic potential surface representation showed that the surface of the GABA allosteric site accommodated compound 1 without any sterric collision (Figure 5).
Compared to the selective inhibitor granisetron, compound 1 exhibited a similar docking pose and lied in the same channel as the potent inhibitor. However, compound 1 lacked some key interactions including the Π-cation interaction with R55 as well as the hydrogen bonding of the protonated tropane ring with W 53 backbone. The hydrogen bonding with the water molecule is less likely to happen in case of compound 1 as the carbonyl oxygen is situated further at a distance of 4.6 Ȧ compared to 2.6 Ȧ in case of granisetron (Figure 7). Therefore, compound 1 represents a potential but not a potent and selective blocker of 5HT3
Figure 5:Compound 1 GABA modulator binding to GABA allosteric site represented as an electrostatic potential surface
Neuronal 5-hydroxytryptamine (5-HT, serotonin) signaling pathways are some of the most complex in the human body. There are at least 15 5-HT receptors grouped into seven distinct signaling families and these are involved in a diverse array of complex brain functions, such as physiological, emotional and cognitive control, including regulation of appetite, sleep, sexual behaviour, anxiety, learning and memory.24 All 5HT receptors are G-protein coupled, except for the 5-HT3 receptor (5-HT3R),which is a pentameric ligand-gated ion channel. This receptor has been implicated in hippocampusdependent memory formation, acts in anxiety, and contributes to
Figure 7:Compound 1 (blue) and granisetron binding to engineered 5-HTBP;distances in Ǻ are indicated by dotted lines for potential hydrogen bonding.
In conclusion, the results of the comparative docking studies indicated that the CNS activity of compound 1 is possibly
through GABA receptor modulation rather than serotonin receptors inhibition. Screening for anxiolytic activity in the light dark transition box revealed that treatment with diazepam (1 mg/kg, i.p.) or the test compound1 increased the time spent by mice in the light area of the dark light transition (Figure 8). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), before compound 1 significantly decreased the cumulative time spent in the light area compared to single treatment with compound 1 (Figure 8A). Hence, blockage of GABA-A receptors attenuated the anxiolytic activity of compound 1. Meanwhile, pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.), before compound 1 did not produce a change in the time spent in the white area compared to single treatment with compound 1 (Figure 8B), ruling out the involvement of 5-HT3 receptors in the anxiolytic activity of compound 1.
Figure 9: Effect of diazepam and compound1 on percent time spent in the open arm in the mouse elevated plus maze. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the percent time. B) Effect of pretreatment with the 5HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on percent time.Mice were screened for anxiolytic activity in the elevated plus maze for 3 min. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P < 0.05. *Compared to vehicle group, #Compared to diazepam, $Compared to compound 1, n = 6.
Figure 8: Effect of diazepam and compound 1 on the time spent in the light area in the dark-light transition test. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the recorded time. B) Effect of pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on the recorded time. Mice were screened for anxiolytic activity in the dark-light transition test for 3 minutes. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P<0.05. *Compared to vehicle group, #Compared to diazepam, $ Compared to compound 1, n = 6.
On the other hand, screening of the anxiolytic activity in the elevated plus maze indicated that the test compound 1 as well as diazepam (1 mg/kg) increased the open arm time % compared to the vehicle treated mice (Figure 9A&B). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), before compound 1 significantly decreased the percent open arm time compared to single treatment with compound 1 (Figure 9A). Meanwhile, pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.), before compound 1 did not produce a change in the percent open arm time compared to single treatment with compound 1 (Figure 9B).
The ability of the test compound 1 to prolong time to death after an acute dose of pentylenetetrazole (70 mg/kg, i.p.) was used as an indicator for their anticonvulsant activity. The test compounds successfully antagonized the lethality of pentylenetetrazole; time to death recorded with the test compound 1 was significantly higher than that recorded in the vehicle group. Importantly, the time to death recorded with compound 1 was significantly higher than that reported with an equivalent dose of diazepam (911±87 vs. 536±12, P<0.05, Figures 10A&B). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), significantly attenuated the anticonvulsant effect of compound 1 (Figure 10A). However, pretreatment with the 5-HT3 receptorblocker, ondansetron(1 mg/kg, i.p.) did not affect the anticonvulsant effect of compound 1 (Figure 10B). This indicated that the anticonvulsant activity of compound 1 is, at least in part, mediated by GABA-A receptors. In conclusion, a new ceramide was isolated from red soft corals. The in vivo testing showed good anxiolytic and CNS depressing activity compared to the standards. Competitive in vivo blocking assays showed that GABA-A receptors could be a preferential target for this compound rather than 5-HT3 receptors. The molecular modeling studies supported the in vivo findings by showing a better interaction of the isolated ceramide with GABA-A receptors compared to the 5-HT3 receptors.
8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18.
19. Figure 10: Effect of diazepam and the compound 1 on the time to death after injection of pentylenetetrazole. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the time to death. B) Effect of pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on the time to death. Mice were screened for anticonvulsant activity for 20 min. PTZ: pentylenetetrazole. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P < 0.05. *Compared to vehicle group, #Compared to diazepam, $Compared to compound 1, n = 6.
Acknowledgments We thank Dr. Tarek Temraz, marine science department, Suez Canal University for identification of the soft coral. We are also thankful to Dr. BaharthiAvula, National Center for Natural Products Research, School of Pharmacy University of Mississippi for HRMS data, References and notes 1. 2. 3. 4. 5. 6.
7.
Carte´, B. K. , Biosceinces, 1996, 46, 271. Donia, M.; Hamann, M. T. The Lancet. 2003, 3, 338. Jeng, M.S.; Huang, H. D.; Dai, C.F; Hsiao, Y.C.; Benayahu, Y. Coral Reefs, 2011, 30, 925. Blunt J.W.; Copp B.R.; Keyzers R.A.; Munro M.H.; Prinsep M.R. Nat. Prod. Rep. 2013, 30, 237. Fabricus, K. and Alderslade, P. Soft corals and Sea Fans. 1st Ed., Australian Institute of Marine Science press, Queensland. 2001, pp 1-41. Eltahawy, N. A.; Ibrahim, A. K.; Radwan, M. M.; ElSohly M. A.; Hassanean, H. A.; Ahmed, S. A.Tetrahedron Lett.2014, 55, 3984. Grg, H. S.; Agrawal, S. J. Nat. Prod.1995, 58, 442.
20.
Hattori, T.; Adachi, K.; Shizuri, Y. J. Nat. Prod.1998, 61, 823. Muralidhar, P.; Krishna, N.; Kumar, M. M.; Rao, C. B.; Rao, D. V. Chem. Pharm. Bull.2003, 51, 1193. Shin, J.; Seo, Y. J. Nat. Prod.1995, 58, 442. Chebaane, K.; Guyot, M. Tetrahedron Lett.1986, 27, 1495. Chakrabarty, M.; Batabyal, A.; Barua, A. K. J. Nat.Prod.1994, 57, 393. Asai, N.; Fusetani, N.; Matsunaga, S.; Sasaki, J. Tetrahedron 2000, 56, 9895. Inagaki, M.; Isobe, R.; Kawano, Y.; Miyamoto, T.; Komori, T.; Higuchi, R. Eur. J.Org. Chem.1998, 1, 129. Lukasv, A.; Bultel-Ponce, V.; Longeon, A.; Guyot, M. J. Nat. Prod.2000, 63, 799. Hannun, Y.A. and Obeid, L.M. "Principles of bioactive lipid signalling: lessons from sphingolipids". Nature Reviews Molecular Cell Biology, 2008, 9 (2): 139. Ahmed, S.A.; Khalifa, S.I.; Hamann, M.T. J. Nat. Prod.2008, 71, 4. Mielke, M.M.1; Bandaru, V.V.; Haughey, N.J.; Xia, J.; Fried, L.P.; Yasar, S.; Albert, M.; Varma, V.; Harris, G.; Schneider, E.B.; Rabins, P.V.; Bandeen-Roche, K.; Lyketsos, C.G.; Carlson, M.C. Serum ceramides increase the risk of Alzheimer disease: the Women's Health and Aging Study II. Neurology.2012, 14;79(7):633. Mosbechl, M.; Olsen1, A.S.; Neess, D.; David, O.B.; Klitten, L.L.; Larsen, J.; Sabers, A.; Vissing, J.; Nielsen, J.E.; Annals of Clinical and Translational Neurology 2014; 1(2): 88. Ormond, R.F.G.; Edwards, A.J. IUCN/Pergamon Press,Oxford, 1987, pp 251–287
21. Inagakia, M.; Isobeb, R.; Kawanoa, Y.; Miyamotoa, T.; Komoria, T.; Higuchi, R., Eur. J. Org. Chem. 1998, 129. 22. Mancini, l.; Guella, G; Debitush, C.; Pietra, F. Helv. Chim. Acta. 1994, 77, 51. 23. Kwon, H. C.; Lee, K. C.; Cho, O. R.; Jung, I. Y.; Cho, S. Y.; Kim, S. Y.; Lee, K. R. J. Nat. Prod., 1991, 66, 466. 24. Spurny, R.; Ramerstorfer, J.; Price, K.; Brams, M.; Ernst, M.; Nury, H.; Verh;eij, M.; Legrand, P.; Bertrand, D.; Bertrand, S.; Dougherty, D.A.; de Esch, I.J.; Corringer, P.J.; Sieghart, W.; Lummis, S.C.; Ulens, C. Proc Natl Acad Sci U S A. 2012, 109(44), 3028. 25. Berger, M., Gray, J.A., Roth, B.L. Annu Rev Med, 2009, 60: 355. 26. Thompson, A.J.; Lummis, S.C. ExpertOpinTher Targets, 2007, 11: 527. 27. Walstab, J.; Rappold, G.; Niesler, B. PharmacolTher, 2010, 128: 146. 28. Kesters, D., Thompson, A.J., Brams, M., Van Elk, R., Spurny, R., Geitmann, M., Villalgordo, J.M., Guskov, A., Helena Danielson, U., Lummis, S.C.R., Smit, A.B., Ulens, C., Embo Rep., 2013, 14, 49.
Supplementary Material 1D and 2D NMR spectral data of 1is available.
Graphical Abstract
Mechanism of Action of Antiepileptic Ceramide
Leave this area blank for abstract info.
from Red Sea Soft Coral Sarcophyton auritum Nermeen A. Eltahawya, Amany K. Ibrahima, Mohamed M. Radwanb, c, Sawsan Zaytond, Mohamed Gomaae, b,f
a
a, *
Mahmoud A. ElSohly , Hashim A. Hassanean , Safwat A. Ahmed
3'
13'
2' 4'
1' O
NH
HO
1
2
3 OH
.
5 4
6
7 21
S0960-894X(15)00874-4 http://dx.doi.org/10.1016/j.bmcl.2015.08.039 BMCL 23033
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
2 May 2015 7 August 2015 14 August 2015
Please cite this article as: Eltahawy, N.A., Ibrahim, A.K., Radwan, M.M., Zayton, S., Gomaa, M., ElSohly, M.A., Hassanean, H.A., Ahmed, S.A., Mechanism of Action of Antiepileptic Ceramide from Red Sea Soft Coral Sarcophytonauritum, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.08.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bioorganic & Medicinal Chemistry Letters
Mechanism of Action of Antiepileptic Ceramide from Red Sea Soft Coral Sarcophytonauritum Nermeen A. Eltahawya, Amany K. Ibrahima, Mohamed M. Radwanb, c,SawsanZaytond, Mohamed Gomaa e, Mahmoud A. ElSohlyb,f, Hashim A. Hassaneana, Safwat A. Ahmeda, * a
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA. c Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt. d Department of Pharmacology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. e Department of Medicinal chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. f Department of Pharmaceutics and Drug Delivery, School of Pharmacy, The University of Mississippi, University, MS 38677. b
*
To whom correspondence should be addressed. Dr. Safwat Ahmed, Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, Phone: 01092638387, Fax: (+20)064-3230741, e-mail: [email protected].
A RT I C L E I N F O
A BS T RA C T ~ U I O /
Article history: Received Revised Accepted Available online
Chemical investigation of the Red Sea soft coral Sarcophytonauritum led to the isolation and structure elucidation of a new ceramide N-((2S,3R,4E,6E)-1,3-dihydroxyhenicosa-4,6-dien-2yl)tridecanamide (1). Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. The anticonvulsant activity of the isolated ceramide was measured in vivo using the pentylenetetrazole (PTZ)-induced seizure model, where it successfully antagonized the lethality of pentylenetetrazole in mice. In addition, the isolated ceramide showed good anxiolytic activity when used in the light-dark transition box and the elevated plus maze compared to diazepam. The molecular modeling studies for the antiepileptic and antianxiety mechanism of the isolated ceramide suggested a CNS depressing activity possibly through GABA and serotonin receptors modulation. The pharmacological activity of the ceramide involved agonistic activity on GABA-A receptors but not 5HT3 receptors.
Keywords: Sarcophytonauritum ceramide Anticonvulsant Anxiolytic GABA receptors 5HT3 receptors
The marine environment is an exceptional reservoir of bioactive natural products, many of which exhibit structural/chemical features not found in terrestrial natural products1. In recent years, many bioactive compounds have been isolated from various marine organisms like tunicates, sponges, soft corals, sea hares, nudibranchs, bryozoans, sea slugs and marine organisms.2 Soft corals or Alcyonacea are an order of corals which do not produce calcium carbonate skeletons. Soft corals contain minute, spiny skeletal elements called sclerites, useful in species identification. Sclerites give these corals some degree of support and give their flesh a spiky, grainy texture that deters predators.3An abundance of unique secondary metabolites including sesquiterpenoids, diterpenoids, steroids, ceramides and other chemical compounds have been isolated and identified from various species of soft corals.4 Sarcophyton is a widespread genus of corals extending from Eastern Africa and the Red sea in the west to Polynesia in the east. About 14 species of the 36 known Sarcophyton species are found in the Red Sea.5
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There are few studies about chemical constituents of the marine soft coral Sarcophytonauritum,from which cytotoxic cembranoids have been isolated.6 Many marine invertebrates are rich sources of ceramides that differ in structure and biological properties from those of terrestrial organisms. Unusual ceramides have been isolated from sponges7-9, coelenterata10-12, crabs13, sea stars14, and ascidia15. As bioactive lipids, ceramides have been implicated in a variety of physiological functions including apoptosis, cell growth arrest and cell senescence.16 Ceramides are precursors of complex sphingolipids (SLs), which are important for normal functioning of both the developing and mature brain. Altered SL levels have been associated with many neurodegenerative disorders, including epilepsy and Alzeheimer disease.17-19 In our continuing search for new compounds isolated from the soft corals inhabited in various locations of the Red Sea20, we were able to isolate a new ceramide, compound 1 (Figure 1), from the Red Sea soft coralSarcophytonauritum. Compound 1 was screened for anxiolytic activity using the lightdark transition box and elevated plus maze. It was also screened forin vivo anticonvulsant activity using the pentylenetetrazole
(PTZ)-induced seizure model. Molecular modeling studies were also performed to further substantiate the proposed mechanism of action of this ceramide.It suggested a CNS depressing activity possibly through a GABA and serotonin receptors modulation. Moreover, the study includes assessment of the mechanism of anxiolytic activity for the isolated ceramide.
Figure 1: Compound 1
The soft coral (5 kg, wet weight) was freeze dried and then exhaustively extracted with MeOH/CH2Cl2 (1:1) for 5 days. The extract was concentrated till drying (150 g) under vacuum, then fractionated on vacuum liquid chromatography (VLC) using silica gel, (mesh 230–400 nm,) and step gradient elution with a non-polar solvent (n-Hexane) with increasing the polarity using CH2Cl2 then MeOH to give nine fractions(1-9). Fraction 5 (25% MeOH in CH2Cl2) was chromatographed using silica gel 230 x 400 mesh (40-63 µm) to give the ceramide ,eluted with 60% CH2Cl2 in hexane, and it was subjected to final purification on Sephadex LH-20 column using MeOH: CH2Cl2 (1:1) as an eluent, where compound 1 was obtained in pure form (5mg). Compound 1 was obtained as white powder, [α]D22= +3.1(c=0.33, CHCl3); IR (film) 2942, 1743, 1465, 1377 cm-1. The HRESIMS exhibited an [M+H]+ ion at m/z536.5043 (calcd536.5043), indicating the molecular formula of C34H66NO3 and three degrees of unsaturation as supported by NMR data. The 13 C-NMR spectrum, (table 1), showed 31 carbon resonances. Characteristic resonances of a 2-amino-1,3-diol unit of the hydrocarbon chain were observed at δC57.3 (C-2), 62.6 (C-1) and 73.7 (C-3). Also, there are four observed resonances for olifinic carbons at δC130.4 (C-4), 133.2 (C-5), 132.1 (C-6) and 131.6 (C7). In addition, there is a resonance at δC14.5 assigned for the two terminal methyl groups (C-21 and C-13') and at δC174.0 assigned for the amide carbonyl (C-1'). The 1H-NMR spectrum, (table 1), showed resonances of an amide proton doublet at δH 8.37 (d, J= 9.2 Hz) and protons of a long methylene chain at δH1.28, indicating a sphingolipid (SL) skeleton. Also characteristic resonances of a 2-amino-1,3-diol unit of the hydrocarbon chain were observed at δ H 4.77 (m),[4.31 (dd, J= 8.0, 4.8 Hz), 4.47 (dd, J= 8.0, 4.8 Hz)] and 4.86 (m) assigned for H-2, H2-1 and H-3 respectively and resonances for four olefinic protons at δH5.51 (m) assigned for (H-4 and H-7) , 6.01 (m) assigned for H-5 and 6.11 (m) for H-6.In addition, resonancescorresponding to aliphatic hydrocarbons at δH 0.88 (t, J= 6.8 Hz) assigned for H-21 and H-13', 1.28 (overlapped H, m), 1.39 (m) assigned for H-3' and 2.48 (t, J=7.6 Hz) assigned for H2'. The position of the double bonds were confirmed through the 1H-1H COSY spectrum, as there were correlations between H-3/H-4, H-4/H-5, H-5/H-6, H-6/H-7 and H-7/H2-8, as well as the HMBC spectrum which showed correlations from H3/C-5 (3JCH), H-4/C-2 (3JCH), H-6/C-4 (3JCH), H2-8/C-6 (3JCH) leading to the assignment of the C-4/C-5 and C-6/C-7 double bonds (Figure 2). The position and geometry of the double bonds were confirmed by 1H-1H COSY analysis and coupling constant data. The J4,5 value of (15.0 Hz) indicated the trans geometry of these double bonds. On the other hand, it is known that the geometry of the double bond in a long chain alkene can be determined from the 13C-NMR chemical shift of the methylene carbon atom next to the olefinic carbon atom. The carbon signal is observed near to
27 in the (Z) type and near to 32 in the (E) type.20 Therefore, compound 1was assigned as a 4E,6Esphingadiene type ceramide. GC-MS analysis of the fatty acid methyl ester of compound 1 was carried out after hydrolysis. The GC-MS analysis exhibited a peak at molecular ion of m/z (228) corresponding to a C13 fatty acid methyl ester which indicated the presence of only one terminal fatty acid tridecanoic (C13:0) acid. The configuration of compound 1 moieties was assigned through comparison of 13C NMR with analogs as reported in the literature. The chemical shifts of C-1 (δc 62.5), C2 (δc 54.6) and C-3 (δc 74.5) in CDCl3 were very similar to those of the neurotrophic ceramide, (4E,6E,2S,3R)-2-N-eicosanoyl-4,6tetradecasphingadienine21. Further confirmation of the absolute configuration of 1 was determined via the Mosher ester analysis protocol22.This evidence indicated the absolute configurations at C-2 and C-3 to be 2S and 3R, respectively.
Figure 2: Selected 1H-1H COSY (─), HMBC correlations (→) of
compound 1 Table 1.1H and 13C NMR spectral Data for compound 1 (400 MHz, δ in ppm, J in Hz, C5D5N) *
compound 1
No. 1 2 3 4 5 6 7 8 9-16
δH (No. of H, mult.,JHz) 2H; 4.31 (dd, 8.0, 4,8), 4.47 (dd, 8.0, 4,8) 1H, 4.77 (m) 1H, 4.86 (m) 1H, 5.51 (m) 1H, 6.01 (m) 1H, 6.11 (m) 1H, 5.51 (m) 2H, 2.03(m) 2H, 1.28 (m)
δC 62.6 57.3 73.7 130.4 133.2 132.1 131.6 32.6 30.5
17 2H, 1.28 (m) 30.0 18 2H, 1.28 (m) 37.4 19 2H, 1.28 (m) 30.2 20 2H, 1.28 (m) 23.4 21 3H, 0.88 (t, 6,8) 14.5 1' 174.0 2' 2H, 2.48 (t, 7.6) 33.4 3' 3H, 1.39 (m) 26.9 4'-10' 2H, 1.28 (m) 30.5 11' 2H, 1.28 (m) 32.6 12' 2H, 1.28 (m) 23.4 13' 3H, 0.88 (t, 6.8) 14.5 NH 8.37 (d, 9.2) * Chemical shifts (δ) are in ppm. Coupling constants (J) are in Hz
GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. To date, the GABA(A) receptors have not been crystallized. However, X-ray structure of a pentameric ligand gated ion channel from Erwiniachrysanthemi (ELIC) in complex with GABA and flurazepam has recently been deposited in the PDB (PDB code 2YOE).23This prokaryotic homolog ELIC
Compound 1 GABA modulator fitted well in the GABA allosteric site and established several hydrophobic and electrostatic interactions. Residues involved in ligand binding at the allosteric site include VAL73, PRO74, ASN60, ASN89, ILE39, LEU76, ALA75, THR87, GLY88, PRO85, SER84, PHE78. TYR102 may also play a role through potential hydrogen bonding withcompound 1 secondary OH (Figure 4).
schizophrenia and drug abuse.25, 26 Antagonists of the 5-HT3R, such as granisetron and ondansetron, are in clinical use as antiemetics to suppress nausea and vomiting induced by general anaesthetics, chemotherapeutics and radiotherapy. 25, 26 Docking of compound 1 in 5HT-1B receptors (PDB code 4IAQ) did not show initial promising results. The 5-HT3receptor differs markedly in structure and mechanism from the other 5-HT receptor subtypes, which are all G-protein-coupled. Therefore, we decided to study the possible binding pattern of compound 1 to 5HT-3 receptor. The 5-HT3 receptor is a member of the super family of ligand-gated ion channels, and consists of 5 subunits arranged around a central ion conducting pore, which is permeable to sodium, potassium, and calcium ions. Binding of the neurotransmitter 5-hydroxytryptamine to the 5-HT3 receptor opens the channel, which, in turn, leads to an excitatory response in neurons. To date the crystal structure of 5HT-3 has not been resolved. The crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor (PDB code 2YME) was used in our molecular modeling study to assess the possible mechanism of the CNS inhibitory activity of compound 1 as a potential HT-3 inhibitor.27, 28 The results showed that compound 1 docked well in the active site and established hydrophobic interactions with W53, W145, Y193, Y186, D162. A potential hydrogen bonding is also noted between compound 1 carbonyl oxygen and the C188 SH (Figure 6).
Figure 4: Compound 1 GABA modulator binding to GABA allosteric site;distances in Ǻ are indicated by green lines for potential hydrogen bonding.
Figure 6: Compound 1 binding to engineered 5-HTBP;distances in Ǻ are indicated by dotted lines for potential hydrogen bonding.
is also activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors and therefore represents a good target for GABA(A) molecular modeling studies. The docking studies revealed that the docking pattern of compound 1 is very close to that of the co-crystallized benzodiazepine (Flurazepam) suggesting a possible similar mode of action (Figure 3).
Figure 3:Compound 1 GABA modulator (yellow) and Flurazepam (red) bound to GABA allosteric site. Co-crystallised GABA occupying the GABA active sites on the opposite side of the receptors.
The electrostatic potential surface representation showed that the surface of the GABA allosteric site accommodated compound 1 without any sterric collision (Figure 5).
Compared to the selective inhibitor granisetron, compound 1 exhibited a similar docking pose and lied in the same channel as the potent inhibitor. However, compound 1 lacked some key interactions including the Π-cation interaction with R55 as well as the hydrogen bonding of the protonated tropane ring with W 53 backbone. The hydrogen bonding with the water molecule is less likely to happen in case of compound 1 as the carbonyl oxygen is situated further at a distance of 4.6 Ȧ compared to 2.6 Ȧ in case of granisetron (Figure 7). Therefore, compound 1 represents a potential but not a potent and selective blocker of 5HT3
Figure 5:Compound 1 GABA modulator binding to GABA allosteric site represented as an electrostatic potential surface
Neuronal 5-hydroxytryptamine (5-HT, serotonin) signaling pathways are some of the most complex in the human body. There are at least 15 5-HT receptors grouped into seven distinct signaling families and these are involved in a diverse array of complex brain functions, such as physiological, emotional and cognitive control, including regulation of appetite, sleep, sexual behaviour, anxiety, learning and memory.24 All 5HT receptors are G-protein coupled, except for the 5-HT3 receptor (5-HT3R),which is a pentameric ligand-gated ion channel. This receptor has been implicated in hippocampusdependent memory formation, acts in anxiety, and contributes to
Figure 7:Compound 1 (blue) and granisetron binding to engineered 5-HTBP;distances in Ǻ are indicated by dotted lines for potential hydrogen bonding.
In conclusion, the results of the comparative docking studies indicated that the CNS activity of compound 1 is possibly
through GABA receptor modulation rather than serotonin receptors inhibition. Screening for anxiolytic activity in the light dark transition box revealed that treatment with diazepam (1 mg/kg, i.p.) or the test compound1 increased the time spent by mice in the light area of the dark light transition (Figure 8). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), before compound 1 significantly decreased the cumulative time spent in the light area compared to single treatment with compound 1 (Figure 8A). Hence, blockage of GABA-A receptors attenuated the anxiolytic activity of compound 1. Meanwhile, pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.), before compound 1 did not produce a change in the time spent in the white area compared to single treatment with compound 1 (Figure 8B), ruling out the involvement of 5-HT3 receptors in the anxiolytic activity of compound 1.
Figure 9: Effect of diazepam and compound1 on percent time spent in the open arm in the mouse elevated plus maze. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the percent time. B) Effect of pretreatment with the 5HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on percent time.Mice were screened for anxiolytic activity in the elevated plus maze for 3 min. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P < 0.05. *Compared to vehicle group, #Compared to diazepam, $Compared to compound 1, n = 6.
Figure 8: Effect of diazepam and compound 1 on the time spent in the light area in the dark-light transition test. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the recorded time. B) Effect of pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on the recorded time. Mice were screened for anxiolytic activity in the dark-light transition test for 3 minutes. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P<0.05. *Compared to vehicle group, #Compared to diazepam, $ Compared to compound 1, n = 6.
On the other hand, screening of the anxiolytic activity in the elevated plus maze indicated that the test compound 1 as well as diazepam (1 mg/kg) increased the open arm time % compared to the vehicle treated mice (Figure 9A&B). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), before compound 1 significantly decreased the percent open arm time compared to single treatment with compound 1 (Figure 9A). Meanwhile, pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.), before compound 1 did not produce a change in the percent open arm time compared to single treatment with compound 1 (Figure 9B).
The ability of the test compound 1 to prolong time to death after an acute dose of pentylenetetrazole (70 mg/kg, i.p.) was used as an indicator for their anticonvulsant activity. The test compounds successfully antagonized the lethality of pentylenetetrazole; time to death recorded with the test compound 1 was significantly higher than that recorded in the vehicle group. Importantly, the time to death recorded with compound 1 was significantly higher than that reported with an equivalent dose of diazepam (911±87 vs. 536±12, P<0.05, Figures 10A&B). Pretreatment with the GABA-A blocker, bicuculline (0.1 mg/kg, i.p.), significantly attenuated the anticonvulsant effect of compound 1 (Figure 10A). However, pretreatment with the 5-HT3 receptorblocker, ondansetron(1 mg/kg, i.p.) did not affect the anticonvulsant effect of compound 1 (Figure 10B). This indicated that the anticonvulsant activity of compound 1 is, at least in part, mediated by GABA-A receptors. In conclusion, a new ceramide was isolated from red soft corals. The in vivo testing showed good anxiolytic and CNS depressing activity compared to the standards. Competitive in vivo blocking assays showed that GABA-A receptors could be a preferential target for this compound rather than 5-HT3 receptors. The molecular modeling studies supported the in vivo findings by showing a better interaction of the isolated ceramide with GABA-A receptors compared to the 5-HT3 receptors.
8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18.
19. Figure 10: Effect of diazepam and the compound 1 on the time to death after injection of pentylenetetrazole. A) Effect of pretreatment with the GABA-A receptor blocker, bicuculline (0.1 mg/kg, i.p.) on the time to death. B) Effect of pretreatment with the 5-HT3 receptor blocker, ondansetron (1 mg/kg, i.p.) on the time to death. Mice were screened for anticonvulsant activity for 20 min. PTZ: pentylenetetrazole. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison’s test at P < 0.05. *Compared to vehicle group, #Compared to diazepam, $Compared to compound 1, n = 6.
Acknowledgments We thank Dr. Tarek Temraz, marine science department, Suez Canal University for identification of the soft coral. We are also thankful to Dr. BaharthiAvula, National Center for Natural Products Research, School of Pharmacy University of Mississippi for HRMS data, References and notes 1. 2. 3. 4. 5. 6.
7.
Carte´, B. K. , Biosceinces, 1996, 46, 271. Donia, M.; Hamann, M. T. The Lancet. 2003, 3, 338. Jeng, M.S.; Huang, H. D.; Dai, C.F; Hsiao, Y.C.; Benayahu, Y. Coral Reefs, 2011, 30, 925. Blunt J.W.; Copp B.R.; Keyzers R.A.; Munro M.H.; Prinsep M.R. Nat. Prod. Rep. 2013, 30, 237. Fabricus, K. and Alderslade, P. Soft corals and Sea Fans. 1st Ed., Australian Institute of Marine Science press, Queensland. 2001, pp 1-41. Eltahawy, N. A.; Ibrahim, A. K.; Radwan, M. M.; ElSohly M. A.; Hassanean, H. A.; Ahmed, S. A.Tetrahedron Lett.2014, 55, 3984. Grg, H. S.; Agrawal, S. J. Nat. Prod.1995, 58, 442.
20.
Hattori, T.; Adachi, K.; Shizuri, Y. J. Nat. Prod.1998, 61, 823. Muralidhar, P.; Krishna, N.; Kumar, M. M.; Rao, C. B.; Rao, D. V. Chem. Pharm. Bull.2003, 51, 1193. Shin, J.; Seo, Y. J. Nat. Prod.1995, 58, 442. Chebaane, K.; Guyot, M. Tetrahedron Lett.1986, 27, 1495. Chakrabarty, M.; Batabyal, A.; Barua, A. K. J. Nat.Prod.1994, 57, 393. Asai, N.; Fusetani, N.; Matsunaga, S.; Sasaki, J. Tetrahedron 2000, 56, 9895. Inagaki, M.; Isobe, R.; Kawano, Y.; Miyamoto, T.; Komori, T.; Higuchi, R. Eur. J.Org. Chem.1998, 1, 129. Lukasv, A.; Bultel-Ponce, V.; Longeon, A.; Guyot, M. J. Nat. Prod.2000, 63, 799. Hannun, Y.A. and Obeid, L.M. "Principles of bioactive lipid signalling: lessons from sphingolipids". Nature Reviews Molecular Cell Biology, 2008, 9 (2): 139. Ahmed, S.A.; Khalifa, S.I.; Hamann, M.T. J. Nat. Prod.2008, 71, 4. Mielke, M.M.1; Bandaru, V.V.; Haughey, N.J.; Xia, J.; Fried, L.P.; Yasar, S.; Albert, M.; Varma, V.; Harris, G.; Schneider, E.B.; Rabins, P.V.; Bandeen-Roche, K.; Lyketsos, C.G.; Carlson, M.C. Serum ceramides increase the risk of Alzheimer disease: the Women's Health and Aging Study II. Neurology.2012, 14;79(7):633. Mosbechl, M.; Olsen1, A.S.; Neess, D.; David, O.B.; Klitten, L.L.; Larsen, J.; Sabers, A.; Vissing, J.; Nielsen, J.E.; Annals of Clinical and Translational Neurology 2014; 1(2): 88. Ormond, R.F.G.; Edwards, A.J. IUCN/Pergamon Press,Oxford, 1987, pp 251–287
21. Inagakia, M.; Isobeb, R.; Kawanoa, Y.; Miyamotoa, T.; Komoria, T.; Higuchi, R., Eur. J. Org. Chem. 1998, 129. 22. Mancini, l.; Guella, G; Debitush, C.; Pietra, F. Helv. Chim. Acta. 1994, 77, 51. 23. Kwon, H. C.; Lee, K. C.; Cho, O. R.; Jung, I. Y.; Cho, S. Y.; Kim, S. Y.; Lee, K. R. J. Nat. Prod., 1991, 66, 466. 24. Spurny, R.; Ramerstorfer, J.; Price, K.; Brams, M.; Ernst, M.; Nury, H.; Verh;eij, M.; Legrand, P.; Bertrand, D.; Bertrand, S.; Dougherty, D.A.; de Esch, I.J.; Corringer, P.J.; Sieghart, W.; Lummis, S.C.; Ulens, C. Proc Natl Acad Sci U S A. 2012, 109(44), 3028. 25. Berger, M., Gray, J.A., Roth, B.L. Annu Rev Med, 2009, 60: 355. 26. Thompson, A.J.; Lummis, S.C. ExpertOpinTher Targets, 2007, 11: 527. 27. Walstab, J.; Rappold, G.; Niesler, B. PharmacolTher, 2010, 128: 146. 28. Kesters, D., Thompson, A.J., Brams, M., Van Elk, R., Spurny, R., Geitmann, M., Villalgordo, J.M., Guskov, A., Helena Danielson, U., Lummis, S.C.R., Smit, A.B., Ulens, C., Embo Rep., 2013, 14, 49.
Supplementary Material 1D and 2D NMR spectral data of 1is available.
Graphical Abstract
Mechanism of Action of Antiepileptic Ceramide
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from Red Sea Soft Coral Sarcophyton auritum Nermeen A. Eltahawya, Amany K. Ibrahima, Mohamed M. Radwanb, c, Sawsan Zaytond, Mohamed Gomaae, b,f
a
a, *
Mahmoud A. ElSohly , Hashim A. Hassanean , Safwat A. Ahmed
3'
13'
2' 4'
1' O
NH
HO
1
2
3 OH
.
5 4
6
7 21