WO2007107319A1 - Isolation, structure determination, synthesis and bioactivity of damipipecolin and damituricin - Google Patents
Isolation, structure determination, synthesis and bioactivity of damipipecolin and damituricin Download PDFInfo
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- WO2007107319A1 WO2007107319A1 PCT/EP2007/002417 EP2007002417W WO2007107319A1 WO 2007107319 A1 WO2007107319 A1 WO 2007107319A1 EP 2007002417 W EP2007002417 W EP 2007002417W WO 2007107319 A1 WO2007107319 A1 WO 2007107319A1
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- 0 C*1C(C(OI[C@](C*2(C)C)C[C@]2(*)O)=O)=CC(C)=C1 Chemical compound C*1C(C(OI[C@](C*2(C)C)C[C@]2(*)O)=O)=CC(C)=C1 0.000 description 3
- MAHCQKTZGPKCIA-CRCLSJGQSA-N O[C@@H](CC1)C[C@H]2N1C(C(F)(F)F)(C(F)(F)F)OC2=O Chemical compound O[C@@H](CC1)C[C@H]2N1C(C(F)(F)F)(C(F)(F)F)OC2=O MAHCQKTZGPKCIA-CRCLSJGQSA-N 0.000 description 2
- CQLTVLIUJXOOGD-UHFFFAOYSA-N O=C(C(Cl)(Cl)Cl)c1cc(Br)c[nH]1 Chemical compound O=C(C(Cl)(Cl)Cl)c1cc(Br)c[nH]1 CQLTVLIUJXOOGD-UHFFFAOYSA-N 0.000 description 1
- BBFDGMDENAEMKF-UHFFFAOYSA-N O=C(C(Cl)(Cl)Cl)c1ccc[nH]1 Chemical compound O=C(C(Cl)(Cl)Cl)c1ccc[nH]1 BBFDGMDENAEMKF-UHFFFAOYSA-N 0.000 description 1
- DQJBWUHXTBYNKA-VXNVDRBHSA-N O=C(c1cc(Br)c[nH]1)O[C@H](CC1)C[C@H]2N1C(C(F)(F)F)(C(F)(F)F)OC2=O Chemical compound O=C(c1cc(Br)c[nH]1)O[C@H](CC1)C[C@H]2N1C(C(F)(F)F)(C(F)(F)F)OC2=O DQJBWUHXTBYNKA-VXNVDRBHSA-N 0.000 description 1
- NDJQQEYLUZNTCW-HTQZYQBOSA-N OC([C@@H](C1)NCC[C@H]1OC(c1cc(Br)c[nH]1)=O)=O Chemical compound OC([C@@H](C1)NCC[C@H]1OC(c1cc(Br)c[nH]1)=O)=O NDJQQEYLUZNTCW-HTQZYQBOSA-N 0.000 description 1
- YUWKEVJEMKKVGQ-UHFFFAOYSA-N OC(c1cc(Br)c[nH]1)=O Chemical compound OC(c1cc(Br)c[nH]1)=O YUWKEVJEMKKVGQ-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
Definitions
- Pyrrole alkaloids from marine sponges represent one of the most prominent groups of natural products in the marine environment (Faulkner, D. J. Nat. Prod. Rep., 2000, 17, 7).
- the variety of pharmacological activities displayed by such metabolites makes these compounds active leads for the development of new drugs and tools for cell biology (Aiello, A., D'Esposito, M., Fattorusso, E., Menna, M., M ⁇ ller, W. E. G., Perovic-Ottstadt, S., Schroder, H. C. Bioorg. Med Chem., 2006, 14, 17).
- bromopyrrole alkaloids fall under the oroidin class of alkaloids (Forenza, S., Minale, L., Riccio, L., Fattorusso, E. Chem. Comm., 1971, 1129), defined by the signature bromopyrrole carboxamide and aminoimidazole moieties connected through a propyl chain.
- damipipecolin (1) and auricin (2) are bromopyrrole alkaloids, named damipipecolin (1) and accordinguricin (2), from the sponge Axinella damicornis.
- Damipipecolin and combinuricin extend the structural variety of the so far known pyrrole alkaloids since their structures lack the commonly found short linear aliphatic segment linking the common imidazole nucleus.
- the 4- bromopyrrole 2-carboxylic acid is directly condensed with a non-protein cyclic ⁇ - aminoacid, the (2R, 4R)-/r ⁇ ms i -4-hydroxypipecolic acid and (2R, 4R)-ds-N,N'- dimethyl-4-hydroxyproline (D-turicine) in 1 and 2, respectively.
- the D-and L-forms of the betaine turicine, where the carboxilate and hydroxyl functions are cis to one other, and L-betonicine, where these functions are in the trans relationship, are naturally occurring derivatives of 4-hydroxyproline. They have been studied in the acetylcholinesterase system and betonicine, though not turicine, is a competitive inhibitor of the same order of magnitude of choline itself (Friess, S.L., Patchett, A. A., and Witkop, B. J. Am. Chem. Soc. 1957, 79, 459).
- Pipecolic acids hexahydropiperidine-2-carboxylic acids
- Pipecolic acids are frequently encountered in nature with their hydroxylated derivatives and often display interesting and potent biological activity (Moloney, M. G. Nat. Prod. Rep., 1998, 205; Ho, B., Zabriskie, T. M. Bioorg. Med Chem. Lett., 1998, 8, 739; Romeo, J. T., Swain, L. A., Bleecker, A. B. Phytochemistry, 1983, 22, 1615; Vanderhaeghe, H., Janssen, G., Compernolle, F. Tetrahedron Letters, 1971, 28, 268; Clark-Lewis, J. W., Mortimer, P. I. J.
- Pipecolic acid derivatives are also useful synthetic intermediates for the preparation of medicinally important compounds such as peptides (Copeland, T. D., Wondrak, E. M., Toszer, J., Roberts. M. M., Oraszlan, S. Biochem. Biophys. Res. Comm., 1990, 169, 310), immunosuppressants (Dragovich, P. S., Parker, J. E., French, J., Incacuan, M., Kalish, V. J., Kissinger, C. R., Knighton, D. R., Lewis, C. T., Moomaw, E. W., Parge, H. E., Pelltier, L.
- peptides Copeland, T. D., Wondrak, E. M., Toszer, J., Roberts. M. M., Oraszlan, S. Biochem. Biophys. Res. Comm., 1990, 169, 310
- immunosuppressants
- Specimens of the sponge Axinella damicornis are collected and kept frozen until used.
- the fresh thawed sponge (75.2 g dry weight after extraction) is homogenized and treated at room temperature with methanol (3 x 600 ml) and, subsequently, with chloroform (3 x 600 ml).
- the extracts are combined and concentrated in vacuo to give an aqueous suspension which is then partitioned between H 2 O and BuOH.
- the BuOH layer after evaporation of the solvent, is subjected to a medium pressure chromatography (MPLC) over a reversed-phase (RP- 18) column using a gradient elution (H 2 O ⁇ MeOH ⁇ CHCI 3 ).
- MPLC medium pressure chromatography
- RP- 18 reversed-phase
- the fraction eluted with MeOHZH 2 O 8:2 is mainly composed of polar alkaloids.
- This fraction is further separated by HPLC on a preparative RP-18 column (Kromasil, 10 ⁇ m, 250 x 10 mm), with H 2 OZMeOH 7:3 as the eluent, and gives a mixture of compounds 1 and 2.
- the planar structure of damipipecolin (1) was determined by spectroscopic methods.
- the ESI (positive ions) mass spectrum of 1 shows two intense pseudomolecular ion peaks at m/z 317 and 319 [M+H] + in the ratio 1:1, suggesting that 1 is a monobromo compound.
- the 13 C-NMR spectrum (CD 3 OD) contains 11 signals, composed of 3 methylenes, 4 methines and 4 unprotonated carbons, according to DEPT and HSQC experiments.
- the combined NMR and mass spectral data of 1 are compatible with the molecular formula Cj 1HnN 2 O 4 Br, which implies six degree of unsaturation. From 13 C-NMR data it is evident that four of the elements of unsaturation are present as double bonds, two of them being carbonyl groups, one as part of a conjugated ester and the other as a free acid; the molecule must be thus bicyclic.
- the 1 H-NMR spectrum (CD 3 OD) of 1 reveals the presence of two aromatic signals at ⁇ 6.92 and 7.01, a deshielded methylene signal ( ⁇ 3.17, Ha; 3.20, Hb), two sp 3 deshielded methine signals ( ⁇ 3.74 and 5.31), and two partially overlapped methylene resonances in the region 1.9—2.4 ppm.
- Interpretation of the COSY spectrum illustrates the proton connectivities in 1 and reveals the presence of two distinct spin systems belonging to two nitrogen-containing rings.
- a successive HSQC experiment allowed the inventors to assign the resonances of the protonated carbon atoms in the 13 C NMR spectrum.
- the remaining part of the molecule consisting of C 6 HiONO 2 , is composed of three methylene groups, two deshielded sp 3 methine groups, and a carboxyl function.
- a large spin system is observed, corresponding to the segment H-272H-6'.
- the proton signal at ⁇ 5.31 (H-4 1 ) of this spin system is correlated to a signal at ⁇ 67.8, clearly indicating that it is due to a proton linked to an oxygen-bearing carbon.
- a nitrogen atom is inferred to be attached to both C-2 1 and C-6 1 from their 13 C chemical shifts ( ⁇ 56.4 and 40.2), and a cross peak, present in the HMBC spectrum of 1 between the resonance for H-2' (adjacent to N) and the carbonyl resonating at ⁇ 179.9, suggests the carboxylic function to be located at C-2 1 .
- a framework of 4-substituted piperidine-2-carboxylic acid (pipecolic acid) can be deduced for the structure of the CeHi O NO 2 unit of damipipecolin.
- the oxymethine proton on C-4 1 ( ⁇ 5.31) reveals an HMBC long-range correlation with the C-6 carbon ( ⁇ 159.9), thus indicating that C-4' is connected with the bromopyrrolecarboxylic acid moiety through an ester linkage.
- the relative stereochemistry of the two substituents on the pipecolic acid ring can be defined by 1 H- 1 H coupling constants analysis.
- the absolute stereochemistry of the amino acidic portion can be clarified through acid hydrolysis of 1 performed with 1 N HCl in H 2 O at 8O 0 C overnight.
- the ESI (positive ions) mass spectrum of reverseuricin (2) shows two intense pseudomolecular ion peaks at m/z 331 and 333 [M+H] + in the ratio 1 :1.
- the 13 C-NMR spectrum (CD 3 OD) contains twelve carbon signals, which are identified, through DEPT and HSQC experiments, as two methyls, two sp 3 methylenes, two sp 2 methines, two deshielded sp 3 methines, and four unprotonated carbons. These NMR and mass spectral data are compatible with the molecular formula Ci 2 HiSN 2 O 4 Br, implying six elements of unsaturation.
- the crucial step of the proposed strategy consists of a Mitsunobu reaction (Mitsunobu, O. Synthesis, 1981, 1; Hughes, D. L. Org. React, 1992, 42, 335) with the HFA-protected cw-4-hydroxy-D-pipecolic acid derivative 8 and 4-bromopyrrole-2-carboxylic acid (3).
- the Mitsunobu reaction is a particularly useful reaction in organic synthesis for the stereochemical inversion of hydroxyl groups.
- the protocol for the Mitsunobu reaction provides for a mixture of triphenylphosphine, a dialkyl azodicarboxilate, and an acid component (usually a carboxylic acid).
- the 4-bromopyrrole-2-carboxylic acid is used to ensure the acidity of the reaction mixture, and the corresponding ester of HFA-protected ft- ⁇ ns-4-hydroxy-D- pipecolic acid (9) is obtained.
- 4-Bromopyrrole-2-carboxylic acid (3) is prepared by bromination of the commercially available 2-trichloroacetylpyrrole (6), which selectively gives the 4- bromo-2-trichloroacerylpyrroIe (7); hydrolysis of 7 with an aqueous base gives the free acid 3.
- the HFA-protected c/s ⁇ -hydroxy-D-pipecolic acid derivative 8 could be obtained starting from D-aspartic acid and hexafluoroacetone as protecting agent according to the procedure described by Golubev and coworkers (Golubev, A., Sewald, N., Burger, K. Tetrahedron Lett., 1995, 36, 2037).
- Serotonin (5-hydroxytryptamin, 5-HT) belongs to the class of biogenic amines (indolamines or monoamine transmitters). Within the CNS the serotonergic neurons can be found in nearly every brain area Serotonin receptors can be classified in three different groups: (Q the transporter (5-HT "uptake site"), (if) the ligand-gated ion- channel (5-HT 3 ) and (Ui) the largest group of the G protein-coupled receptors (Boess, F. G., Martin, I. L. Neuropharmacology, 1994, 33, 275).
- 5-Hydroxytryptamin modulates several biological function in the CNS (Maeda, T., Fujimiya, M., Kitahama, K., Imai, H., Kimura, H. Arch. Histol.
- Cytol 1989, 52 (Suppl), 113); it influences processes related to the memory, and learning, sexual behaviour, as well as feeding behaviour (Brunelli, M., Garcia-Gil, M., Mozzachiodi, R., Scuri, R., Zaccardi, M.L Arch. Ital. Biol. , 1997, 135, 15). It seems also be involved in regulating aggressive behaviour. Also release of adenocorticotropin (ACTH), corticosterone, prolactin and gonadotropins can be activated by serotonin (Fuller, R. W. Neuropsychopharmacology, 1990, 3, 495).
- ACTH adenocorticotropin
- corticosterone corticosterone
- prolactin prolactin
- gonadotropins can be activated by serotonin (Fuller, R. W. Neuropsychopharmacology, 1990, 3, 495).
- 5-HT 1 receptors are implicated in the pathogenesis of many disorders and Ln contrast to the receptors of the 5-HT 2 and 5-HT 4 they display a high affinity for serotonin, they become an important target for drug therapy (Passchier, J., van Waarde, A. Eur. J. Nucl. Med., 2001, 28, 113).
- Preincubation of neurons with 10 ⁇ g/ml of the compound 1 and compound 2 induces no or small (-22% reduction) decrease in Ca 2+ influx after addition of 200 ⁇ M serotonin. Reduction of the concentration of 1 and 2 to 1 and 0.1 ⁇ g/ml resulted in a strong decrease of the Ca 2+ influx in neurons.
- Compound 1 is a potent antagonist of serotonin in the concentration of 1 ⁇ g/ml ( ⁇ 70% reduction), while the effect of serotonin on neurons was completely blocked after the addition of 0.1 ⁇ g/ml of DAM ( ⁇ 91% reduction), hi contrast, compound 2 is a less potent antagonist of serotonin at the concentration of 1 ⁇ g/ml ( ⁇ 60% reduction) and 0.1 ⁇ g/ml of DAM (-70% reduction).
- Different types of serotonin receptors exist in brain tissue. Compounds that interact with 5-HT receptors have important therapeutic applications.
- compound 1 is a new promising serotonin antagonist with a potential in the therapy of emesis induced by chemotherapy, psychosis, different phobia and mood fluctuation disorders.
- Compound 2 has also antagonistic effects on serotonin receptors.
- the amino acids L-glutamate and L-aspartate are the most abundant key excitatory neurotransmitters in the central nervous system (CNS); glutaminergic neurons are especially prominent in the cerebral cortex (Arai, Y., Mizuguchi, M., Takashima, S. Anat. Embryol. (Berl), 1997, 195, 65).
- CNS central nervous system
- glutaminergic neurons are especially prominent in the cerebral cortex (Arai, Y., Mizuguchi, M., Takashima, S. Anat. Embryol. (Berl), 1997, 195, 65).
- the biological effects of these excitatory amino acids are manifold.
- glutamate provides beneficial effect in the regulation of neuronal function, growth and differentiation
- excitatory amino acids can be harmful to brain tissue.
- the excitotoxic effect is related to the massive influx of Na + , K + , and Ca 2+ in neurons as a consequence of the sustained activation of glutamate receptors (Arundine, M., Tymianski, M. Cell Calcium, 2003, 34, 325; Stys, P.K. Curr. MoI Med., 2004, 4, 113).
- preincubation of neurons with 10, 1, and 0.1 ⁇ g/ml of compound 1 or compound 2 induces no significant decrease in Ca 2+ entry in the presence of glutamate or NMDA.
- compound 1 and compound 2 are not binding on glutamate or NMDA receptor.
- ESI mass spectra can be obtained by using for example an API 2000 mass spectrometer.
- High-resolution FAB mass spectra (glycerol matrix) can be performed for example on a VG Prospec (FISONS) mass spectrometer.
- Optical rotations can be measured using for example a Perkin-Elmer 192 polarimeter.
- CD spectra can be recorded for example on a J-710 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a J-710 for Windows software (Jasco).
- NMR experiments can be performed for example on a Bruker AMX-500 spectrometer; chemical shifts are referred to the residual solvent signal.
- MLC Medium-pressure liquid chromatographies
- HPLC High-performance liquid chromatography
- UV spectra can be recorded for example on a Sbimadzu UV- 1204 instrument.
- PC 12 cells are grown in Dulbecco's modified Eagle's medium [DMEM]/10% (v/v) fetal calf serum (FCS)/5% (v/v) horse serum.
- HEK cells are grown in Dulbecco's modified Eagle's medium [DMEM]/10% (v/v) FCS/4.5 g/l glucose.
- PC 12 and HEK cells are passaged twice per week at a 1:10 ratio.
- L5178y and HeLa cells are maintained in Roswell Park Memorial Institute medium [RPMI] 1640 supplemented with 10% (v/v) FCS.
- the cells are subcultured twice weekly at a 1:160 (L5178y cells) and 1:10 ratio (HeLa cells). All cells are kept in an atmosphere of 95% air and 5% CO 2 at 37°C.
- PC 12, L5178y, and HeLa cells are incubated for 72 h in the presence of different concentrations (0.1; 0.3; 1; 3, and 10 ⁇ g/ml) of compound 1 or compound 2.
- the final volumes are 200 ⁇ l.
- Compound 1 is dissolved in DMSO (stock solution 10 mg/ml) and stored at -20 0 C.
- the compound 2 is dissolved in H 2 O (stock solution 5 mg/ml) and stored at -20 0 C.
- the viability of the cells is determined using the MTT colorimetric assay system (Scudiero, D. A., Shoemaker, R. H., Paull, K. D., Monks, A., Tierney, S., Nofciger, T.
- Rat cortical cell cultures are prepared from the brains of 17- to 18-day-old Wistar rat embryos, as described (Freshney, R. I. In: Culture of Animal Cells. A Manual of Basic Technique; Freshney, R. L, Ed., Alan R. Liss Inc.: New York, 1987; pp. 257-288; Perovic, S., Schleger, C, Pergande, G., Iskric, S., Ushijima, H., Rytik, P., M ⁇ ller, W. E. G. Eur. J. Pharmacol. (MoI Pharmacol. Sec), 1994, 288, 27).
- HBSS Hanks' balanced salt solution
- Brain tissue is dissociated in HBSS using 0.025% (w/v) trypsin (10 min; 37°C); the proteolytic reaction is stopped by addition of 10% FCS.
- FCS 10% (v/v) FCS.
- the cells are seeded into poly-L-lysine (5 ⁇ g/ml, 300 ⁇ l/cm 2 )-coated plastic dishes at a concentration of 2.0 x 10 5 cells/cm 2 .
- DMEM/HG/10% (v/v) FCS is removed and the cells are cultivated further in a DMEM/HG serum-free medium supplemented with 0.1% (w/v) bovine serum albumin (BSA), 2 mM L- ghitarnine, 100 ⁇ g/ml transferrin, 100 mU/1 insulin, 16 ⁇ g/ml putrescine, 6.3 ng/ml of progesterone, and 5.2 ng/ml Na 2 SeO 3 .
- BSA bovine serum albumin
- cells are cultivated on poly-L-lysine coated borosilicate coverglass (Nunc).
- An inverted-stage microscope for example Olympus LX70with objective UApo40X/340 is used for the fluorescence measurements.
- the cells are alternately illuminated with light of wavelengths 340 and 380 nm.
- An additional 0.25 ND filter is used at 380 nm.
- the fluorescence emissions at 510 nm are monitored by an intensified CCD camera, for example model C2400-87 (Hamamatsu, Herrsching, Germany). Images are then digitized with a computerized imaging system (for example Argus-50, Hamamatsu).
- the [Ca 2+ J j is determined by measuring the fluorescence ratio of the Ca 2+ - indicator dye fura-2-AM at 340 and 380 nm (Grynkiewicz, G., Poenie, M., Tsien, R. Y. J. Biol. Chem., 1985, 260, 3440). Neurons are loaded with 4 ⁇ M fura-2- acetoxymethyl (AM) ester and PC 12, HeLa and HEK cells with 10 ⁇ M fiira-2-AM in DMEM/HG serum-free medium, supplemented with 1% (w/v) BSA at 37 0 C for 60 min.
- a calcium calibration curve is prepared according to the method of Grynkiewicz (Grynkiewicz, G., Poenic, M., Tsien, R. Y. J. Biol. Chem., 1985, 260, 3440).
- One ratio value 340/380nm equals 228 nM [Ca 2+ ],.
- Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO 3 , 5.6 mM glucose and 10 mM Hepes; pH 7.4) without Ca 2+ and Mg 2+ is used as incubation medium in all set of experiments.
- a stock solution of 10 mg/ml of compound 1 dissolved in DMSO and of 5 mg/ml of compound 2 dissolved in H 2 O are used. All serial dilution are done in DMSO (compound 1) or H 2 O (compound 2).
- neurons are stimulated with (compound 1) or without (compound 2) 0.1% (v/v) DMSO after 5 min and with 200 ⁇ M of serotonin (5-HT), N-methyl-D-aspartic acid (NMDA) or L-glutamic acid (L-GIu) and 2.5 mM CaCl 2 after 10 min from the beginning of the measurements.
- 5-HT serotonin
- NMDA N-methyl-D-aspartic acid
- L-GIu L-glutamic acid
- CaCl 2 2.5 mM CaCl 2 after 10 min from the beginning of the measurements.
- the primary neurons are first preincubated with 10, 1, and 0.1 ⁇ g/ml of the compound 1 or compound 2 (5 min) and after 10 min 5-HIVCaCl 2 , NMDAZCaCl 2 or L-Glu/CaCl 2 are added to the neurons.
- PC 12 and HeLa cells are first preincubated with 1 ⁇ g/ml of compound 1 or compound 2 (5 min) and after 10 min 5-HT/CaCl 2 are added to the cells.
- the peincubation of the cells is done with 10, 1 and 0.1 ⁇ g/ml of the compound 1 or compound 2 (5 min) and after 10 min 5- HT/CaCl 2 are added to the cells.
- the cells treated with or without 0.1% (v/v) DMSO and 200 ⁇ M 5-HT with 2.5 mM CaCl 2 are used.
- DAM-I compound 1; DAM-2: compound 2. able 1. 1 H and 13 C NMR data of compounds 1, 2, 4-hydroxypipecolic acid (4), and D-turicine (5).
Abstract
Two new bromopyrrole alkaloids, compounds (1) and (2), have been isolated from the Mediterranean sponge Axinella damicυrnis, and their structure established through spectroscopic methods. Compounds (1) and (2 )display a modulating effect of the serotonin receptor activity and neuroprotective activity in vitro.
Description
APPLICATION FOR PATENT
ISOLATION, STRUCTURE DETERMINATION, SYNTHESIS AND BIOACTPyITY OF DAMIPIPECOLIN AND DAMITURICIN
SPECIFICATION
Background of Invention
Pyrrole alkaloids from marine sponges represent one of the most prominent groups of natural products in the marine environment (Faulkner, D. J. Nat. Prod. Rep., 2000, 17, 7). The variety of pharmacological activities displayed by such metabolites makes these compounds active leads for the development of new drugs and tools for cell biology (Aiello, A., D'Esposito, M., Fattorusso, E., Menna, M., Mϋller, W. E. G., Perovic-Ottstadt, S., Schroder, H. C. Bioorg. Med Chem., 2006, 14, 17). Most of the bromopyrrole alkaloids fall under the oroidin class of alkaloids (Forenza, S., Minale, L., Riccio, L., Fattorusso, E. Chem. Comm., 1971, 1129), defined by the signature bromopyrrole carboxamide and aminoimidazole moieties connected through a propyl chain.
Summary of Invention
In the frame of their research on bioactive substances from Mediterranean Axinella sponges, the inventors have isolated two bromopyrrole alkaloids, named damipipecolin (1) and damituricin (2), from the sponge Axinella damicornis.
Damipipecolin and damituricin extend the structural variety of the so far known pyrrole alkaloids since their structures lack the commonly found short linear aliphatic segment linking the common imidazole nucleus. In these compound, the 4- bromopyrrole 2-carboxylic acid is directly condensed with a non-protein cyclic α- aminoacid, the (2R, 4R)-/r<msi-4-hydroxypipecolic acid and (2R, 4R)-ds-N,N'- dimethyl-4-hydroxyproline (D-turicine) in 1 and 2, respectively. The D-and L-forms of the betaine turicine, where the carboxilate and hydroxyl functions are cis to one other, and L-betonicine, where these functions are in the trans relationship, are naturally occurring derivatives of 4-hydroxyproline. They have been studied in the acetylcholinesterase system and betonicine, though not turicine, is a competitive inhibitor of the same order of magnitude of choline itself (Friess, S.L., Patchett, A. A., and Witkop, B. J. Am. Chem. Soc. 1957, 79, 459). Pipecolic acids (hexahydropiperidine-2-carboxylic acids) are frequently encountered in nature with their hydroxylated derivatives and often display interesting and potent biological activity (Moloney, M. G. Nat. Prod. Rep., 1998, 205; Ho, B., Zabriskie, T. M. Bioorg. Med Chem. Lett., 1998, 8, 739; Romeo, J. T., Swain, L. A., Bleecker, A. B. Phytochemistry, 1983, 22, 1615; Vanderhaeghe, H., Janssen, G., Compernolle, F. Tetrahedron Letters, 1971, 28, 268; Clark-Lewis, J. W., Mortimer, P. I. J. Chem. Soc, 1961, 189). Pipecolic acid derivatives are also useful synthetic intermediates for the preparation of medicinally important compounds such as peptides (Copeland, T. D., Wondrak, E. M., Toszer, J., Roberts. M. M., Oraszlan, S. Biochem. Biophys. Res. Comm., 1990, 169, 310), immunosuppressants (Dragovich, P. S., Parker, J. E., French, J., Incacuan, M., Kalish, V. J., Kissinger, C. R., Knighton, D. R., Lewis, C. T., Moomaw, E. W., Parge, H. E., Pelltier, L. A. K., Prince, T. J., Showalter, R. E., Tatlock, J. H., Tucker, K. D., Villafranca, J. E. J. Med. Chem. 1996, 39, 1872), enzyme inhibitors (Ho, B., Zabriskie, T. M. Bioorg. Med. Chem. Lett, 1998, 8, 739; Gillard, J., Abraham, A., Anderson, P. C, Beaulieu, P. L., Bogri, T., Bousquet, Y., Grenier, L., Guse, Y., Lavellee, P. J. Org. Chem., 1996, 61, 2226) and NMDA antagonists (Skiles, J. W., Giannousis, P. P., Fales, K. R. Bioorg. Med. Chem. Lett., 1996, 6, 963).
Compounds 1 and 2 were found to display modulating effect of the serotonin receptor activity in vitro.
Isolation of compounds 1 and 2
Specimens of the sponge Axinella damicornis are collected and kept frozen until used. For the extraction, the fresh thawed sponge (75.2 g dry weight after extraction) is homogenized and treated at room temperature with methanol (3 x 600 ml) and, subsequently, with chloroform (3 x 600 ml). The extracts are combined and concentrated in vacuo to give an aqueous suspension which is then partitioned between H2O and BuOH. The BuOH layer, after evaporation of the solvent, is subjected to a medium pressure chromatography (MPLC) over a reversed-phase (RP- 18) column using a gradient elution (H2O → MeOH → CHCI3). Following this procedure, the fraction eluted with MeOHZH2O 8:2 is mainly composed of polar alkaloids. This fraction is further separated by HPLC on a preparative RP-18 column (Kromasil, 10 μm, 250 x 10 mm), with H2OZMeOH 7:3 as the eluent, and gives a mixture of compounds 1 and 2. Final separation and purification of the two compounds is achieved by HPLC on a RP-18 column (Luna, 5 μm, 250 x 4.6 mm), using MeOHZH2O 8:1 as the eluent, thus obtaining damipipecolin [1, [αjo 20 = + 5.4 (H2O, c = 0.001), 4 mg], and damituricin [2, [α]D 20 = + 10.7 (H2O, c = 0.0013), 3 mg], both in a pure form.
Structural elucidation of damipipecolin
The planar structure of damipipecolin (1) was determined by spectroscopic methods. The ESI (positive ions) mass spectrum of 1 shows two intense pseudomolecular ion peaks at m/z 317 and 319 [M+H]+ in the ratio 1:1, suggesting that 1 is a monobromo compound. The 13C-NMR spectrum (CD3OD) contains 11 signals, composed of 3 methylenes, 4 methines and 4 unprotonated carbons, according to DEPT and HSQC experiments. The combined NMR and mass spectral data of 1 are compatible with the molecular formula Cj 1HnN2O4Br, which implies six degree of unsaturation. From 13C-NMR data it is evident that four of the elements of unsaturation are present as double bonds, two of them being carbonyl groups, one as part of a conjugated ester and the other as a free acid; the molecule must be thus bicyclic.
The 1H-NMR spectrum (CD3OD) of 1 reveals the presence of two aromatic signals at δ 6.92 and 7.01, a deshielded methylene signal (δ 3.17, Ha; 3.20, Hb), two
sp3 deshielded methine signals (δ 3.74 and 5.31), and two partially overlapped methylene resonances in the region 1.9—2.4 ppm. Interpretation of the COSY spectrum illustrates the proton connectivities in 1 and reveals the presence of two distinct spin systems belonging to two nitrogen-containing rings. A successive HSQC experiment allowed the inventors to assign the resonances of the protonated carbon atoms in the 13C NMR spectrum. The presence of a 4-bromopyrrole-2-carboxylic acid derivative moiety is evident from the chemical shifts and the multiplicity of the aromatic proton signals, which constituted one of the two spin system, and from a 13C NMR pattern of resonances (δ 124.5, 97.8, 117.7, 123.0, 159.9). This hypothesis is fully corroborated by comparison of the NMR data of 1 with those reported in literature for other bromopyrrole alkaloids (Foley, L. H., Habgood, G. J., Gallagher, K. S. Mag. Res. Chem., 1988, 26, 1037); this unit accounts for two CC double bonds, the ester function and one ring.
The remaining part of the molecule, consisting of C6HiONO2, is composed of three methylene groups, two deshielded sp3 methine groups, and a carboxyl function. Following the COSY correlations in the spectrum recorded in MeOD, starting from the deshielded methine signal at δ 3.74 (m), a large spin system is observed, corresponding to the segment H-272H-6'. In the HSQC spectrum, the proton signal at δ 5.31 (H-41) of this spin system is correlated to a signal at δ 67.8, clearly indicating that it is due to a proton linked to an oxygen-bearing carbon. A nitrogen atom is inferred to be attached to both C-21 and C-61 from their 13C chemical shifts (δ 56.4 and 40.2), and a cross peak, present in the HMBC spectrum of 1 between the resonance for H-2' (adjacent to N) and the carbonyl resonating at δ 179.9, suggests the carboxylic function to be located at C-21. On the basis of the combination of these NMR results, a framework of 4-substituted piperidine-2-carboxylic acid (pipecolic acid) can be deduced for the structure of the CeHiONO2 unit of damipipecolin. The oxymethine proton on C-41 (δ 5.31) reveals an HMBC long-range correlation with the C-6 carbon (δ 159.9), thus indicating that C-4' is connected with the bromopyrrolecarboxylic acid moiety through an ester linkage. The relative stereochemistry of the two substituents on the pipecolic acid ring can be defined by 1H-1H coupling constants analysis. The C-21 proton, a α proton of the pipecolic acid resonating at δ 3.74, appears as a double doublet with a large coupling of 12 Hz and a small coupling of 3 Hz to the protons of the adjacent C-3'
methylene, whereas H-4' (brs) shows small J values to the protons of the adjacent C-31 and 5' methylenes. Assuming that the piperidine ring is in a chair-like conformation, a trans relative configuration of the 4-substituted pipecolic acid, where H-21 and H-4' occupy an axial and an equatorial position, respectively, is consistent with the observed coupling pattern. The absolute stereochemistry of the amino acidic portion can be clarified through acid hydrolysis of 1 performed with 1 N HCl in H2O at 8O0C overnight. The reaction mixture is evaporated under nitrogen and then subjected to HPLC purification using a RP- 18 Luna 5 μ (250 x 3 mm) column with H2OMeOH 8:2 as the eluent, yielding 1 mg of pure 4-bromopyrrole 2-carboxylic acid (3) and (+)- /raw-4-hydroxypipecolic acid (4, [α]D 20 = + 23, c = 0.001, H2O; ESIMS : m/z 146 [M+H]+; 1H and 13C-NMR are reported in Table 1).
Both compounds can be easily identified by comparison of their spectroscopic data with those reported in literature; in particular, the NMR data of 4- hydroxypipecolic acid are identical to those reported for the (2R, AR)-trans isomer (Agami, C, Couty, F., Poursoulis, M., Vaissermann, J. Tetrahedron, 1992, 48, 431), thus confirming the relative stereochemistry proposed for damipipecolin. Both the sign and value of the optical rotation of the amino acid 4 matches well with that of (2R, 4i?)-4-hydroxypipecolic acid reported previously (Clark-Lewis, J. W., Mortimer, P. I. J. Chem. Soc, 1961, 189) thus indicating that the absolute stereochemistry of the aminoacidic portion of damipipecolin is (2R, AR).
Structural elucidation of damituricin
The ESI (positive ions) mass spectrum of damituricin (2) shows two intense pseudomolecular ion peaks at m/z 331 and 333 [M+H]+ in the ratio 1 :1. The 13C-NMR spectrum (CD3OD) contains twelve carbon signals, which are identified, through DEPT and HSQC experiments, as two methyls, two sp3 methylenes, two sp2 methines, two deshielded sp3 methines, and four unprotonated carbons. These NMR and mass
spectral data are compatible with the molecular formula Ci2HiSN2O4Br, implying six elements of unsaturation. From 13C-NMR data, the presence of two carbon-carbon double bonds and two carbonyl groups is evident; thus, also molecule 2 must be bicyclic. The comparison of 1H and 13C-NMR spectra of 1 and 2 reveals some similarities. Particularly, the aromatic proton and carbon resonances present in the spectra of 2 (δc: 124.3, 97.1, 117.5, 122.2; δH: 6.95, 7.05) are almost identical to those of 1, allowing the inventors to identify in 2 the same 4-bromopyrrole-2-carboxylic acid moiety as in 1, connected via an ester bond (δc 159.2) to a different cyclic unit. Analysis of 1H-NMR spectrum (CD3OD) of 2, integrated with data obtained from the HSQC experiment, shows, in addition to pyrrole signals, the presence of two three proton singlet at δ 3.43 and 3.34, attributable to N-linked methyls of a quaternary ammonium compound, also on the basis of their deshielded carbons (δ 52.3 and 46.7). Additional features of 1H-NMR spectrum are two AB system due to methylene groups (2H5: δ 3.92, Ha; 3.98, Hb. 2H3: δ 2.64, Ha; 3.10, Hb), and two sp3 deshielded methine signals (δ 4.19 and 5.56). These signals are connected, by interpretation of COSY spectrum, in an only large spin system belonging, also according to mass data, to an oxygenated nitrogen-containing ring, as deduced by the following HMBC results. In the HMBC spectrum, both the N-methyl protons are long range correlated to the methylene carbon at δ 72.0 (C-4') and the methine at δ 75.7 (C-2'); thus, the same nitrogen atom was inferred to be linked to these carbons and to methyl carbons. A further key cross peak is observed in the HMBC spectrum between the proton resonating δ 4.19 (H-21) and the carbonyl resonating at δ 169.0; it clearly indicates that a carboxylate function is linked at C-2\ On the basis of these results, and according to the whole series of COSY, HSQC and HMBC correlations, an N,N'-dimethyl-4- hydroxypyrrolidine-2-carboxylic acid (4-hydroxyproline betaine) unit is deduced to be present in 2. An HMBC correlation between the oxymethine proton at δ 5.56 (H-4') and the ester carbonyl at δ 159.2 suggests that this unit is connected to the bromopyrrolecarboxylic acid moiety through an ester linkage. Crystal structures of the two diastestereoisomeric betonicine (trans-NJV- dimethyl-4-hydroxy-L-proline, levorotatory) and turicine (c«-N,N'-dimethyl-4- hydroxy-D-proline, dextrorotatory) (Kueng A., Trier, G., Z. physiol. Chem., 1913, 85,
209), have been determined as their hydrochlorides (Jones, G.P., Naidu, B.P., Paleg, L. G., and Tienik, E.R.T. Acta Cryst., 1988, C44, 2208). Taking into account these reported data, the relative cw-stereochemistry to the betaine unit present in the molecule of 2 can be assigned on the basis of the data obtained from a ROESY spectrum (CD3OD). In fact, an intense cross peak is observed between H-2' (δ 4.19) and H-41 (δ 5.56), which is consistent only with a relative cw-orientation of the carboxylate group and the acylic portion. The absolute stereochemistry of the amino acidic portion of 2 can be clarified through acid hydrolysis of 2. It can be performed following the same procedure reported for 1 and gives 4-bromopyrrole 2-carboxylic acid (3) and (+)-cz>N,N'-dimethyl-4-hydroxy-D-proline (turicine, 5, [α]o 20 = + 20.3, c = 0.001, H2O; ESIMS : m/z 182 [M+Na]+; 1H and 13C-NMR are reported in Table 1), easily identified by comparison of their spectroscopic data with reported in literature (Kueng A., Trier, G., Z. physiol. Chem., 1913, 85, 209; Jones, G.P., Naidu, B.P., Paleg, L. G., Tienik, E.R.T., and Snow, M. R. Phytochemistry, 1987, 26, 3343). On the basis of this result, the inventors assign the (2i?, 4R) absolute stereochemistry to the amino acidic portion of damituricin.
Synthesis of damipipecolin and its analogues
A synthetic access to the new alkaloid damipipecolin has been designed. The crucial step of the proposed strategy consists of a Mitsunobu reaction (Mitsunobu, O. Synthesis, 1981, 1; Hughes, D. L. Org. React, 1992, 42, 335) with the HFA-protected cw-4-hydroxy-D-pipecolic acid derivative 8 and 4-bromopyrrole-2-carboxylic acid (3). The Mitsunobu reaction is a particularly useful reaction in organic synthesis for the stereochemical inversion of hydroxyl groups. The protocol for the Mitsunobu reaction provides for a mixture of triphenylphosphine, a dialkyl azodicarboxilate, and an acid component (usually a carboxylic acid). In the synthesis being part of this invention, the 4-bromopyrrole-2-carboxylic acid is used to ensure the acidity of the reaction mixture, and the corresponding ester of HFA-protected ft-αns-4-hydroxy-D- pipecolic acid (9) is obtained. Final deprotection of the amino and carboxylic functions, achieved on treatment with .-PrOHZH2O, affords damipipecolin 1 (Scheme 1). 4-Bromopyrrole-2-carboxylic acid (3) is prepared by bromination of the commercially available 2-trichloroacetylpyrrole (6), which selectively gives the 4- bromo-2-trichloroacerylpyrroIe (7); hydrolysis of 7 with an aqueous base gives the
free acid 3. The HFA-protected c/s^-hydroxy-D-pipecolic acid derivative 8 could be obtained starting from D-aspartic acid and hexafluoroacetone as protecting agent according to the procedure described by Golubev and coworkers (Golubev, A., Sewald, N., Burger, K. Tetrahedron Lett., 1995, 36, 2037).
Scheme 1
The above reported procedure can easily be adopted for the preparation of other damipipecolin derivatives. As reported in scheme 2, starting from unsubstituted or diversely substituted pyrrole-2 carboxylic acids, a series of analogues could be obtained, differing in the substitution pattern on the pyrrole moiety. Alternatively, esterification of pyrrole-2-carboxylic acid chloride derivatives with HFA-protected
cw-4-hydroxy-L-pipecolic acid derivative 8 gives the series of the corresponding cis- 4-hydroxy-L-pipecolic acid derivatives (Scheme 2).
H20//-Pr0H
Scheme 2
Synthesis of damituricin and its analogues The synthesis of damituricin can be accomplished starting from commercially available cis-4-hydroxy-D-proline (10). Compound 10 is converted into the corresponding betaine (11) by reaction of methyl iodide with the silver salt of the amino acid in methanol at room temperature (Patchett, A.A., and Witkop, B. J Am.
Chem. Soc. 1957, 79, 185). After protection of the 4-hydroxyl group of 11 as its benzyl ether, the resulting amino acid 12 is converted into the corresponding tert-butyl ester 13 by treatment with perchloric acid and fer/-butyl acetate (Dolence, E.K., Lin, C, and Miller, MJ. J. Med. Chem., 1991, 34, 956). Cleavage of the beri2yl ether is then achieved by catalytic hydrogenolysis and provide cw-N,N'-dimethyl-4-hydroxy- D-proline tert-butyl ester (14) (Scheme 3).
te
Scheme 3
The 4-bromopyrrole-2-carboxylic acid (3) is prepared according to the procedures reported in Scheme 1 and then converted in the corresponding chloride 15 as reported in Scheme 2. Reaction of 14 with 15, followed by removal of the tert-buty\ ester group by brief treatment (1 h) with trifluoroacetic acid gives damituricin 2 in its protonated form. (Scheme 4).
Scheme 4
A series of analogues of damituricin, differing in the substitution pattern on the pyrrole moiety, could be obtained with the same procedure, by reaction of unsubstituted or diversely substituted pyrrole-2-carboxylic acid chlorides with D- turicine tert-butyl ester (14). Furthermore, esterification of the diversely substituted pyrrole-2-carboxylic acid chlorides with the tert-butyl esters of the isomeric betaines betonicine (17) or L-turicine (18), gives the corresponding diastereomers and enantiomers of these analogues (Scheme 5). Compounds 17 and 18 can be prepared starting from tomy-4-hydroxy-L-proline and cw-4-hydroxy-L-proline (both commercially available), respectively, following the procedure reported in Scheme 3.
TFA
TFA CH2CI2
40
1h 1h
Cytotoxic effect Scheme 5
The cell viability tests using the MTT assay (Bringmann, G., Lang, G., Mϋhlbacher, J., Schaumann, K., Steffens, S., Rytik, P. G., Hentschel, U., Morschhauser, J., Brun,
R., Mϋller, W. E. G. In: Marine Molecular Biotechnology; Mϋller, W. E. G., Ed.;
Springer-Press: Berlin, 2003; pp. 231-253) reveals that compound 1 and compound 2
are not toxic at a concentration of 10 μg/ml or less for all cell lines tested (PC 12, HeLa, and L5178y cells).
Effect of compound 1, compound 2, and serotonin on the Ca^-level in primary neurons
Stimulation of the neurons with 200 μM serotonin (5-hydroxytryptamin; 5-HT) and 2.5 mM CaCl2 results in a significant (p < 0.001) increase in intracellular free calcium level ([Ca2+]O Δ ratio 1.318 (set to 100%). The changes are expressed as 340/380 nm ratio values. As shown in Figure 1 preincubation of the neurons for 5 min with 10 μg/ml of compound 1 (D) induces no significant changes in the [Ca2+]j after addition of 5- HT/CaC-2. The [Ca2+]i values increases to 114.9%. In the comparable experiment with compound 2 (■) a small decrease in the [Ca2+Ji values to 78.1% was detected. hi contrast, preincubation (5 min) of neurons with 10- and 100-times (1 and 0.1 μg/ml) lower dose of compound 1 and 2 induces a significant (p < 0.001) reduction of the [Ca2+Jj influx after addition of 200 μM 5-HT and 2.5 mM CaCl2 (Figure 1). The [Ca2+J1 values decrease to 32.8/41.2% (1 μg/ml; compound 1 and compound 2, respectively) and to 9.6/29.1% (0.1 μg/ml; compound 1 and compound 2, respectively).
Effect of compound 1, compound 2, and serotonin on the Ca2+-IeVeI in PC12, HeLa and HEK cells
The same experiments with PC 12, HeLa and HEK cells give the following results. Treatment of PC12, HeLa and HEK cells with 200 μM 5-HT and 2.5 mM CaCl2 results only in a small increase in [Ca2+Jj (expressed as 340/380 nm ratio values); Δ ratio 0.096 (PC12 cells), Δ ratio 0.037 (HeLa cells), and Δ ratio 0.115 (HEK cells). The control values are set to 100% (Figure 3A).
As shown in Figure 3 A preincubation of PC 12 cells for 5 min with 1 μg/ml of compound 1 (D) does not induce a significant change of [Ca2+Jj after addition of 5- HT/CaC.2, while preincubation of HeLa cells with compound 1 under the same conditions results in a strong (2.7-fold) increase in [Ca2+J,. In parallel experiment preincubation of PC 12 cells for 5 min with 1 μg/ml of compound 2 (■) generates a significant change of [Ca2+Jj after addition of 5-HT/CaCl2. The [Ca2+Jj decreased to
43.8%. At the same time preincubation of HeLa cells with compound 2 under the same conditions causes no changes in [Ca2+]-,.
Preincubation of HEK cells with 10 μg/ml of compound 1 resulted after addition of 5-HTZCaCi2 in a significant (p < 0.001) increase in [Ca2+J1 to 280.9% (Figure 3B; D). Preincubation of the cells with 10 μg/ml of compound 2 resulted after addition of serotonin/CaCh in small increase in [Ca2+]J to 147.0% (■). In response to control no significant changes in the [Ca2+]; were measured after treatment of the cells with 1 and 0.1 μg/ml of compound 1 or compound 2, respectively (Figure 3B) following application of 5-HTVCaCh.
Effect of compound 1, compound 2, and L-glutamic acid on the Ca2+-level in primary neurons
Stimulation of neurons with 200 μM L-glutamic acid (L-GIu) and 2.5 mM CaCl2 results in a significant (p < 0.001) increase in [Ca2+Jj from 0.704 ± 0.012 to 2.533 ± 0.058 (Δ ratio 1.829; 100%).
As shown on Figure 2 preincubation (5 min) of the neurons with 10, 1, and 0.1 μg/ml of compound 1 (o) and compound 2 (■) induces no significant decrease in the
[Ca2+], after addition of L-Glu/CaC^. In comparison with the control the changes in
[Ca2+J1 in neurons treated with 10 μg/ml of compound 1 and compound 2 are 96.6% and 98.9%, respectively; with 1 μg/ml of compound 1 and compound 2 are 90.5% and
100.2%, respectively and with 0.1 μg/ml of compound 1 and compound 2 are 83.3% and 109.7%, respectively.
Effect of compound 1, compound 2, and N-methyl-D-aspartic acid on the Ca2+-level in primary neurons
Stimulation of neurons with 200 μM N-methyl-D-aspartic acid (NMDA) and 2.5 mM CaCh results in a significant (p < 0.001) increase in [Ca2+];; Δ ratio 0.868 (set to 100%).
As shown on Figure 4 preincubation (5 min) of the neurons with 10 and 0.1 μg/ml of compound 1 (o) induces no significant decrease in the [Ca2+I1 ^61" addition of NMDA/CaCh. In comparison with the control the changes in [Ca2+] i are between
105.9% (Δ ratio 0.919; 10 μg/ml) and 114.1% (Δ ratio 0.990; 0.1 μg/ml). Only after addition of 1 μg/ml of compound 1 a significant decrease in [Ca2+]; was measured
(70.4%, Δ ratio 0.611). In contrast, pretreatmet of neurons with compound 2 induces only in the dose group of 10 μg/ml (Figure 4; ■) a significant decrease in [Ca2+]) after addition of NMDA/CaCl2. The values decrease to 58.8% (Δ ratio 0.510). No changes are measured after treatment of neurons with i (119.52%) and 0.1 μg/ml (95.0%) of compound 2.
Application in biomedicine and human therapy
Serotonin (5-hydroxytryptamin, 5-HT) belongs to the class of biogenic amines (indolamines or monoamine transmitters). Within the CNS the serotonergic neurons can be found in nearly every brain area Serotonin receptors can be classified in three different groups: (Q the transporter (5-HT "uptake site"), (if) the ligand-gated ion- channel (5-HT3) and (Ui) the largest group of the G protein-coupled receptors (Boess, F. G., Martin, I. L. Neuropharmacology, 1994, 33, 275). On the basis of cloning data until now seven subtypes (5-HT1-5-HT7) have been distinguished (Zifa, E., Fillion, G. Pharmac. Rev., 1992, 44, 401). 5-Hydroxytryptamin modulates several biological function in the CNS (Maeda, T., Fujimiya, M., Kitahama, K., Imai, H., Kimura, H. Arch. Histol. Cytol, 1989, 52 (Suppl), 113); it influences processes related to the memory, and learning, sexual behaviour, as well as feeding behaviour (Brunelli, M., Garcia-Gil, M., Mozzachiodi, R., Scuri, R., Zaccardi, M.L Arch. Ital. Biol. , 1997, 135, 15). It seems also be involved in regulating aggressive behaviour. Also release of adenocorticotropin (ACTH), corticosterone, prolactin and gonadotropins can be activated by serotonin (Fuller, R. W. Neuropsychopharmacology, 1990, 3, 495). Alteration in serotonin function have been linked to anxiety states, affective disorders, hallucinogenic behaviour, motion sickness, eating disorders, sleep disorders and migraine (Johnson, K. W., Phebus, L. A., Cohen, M. L. Prog. Drug Res., 1998, 51, 219). The serotonergic system plays a significant role in the generation of depression. 5-HT reuptake inhibitors are effective antidepressants (Goodnick, P. J., Goldstein, B. J. J. Psychopharmacol, 1998, 12(Suppl. B), 5). Some forms of phobia seem to be associated with an increase in serotonin levels. In the treatment of psychosis, antagonists of 5-HT2 receptor seem to be efficacious (Hollister, L. E. J. Clin. Psychopharmacol., 1994, 14, 50; Love, R. C, Nelson, M. W. Expert. Opin. Pharmacother., 2000, 1, 1441; Ikeguchi, K., Kuroda, A. Eur. Arch. Psychiatry Clin.
Neurosci., 1995, 244, 320). Since 5-HT1 receptors are implicated in the pathogenesis of many disorders and Ln contrast to the receptors of the 5-HT2 and 5-HT4 they display a high affinity for serotonin, they become an important target for drug therapy (Passchier, J., van Waarde, A. Eur. J. Nucl. Med., 2001, 28, 113). Preincubation of neurons with 10 μg/ml of the compound 1 and compound 2 induces no or small (-22% reduction) decrease in Ca2+ influx after addition of 200 μM serotonin. Reduction of the concentration of 1 and 2 to 1 and 0.1 μg/ml resulted in a strong decrease of the Ca2+ influx in neurons. Compound 1 is a potent antagonist of serotonin in the concentration of 1 μg/ml (~70% reduction), while the effect of serotonin on neurons was completely blocked after the addition of 0.1 μg/ml of DAM (~91% reduction), hi contrast, compound 2 is a less potent antagonist of serotonin at the concentration of 1 μg/ml (~60% reduction) and 0.1 μg/ml of DAM (-70% reduction). Different types of serotonin receptors exist in brain tissue. Compounds that interact with 5-HT receptors have important therapeutic applications. In conclusion, compound 1 is a new promising serotonin antagonist with a potential in the therapy of emesis induced by chemotherapy, psychosis, different phobia and mood fluctuation disorders. Compound 2 has also antagonistic effects on serotonin receptors.
The amino acids L-glutamate and L-aspartate are the most abundant key excitatory neurotransmitters in the central nervous system (CNS); glutaminergic neurons are especially prominent in the cerebral cortex (Arai, Y., Mizuguchi, M., Takashima, S. Anat. Embryol. (Berl), 1997, 195, 65). The biological effects of these excitatory amino acids are manifold. On one hand, glutamate provides beneficial effect in the regulation of neuronal function, growth and differentiation, on the other hand, excitatory amino acids can be harmful to brain tissue. The excitotoxic effect is related to the massive influx of Na+, K+, and Ca2+ in neurons as a consequence of the sustained activation of glutamate receptors (Arundine, M., Tymianski, M. Cell Calcium, 2003, 34, 325; Stys, P.K. Curr. MoI Med., 2004, 4, 113). In our experiments, preincubation of neurons with 10, 1, and 0.1 μg/ml of compound 1 or compound 2 induces no significant decrease in Ca2+ entry in the presence of glutamate or NMDA. Thus, compound 1 and compound 2 are not binding on glutamate or NMDA receptor.
Experimental procedures
General
ESI mass spectra can be obtained by using for example an API 2000 mass spectrometer. High-resolution FAB mass spectra (glycerol matrix) can be performed for example on a VG Prospec (FISONS) mass spectrometer. Optical rotations can be measured using for example a Perkin-Elmer 192 polarimeter. CD spectra can be recorded for example on a J-710 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a J-710 for Windows software (Jasco). NMR experiments can be performed for example on a Bruker AMX-500 spectrometer; chemical shifts are referred to the residual solvent signal. Medium-pressure liquid chromatographies (MPLC) can be performed for example on a Buchi 861 apparatus with SiO2 (230-400 mesh) packed columns. High-performance liquid chromatography (HPLC) separations can be achieved for example on a Waters 501 apparatus equipped with an RI detector. UV spectra (MeOH) can be recorded for example on a Sbimadzu UV- 1204 instrument.
Cell lines
PC 12 cells are grown in Dulbecco's modified Eagle's medium [DMEM]/10% (v/v) fetal calf serum (FCS)/5% (v/v) horse serum. HEK cells are grown in Dulbecco's modified Eagle's medium [DMEM]/10% (v/v) FCS/4.5 g/l glucose. PC 12 and HEK cells are passaged twice per week at a 1:10 ratio. L5178y and HeLa cells are maintained in Roswell Park Memorial Institute medium [RPMI] 1640 supplemented with 10% (v/v) FCS. The cells are subcultured twice weekly at a 1:160 (L5178y cells) and 1:10 ratio (HeLa cells). All cells are kept in an atmosphere of 95% air and 5% CO2 at 37°C.
MTT assay
To estimate the IC50 values, PC 12, L5178y, and HeLa cells are incubated for 72 h in the presence of different concentrations (0.1; 0.3; 1; 3, and 10 μg/ml) of compound 1 or compound 2. The final volumes are 200 μl. Compound 1 is dissolved in DMSO (stock solution 10 mg/ml) and stored at -200C. The compound 2 is dissolved in H2O (stock solution 5 mg/ml) and stored at -200C. The viability of the cells is determined using the MTT colorimetric assay system (Scudiero, D. A., Shoemaker, R. H., Paull,
K. D., Monks, A., Tierney, S., Nofciger, T. H., Currens, M. J., Seniff, D., Boyd, M. R. Cancer Res., 1988, 48, 4827). Evaluation is performed in 96- well plates at 595 nm using an ELISA plate reader, after overnight incubation at 37°C.
Calcium measurements on primary neurons, PCl 2, HeLa and HEK cells
Rat cortical cell cultures are prepared from the brains of 17- to 18-day-old Wistar rat embryos, as described (Freshney, R. I. In: Culture of Animal Cells. A Manual of Basic Technique; Freshney, R. L, Ed., Alan R. Liss Inc.: New York, 1987; pp. 257-288; Perovic, S., Schleger, C, Pergande, G., Iskric, S., Ushijima, H., Rytik, P., Mϋller, W. E. G. Eur. J. Pharmacol. (MoI Pharmacol. Sec), 1994, 288, 27). Briefly, after isolation cerebral hemispheres are placed into Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+. Brain tissue is dissociated in HBSS using 0.025% (w/v) trypsin (10 min; 37°C); the proteolytic reaction is stopped by addition of 10% FCS. The single cell suspension is centrifuged and the pellet containing dissociated neuronal cells is resuspended in DMEM/HG (high glucose; 4.5 g/1 glucose), containing 2 mM L-glutamine, 100 mU/1 insulin, and 10% (v/v) FCS. The cells are seeded into poly-L-lysine (5 μg/ml, 300 μl/cm2)-coated plastic dishes at a concentration of 2.0 x 105 cells/cm2. Two days after isolation DMEM/HG/10% (v/v) FCS is removed and the cells are cultivated further in a DMEM/HG serum-free medium supplemented with 0.1% (w/v) bovine serum albumin (BSA), 2 mM L- ghitarnine, 100 μg/ml transferrin, 100 mU/1 insulin, 16 μg/ml putrescine, 6.3 ng/ml of progesterone, and 5.2 ng/ml Na2SeO3.
For determination of the intracellular Ca2+ concentration ([Ca2+Jj), cells are cultivated on poly-L-lysine coated borosilicate coverglass (Nunc). An inverted-stage microscope (for example Olympus LX70with objective UApo40X/340) is used for the fluorescence measurements. The cells are alternately illuminated with light of wavelengths 340 and 380 nm. An additional 0.25 ND filter is used at 380 nm. The fluorescence emissions at 510 nm are monitored by an intensified CCD camera, for example model C2400-87 (Hamamatsu, Herrsching, Germany). Images are then digitized with a computerized imaging system (for example Argus-50, Hamamatsu).
The [Ca2+Jj is determined by measuring the fluorescence ratio of the Ca2+- indicator dye fura-2-AM at 340 and 380 nm (Grynkiewicz, G., Poenie, M., Tsien, R. Y. J. Biol. Chem., 1985, 260, 3440). Neurons are loaded with 4 μM fura-2-
acetoxymethyl (AM) ester and PC 12, HeLa and HEK cells with 10 μM fiira-2-AM in DMEM/HG serum-free medium, supplemented with 1% (w/v) BSA at 370C for 60 min. A calcium calibration curve is prepared according to the method of Grynkiewicz (Grynkiewicz, G., Poenic, M., Tsien, R. Y. J. Biol. Chem., 1985, 260, 3440). One ratio value 340/380nm equals 228 nM [Ca2+],.
Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5.6 mM glucose and 10 mM Hepes; pH 7.4) without Ca2+ and Mg2+ is used as incubation medium in all set of experiments. A stock solution of 10 mg/ml of compound 1 dissolved in DMSO and of 5 mg/ml of compound 2 dissolved in H2O are used. All serial dilution are done in DMSO (compound 1) or H2O (compound 2).
In the first set of experiments neurons are stimulated with (compound 1) or without (compound 2) 0.1% (v/v) DMSO after 5 min and with 200 μM of serotonin (5-HT), N-methyl-D-aspartic acid (NMDA) or L-glutamic acid (L-GIu) and 2.5 mM CaCl2 after 10 min from the beginning of the measurements. In the second set of experiments the primary neurons are first preincubated with 10, 1, and 0.1 μg/ml of the compound 1 or compound 2 (5 min) and after 10 min 5-HIVCaCl2, NMDAZCaCl2 or L-Glu/CaCl2 are added to the neurons.
In the next set of experiments PC 12 and HeLa cells are first preincubated with 1 μg/ml of compound 1 or compound 2 (5 min) and after 10 min 5-HT/CaCl2 are added to the cells. In the case of HEK cells, the peincubation of the cells is done with 10, 1 and 0.1 μg/ml of the compound 1 or compound 2 (5 min) and after 10 min 5- HT/CaCl2 are added to the cells. As a control the cells treated with or without 0.1% (v/v) DMSO and 200 μM 5-HT with 2.5 mM CaCl2 are used.
In all set of experiments the [Ca2+]; level is measured during the whole incubation period (for at least 20 min).
Legends to Figures
Figure 1. Incubation of the neurons with 200 μM serotonin (5-hydroxytryptamin; 5- HT) and 2.5 mM CaCk in. the presence of 10, 1, and 0.1 μg/ml of compound 1 and compound 2. In the first set of experiments (control) neurons were stimulated only with 5-HT and CaC^. In the second set of experiments the effect of 5-HT/CaCl2 on the calcium level in neurons preincubated for 5 min with 10, 1, and 0.1 μg/ml of compound 1 (α) and compound 2 (■) was measured. Experiments were completed in Locke's solution. In all assays [Ca2+J1 level was measured for 20 min. The results are expressed as % of control (assays with serotonin only). DAM-I : compound 1; DAM- 2: compound 2.
Figure 2. Incubation of the neurons with 200 μM L-glutamic acid (L-GIu) and 2.5 mM CaCl2 in the presence of 10, 1, and 0.1 μg/ml of compound 1 (α) and compound 2 (■). The results are expressed as % of control (control n = 103; compound I n = 112; compound 2 n = 96). Further details are given in Figure 1. DAM-I: compound 1; DAM-2: compound 2.
Figure 3. A Incubation of the PC 12, HeLa and HEK cells with 200 μM serotonin (5- HT) and 2.5 mM CaCl2 in the presence of 1 μg/ml of compound 1 (G) and compound 2 (■). B Incubation of the HEK cells with 5-HT/CaC-2 in the presence of 10, 1, and 0.1 μg/ml of compound 1 (o) and compound 2 (■). The results are expressed as % of control (control n = 59; compound 1 n = 86; compound 2 n = 62). Further details are given in Figure 1. DAM-I : compound 1; DAM-2: compound 2.
Figure 4. Incubation of the neurons with 200 μM N-methyl-D-aspartic acid (NMDA) and 2.5 mM CaCh in the presence of 10, 1, and 0.1 μg/ml of compound 1 (α) and compound 2 (■). The results are expressed as % of control (control n = 98; compound 1 n = 110; compound 2 n = 105). Further details are given in Figure 1. DAM-I: compound 1; DAM-2: compound 2.
able 1. 1H and 13C NMR data of compounds 1, 2, 4-hydroxypipecolic acid (4), and D-turicine (5).
DD3OD. b. D2O. c. Overlapped signals
Claims
1. Compound of the general structure 19
19
where Rl to R3 can be H, methyl, ethyl, alkyl, methoxy, ethoxy, alkoxy or halogen and R4 is a piperidine- or pyrrolidine-2-carboxylic acid derivative.
2. Compound according to claim 1 with structure 1:
1
(damipipecolin) or its derivatives, diastereomers, as well corresponding enantiomers and pharmaceutically tolerable salts or solutions of this compound.
3. Compound according to claim 1 with structure 2:
4. Procedure for synthesis of a compound according to claims 1, 2 or 3, by isolating a sponge of the order Axinella, in particular Axinella damicornis.
5. Procedure for synthesis of a compound of the general structure 19 according to claim 1.
6. Procedure according to claim 5, additionally comprising a subsequent synthetic derivatisation of the described compound.
7. Compound of the general structure 19 or structures 1 and 2 according to claims 1, 2, and 3 for prophylaxis or treatment of diseases.
8. Compound of the general structure 19 or structures 1 and 2 according to claims 1, 2 and 3 for prophylaxis or treatment of neurodegenerative diseases.
9 Compound of the general structure 19 or structures 1 and 2 according to claims 1, 2 and 3 for the use as an antiemeticum.
10. Compound of the general structure 19 or structures 1 and 2 according to claims 1, 2 and 3 for the treatment and prevention of emesis induced by chemotherapy.
11. Pharmaceutical composition comprising a compound according to claims 1, 2 and 3 together with suitable additives and supplements.
12. Pharmaceutical composition according to claim 11, wherein the compound is present in form of a depot compound or as a precursor together with a suitable, pharmaceutically tolerable dilution solution or a carrier substance.
13. Pharmaceutical composition according to claim 11 to 12, wherein this composition has additional bioactivity that positively interacts with further compounds and modulates the intracellular calcium concentration.
14. Pharmaceutical composition according to claim 11 or claim 12 in form of tablets, coated tablets (dragees), capsules, drops, suppositories, and preparations for injections or infusions for peroral, rectal or parenteral application.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009152071A1 (en) * | 2008-06-09 | 2009-12-17 | Vanderbilt University | Unnatural dispyrin analogues, preparation and uses thereof |
JP2020519650A (en) * | 2017-05-12 | 2020-07-02 | オスピタル サン ジュアン ダ デウ | Betaine for the prevention of obesity |
-
2007
- 2007-03-19 WO PCT/EP2007/002417 patent/WO2007107319A1/en active Application Filing
Non-Patent Citations (2)
Title |
---|
CAFIERI, FRANCESCO ET AL: "A novel bromopyrrole alkaloid from the sponge Agelas longissima with antiserotonergic activity", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS , 5(8), 799-804 CODEN: BMCLE8; ISSN: 0960-894X, 1995, XP002430513 * |
FATTORUSSO ERNESTO ET AL.: "Novel bioactive bromopyrrole alkaloids from the Mediterranean sponge Axinella verrucosa", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 14, 2006, pages 17 - 24, XP002430512 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009152071A1 (en) * | 2008-06-09 | 2009-12-17 | Vanderbilt University | Unnatural dispyrin analogues, preparation and uses thereof |
JP2020519650A (en) * | 2017-05-12 | 2020-07-02 | オスピタル サン ジュアン ダ デウ | Betaine for the prevention of obesity |
US11464754B2 (en) | 2017-05-12 | 2022-10-11 | Hospital Sant Joan De Deu | Betaine for the prevention of obesity |
JP7184805B2 (en) | 2017-05-12 | 2022-12-06 | オスピタル サン ジュアン ダ デウ | Betaine for prevention of obesity |
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