Chromane-like cyclic prenylflavonoids for the medical intervention in neurological disorders

Abstract

The present invention relates to certain chromane-like cyclic prenylflavonoids, in particular the compounds of formulae (I), (II) and (III) as described and defined herein, for use in the treatment or prevention of a neurological disorder, as well as their use in promoting neuronal differentiation, neurite outgrowth and neuroprotection. ##STR00001##

Claims

1. A method of treating a neurological disorder selected from the group consisting of a spinal cord injury and a peripheral nerves injury, the method comprising the administration of a compound of the following formula (I) or a pharmaceutically acceptable salt or solvate of said compound to a subject in need of such a treatment, ##STR00053## wherein: A is aryl or heteroaryl, wherein said aryl or said heteroaryl is optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCOC.sub.1-6 alkyl, OCO-phenyl, OCO(C.sub.1-6 alkylene)-phenyl, OCOO(C.sub.1-6 alkyl), COC.sub.1-6 alkyl, COOH, COO(C.sub.1-6 alkyl), CONH.sub.2, CONH(C.sub.1-6 alkyl), CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCONH.sub.2, OCONH(C.sub.1-6 alkyl), OCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), NHCO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CO(C.sub.1-6 alkyl), NHCOO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-COO(C.sub.1-6 alkyl), NHCONH.sub.2, N(C.sub.1-6 alkyl)-CONH.sub.2, NHCONH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CONH(C.sub.1-6 alkyl), NHCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN; R.sup.10 is OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), hydrogen, or C.sub.1-6 alkyl; R.sup.11 and R.sup.12 are each independently selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCOC.sub.1-6 alkyl, OCO-phenyl, OCO(C.sub.1-6 alkylene)-phenyl, OCOO(C.sub.1-6 alkyl), COC.sub.1-6 alkyl, CONH.sub.2, CONH(C.sub.1-6 alkyl), CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCONH.sub.2, OCONH(C.sub.1-6 alkyl), OCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), NHCO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CO(C.sub.1-6 alkyl), NHCOO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-COO(C.sub.1-6 alkyl), NHCONH.sub.2, N(C.sub.1-6 alkyl)-CONH.sub.2, NHCONH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CONH(C.sub.1-6 alkyl), NHCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN; each R.sup.13 is independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCOC.sub.1-6 alkyl, OCO-phenyl, OCO(C.sub.1-6 alkylene)-phenyl, OCOO(C.sub.1-6 alkyl), COC.sub.1-6 alkyl, CONH.sub.2, CONH(C.sub.1-6 alkyl), CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCONH.sub.2, OCONH(C.sub.1-6 alkyl), OCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), NHCO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CO(C.sub.1-6 alkyl), NHCOO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-COO(C.sub.1-6 alkyl), NHCONH.sub.2, N(C.sub.1-6 alkyl)-CONH.sub.2, NHCONH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CONH(C.sub.1-6 alkyl), NHCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN; R.sup.14 and R.sup.15 are each independently selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCOC.sub.1-6 alkyl, OCO-phenyl, OCO(C.sub.1-6 alkylene)-phenyl, OCOO(C.sub.1-6 alkyl), COC.sub.1-6 alkyl, CONH.sub.2, CONH(C.sub.1-6 alkyl), CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCONH.sub.2, OCONH(C.sub.1-6 alkyl), OCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), NHCO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CO(C.sub.1-6 alkyl), NHCOO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-COO(C.sub.1-6 alkyl), NHCONH.sub.2, N(C.sub.1-6 alkyl)-CONH.sub.2, NHCONH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CONH(C.sub.1-6 alkyl), NHCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN; R.sup.16 is O, S or N(OH); n is 0, 1, 2 or 3; and each in formula (I) is independently a single bond or a double bond, provided that at least one of the two adjacent bonds is a single bond.

2. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein A is phenyl which is optionally substituted with one or more groups independently selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, OH, O(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCOC.sub.1-6 alkyl, OCO-phenyl, OCO(C.sub.1-6 alkylene)-phenyl, OCOO(C.sub.1-6 alkyl), COC.sub.1-6 alkyl, COOH, COO(C.sub.1-6 alkyl), CONH.sub.2, CONH(C.sub.1-6 alkyl), CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), OCONH.sub.2, OCONH(C.sub.1-6 alkyl), OCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), NHCO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CO(C.sub.1-6 alkyl), NHCOO(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-COO(C.sub.1-6 alkyl), NHCONH.sub.2, N(C.sub.1-6 alkyl)-CONH.sub.2, NHCONH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CONH(C.sub.1-6 alkyl), NHCON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)-CON(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN.

3. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein A is phenyl which is optionally substituted with one group selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), or halogen.

4. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R.sup.10 is OH, or O(C.sub.1-6 alkyl).

5. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R.sup.10 is OCH.sub.3.

6. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R.sup.11 and R.sup.12 are each independently selected from hydrogen, C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN.

7. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein each R.sup.13 is independently selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), NH.sub.2, NH(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)(C.sub.1-6 alkyl), halogen, CF.sub.3, or CN.

8. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R.sup.14 and R.sup.15 are each independently selected from hydrogen, C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), or halogen.

9. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein n is 0.

10. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein n is 2 and each R.sup.13 is independently selected from hydrogen, C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), or halogen.

11. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R.sup.16 is O.

12. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein each in formula (I) is a single bond.

13. The method of claim 1, wherein said compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein one of the two adjacent bonds in formula (I) is a single bond and each one of the other two bonds in formula (I) is a double bond.

14. The method of claim 1, wherein said compound is a compound of the following formula 1a, 1b, 1c, 1d, 1e, 1f, or 1g ##STR00054## ##STR00055## or a pharmaceutically acceptable salt or solvate thereof.

15. The method of claim 1, wherein said subject is a human.

16. The method of claim 1, wherein the neurological disorder is a spinal cord injury.

17. The method of claim 1, wherein the neurological disorder is a peripheral nerves injury.

Description

(1) FIGS. 1A-D: The compounds 1a (ENDF1), 1b (ENDF3), 1c (ENDF8), 1e (ENDF10) and 1f (ENDF11) according to the invention significantly and strongly induce the activity of the neuronal precursor and neuronal differentiation specific DCX promoter. DCX promoter-luciferase transfected mouse embryonic day 16 forebrain (MEF) cells were stimulated for three days with the different substances, as described in Example 17. FF and R luciferase activities were measured and FF/R values were related to the value obtained by stimulation with the control medium. For normalized renilla activities, the R values of the substances tested were related to the R value obtained by stimulation with control medium. Supposing a constant number of transfected cells, the normalized renilla activities provide an approximate measure of the test substances' effect on cell survival and, thus, their relative cytotoxicity. The effects of 10 M of, inter alia, compound 1a (ENDF1) or compound 1b (ENDF3) on DCX promoter induction were significantly higher than those exerted by the reference compounds xanthohumol (p<0.001), ENDF4 (p<0.001, 0.01), tocopherol (p<0.001, 0.01), osajin (p<0.001), chromanol (p<0.001, 0.01), and isoxanthohumol (p<0.001). Compound 3a (ENDF2) also yielded good results (p<0.01, 0.05), as did compounds 1d (ENDF9), 1g (ENDF7), 2a (ENDF5) and 3b (ENDF6). The normalized renilla activity values, as shown in FIG. 1D, point to a favorably low cytotoxicity of the compounds according to the invention as, e.g., the normalized renilla activity of compounds 1a (ENDF1) and 1b (ENDF3) was significantly higher than that exerted by the reference compounds xanthohumol and osajin. Experiments were performed in triplicates or tetraplicates and p-values of 0.05-0.001 were considered to be significant. Data are presented as mean+/SD. Statistical analysis was performed using one-way ANOVA-Tukey post hoc.

(2) FIGS. 2A-C: Compound 1a (ENDF1; denoted as ENDF in the figure) at 10 and 100 M enhances neuronal differentiation. As described in Example 18, MEF cells were incubated for 7 days in DMEM-KO medium in presence of control medium, RA and VPA, and in 10 M or 100 M of compound 1a. Cells were fixed and immunostained for MAP2ab, DCX and DAPI. The percentage of cells expressing MAP2ab and/or DCX was determined. FIG. 2A shows the percentage of DCX positive cells, FIG. 2B indicates the percentage of DCX/Map2ab double positive cells, and FIG. 2C shows the percentage of Map2ab positive cells. Note the elevated number of DCX positive and of MAP2ab positive cells in the cultures treated with compound 1a.

(3) FIGS. 3A-B: Compounds of the invention enhance neuronal differentiation. As described in Example 18, MEF cells were incubated for 7 days in NB+1% FCS medium in the presence of control medium, Retinoic Acid plus Valproic Acid (RA/VPA), the compounds according to the invention 1a (ENDF1), 1b (ENDF3) or 3a (ENDF2), or the reference compounds xanthohumol, isoxanthohumol, ENDF4, 6-prenylnaringenin, 8-prenylnaringenin, chromanol or tocopherol. Cells were fixed and immunostained for DCX, Map2ab and DAPI. The percentage of cells expressing DCX and Map2ab was determined. FIG. 3A shows the relative number of DCX positive cells, and FIG. 3B shows the relative number of DCX/Map2ab double positive cells. The most vigorous result shows compound 1a (p<0.001), while compound 1b (p<0.01) and ENDF4 (p<0.01) also strongly enhance the number of DCX positive cells (FIG. 3A). The effect of compound 3a (p<0.05) is similar to that of 8-prenylnaringenin (p<0.05), 6-prenylnaringenin (p<0.05) and tocopherol (p<0.05) whereas ENDF4 increased slightly more the number of DCX and Map2ab double positive cells (FIG. 3B). The highest number of DCX and Map2ab double positive cells show compounds 1a (p<0.001) and 1b (p<0.001). Good results demonstrate also compound 3a as well as isoxanthohumol, ENDF4, 6-prenylnaringenin and 8-prenylnaringenin with a significance of p<0.01. Chromanol and tocopherol marginally increase the number of double positive cells (p<0.05). In the figure, ***refers to p<0.001, **means p<0.01, and *refers top<0.05.

(4) FIGS. 4A-J: Compound 1a (ENDF1) enhances neurite growth and sprouting in MEF cultures. MEF cells were treated for 3 days (FIGS. 4A to 4D) or 7 days (FIGS. 4E to 4H) with control medium DMEM-KO, RA/VPA or with compound 1a (ENDF1), fixed and stained for DCX and Map2ab. Note the extended neurite length and branching in the compound 1a stimulated culture. The scale in FIGS. 4A to 4H is 30 m. A comparison of neurite growth (FIG. 4I) and sprouting (FIG. 4J) is furthermore shown. MEF cells were treated for 7 days with control medium, RA/VPA or with 10 M or 100 M of compound 1a (ENDF1), fixed and stained for DCX and Map2ab, and the length of DCX positive neurite profiles was measured using Volocity 5.4.1 measurement program. Note the significant increase in neurite length in compound 1a 10 M (p<0.001) and compound 1a 100 M (p<0.001) stimulated MEF cells compared to control and RA+VPA (FIG. 4I). In FIG. 4J, branches/sprouts per 100 m are shown. Note the high density of branches/sprouts in compound 1a treated cultures and the significant difference between compound 1a 100 M (p<0.01) and RA+VPA (p<0.05), as well as control (p<0.01). Experiments were performed in duplicates and p-values of 0.05-0.01 were considered to be significant. Statistical analysis was performed using one-way ANOVA-Tukey post hoc.

(5) FIGS. 5A-G: The compounds of the invention enhance neurite outgrowth in Neuro-2a cells. Neuro-2a cells were treated for 2 days with control medium (FIG. 5A), RA/VPA (FIG. 5B), or with 10 M compound 1a (ENDF1) (FIG. 5C), compound 3a (ENDF2) (FIG. 5D), compound 1b (ENDF3) (FIG. 5E) or ENDF4 (FIG. 5F), fixed and stained for GAP-43. Note that compounds 1a and 1b (ENDF1 and ENDF3) treated cultures show longer growth of neurites as well as more elaborated branches compared to control or RA/VPA treated cultures. Scale: 100 m. Statistical analysis shows a high significance with compounds 1a and 1b (p<0.001) as well as ENDF4 (p<0.01) compared to control (FIG. 5G).

(6) FIGS. 6A-I: Compound 1a (ENDF1) promotes neurite extension in DRG cultures. Dorsal root ganglia E8 (FIGS. 6A to 6D) and E15 (FIGS. 6E to 6H) neurons were treated for 12 to 24 hours with control medium (FIGS. 6A and 6E), 20 ng/ml NGF (FIGS. 6B and 6F), 10 M compound 1a (ENDF1) (FIGS. 6C and 6G) or 10 M compound 1a (ENDF1) plus 20 ng/ml NGF (FIGS. 6D and 6H), fixed and stained for GAP-43. NGF (p<0.01), compound 1a (p<0.05) and NGF plus compound 1a (p<0.01) strongly promote neurite extension as compared to the control (FIG. 6I). Compound 1a (ENDF1) promotes neurite extension to a similar degree as NGF. Scale: 100 m.

(7) FIGS. 7A-B: ENDFs are neuroprotective. PC12 cells were stimulated for 24 hours with different substances, as described in Example 20, and Caspase 3/7 activation as well as LDH release was measured. As determined in the Caspase 3/7 assay (FIG. 7A) and the LDH release assay (FIG. 7B), the cells were partially protected from CoCl.sub.2 300 M induced cell death by NGF, compound 1a (ENDF1), compound 1b (ENDF3) and compound 3a (ENDF2).

(8) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

(9) General Experimental Procedures

(10) Microwave irradiation was carried out with CEM Discover S class single-mode synthesis system interfaced with a laptop PC running CEM synergy software monitoring the reaction. The temperature was checked by an external infrared sensor in the bottom of the cavity. Once the target temperature is reached, the microwave system automatically starts to count down the hold time. For reactions CEM vials 10 ml with snap-on caps were used. The pressure was monitored by a sensor outside the snap-on caps. The upper pressure limit was set to 18 bar. Temperature/pressure recording were attached to CEM synergy reaction files.

(11) Column chromatography for compound purification was performed with silica gel 60 (0.06-0.2 mm) Roth (Karlsruhe) in glass-columns.

(12) The purity of the isolated or synthesized flavonoids was estimated at 95+ % by HPLC-UV.

(13) NMR spectra of unlabeled compounds were recorded on Bruker DBX 400 instrument. Chemical shifts are reported () in ppm and coupling constants (J) in Hz.

(14) The Shimadzu HPLC-System consisted of system controller CBM-20A, two Pumps LC-20AD, Autosampler SIL-20AC with 2 ml sample loop, Oven CTO-20AC and PDA SPD-M20A.

(15) ()--Tocopherol (96%), 2,2,5,7,8-pentamethyl-6-chromanol (97%), 3-methyl-2-butenal (SAFC), 2;3-dichloro-5,6-dicyanobenzoquinone, formic acid, ethylenediamine-N, N-diacetic acid (Fluka) were obtained from Sigma-Aldrich (Steinheim). Osajin was purchased from Chromadex (Irvine).

Example 1: Isolation of Compounds 1a, 1b and 3a from a Xanthohumol-Rich Hop Extract

(16) A xanthohumol-rich (85%) hop extract, i.e. Xanthoflav (DE 102 40 065 A1), supplied in different batches from Hallertauer Hopfenveredelungsgesellschaft m.b.H. was used as source material to get the following minor compounds.

(17) The extract was dissolved in diethyl ether. The solution was washed with water. The organic layer was concentrated in vacuo. The residue was then extracted 24 hours in soxhlet apparatus with heptane. The residue was crystallized from MeOH/H.sub.2O (2/1) to decrease the xanthohumol content.

(18) The purified extract with low xanthohumol concentration was separated by preparative RP-HPLC. The fractions were lyophilized to get the products as solids.

(19) Separation was achieved on a Phenomenex Luna 5 C18 (2); 100 ; 25015 mm with a solvent gradient, starting on injection, from 43% to 91% B (acetonitrile) in A (1% aqueous formic acid) over 34 min, then to 95% B over 1 min, followed by 95% B for 2.5 min at 9.57 ml/min. The compounds were detected at 370 nm and 290 nm. The column oven temperature was 30 C. The column outlet was connected to a 16-way valve (Knauer, Berlin, K-16). The first 2 minutes the column effluent was diverted to waste. Then fractions were collected:

(20) TABLE-US-00001 2.00-7.01 min fraction 2 7.02-7.41 min fraction 3 7.42-8.41 min fraction 4 8.42-8.84 min fraction 5 8.85-9.64 min fraction 6 9.65-10.04 min fraction 7 10.05-11.71 min fraction 8 11.72-12.24 min fraction 9 12.25-15.61 min fraction 10 15.62-16.21 min fraction 11 16.22-16.90 min fraction 12 16.91-19.16 min fraction 13 19.17-20.32 min fraction 14 20.33-36.00 min fraction 15

(21) Compounds 1a (ENDF 1) and 1b (ENDF 3) are found in fraction 15. Compound 3a (ENDF 2) is found in fraction 10.

Example 2: Synthesis of (E)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (Compound 1a; ENDF 1)

(22) ##STR00019##

(23) A solution of xanthohumol (200 mg, 0.56 mmol, 1 eq) and 2,3-Dichloro-5,6-dicyanobenzoquinone (Jain, Gupta, et al. Tetrahedron 1978, 34(24), 3563-3567) (128 mg, 0.56 mmol, 1 eq) in 1 ml benzene (dry) with 2 drops of dioxan was radiated in microwave at 120 C. for 3 min. Dicyanobenzoquinone (DDQ) was separated by filtration and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/hexane: 2/3) to give 109.2 mg (55.4%) of a yellow solid, M=352 g/mol (C.sub.21H.sub.20O.sub.5).

(24) .sup.1H-NMR (CDCl.sub.3):

(25) (ppm): 1.44 (s, 6H, 2CH.sub.3), 3.89 (s, 3H, OCH.sub.3), 5.44 (d, J=11.03 Hz, 1H, H-5), 5.9 (s, 1H, H-5), 6.63 (d, J=11.03 Hz, 1H, H-4), 6.84 (d, J=8.58 Hz, 2H, H-3 & H-5), 7.46 (d, J=8.58 Hz, 2H, H-2 & H-6), 7.7 (s, 2H, H- & H-), 14.60 (s, 1H, OH)

(26) .sup.13C-NMR (CDCl.sub.3):

(27) 192.8 (CO), 162.6 (C-2), 162.5 (C-4), 160.3 (C-6), 157.7 (C-4), 142.4 (C-), 130.3 (C-2 & C-6), 128.5 (C-1), 125.4 (C-5), 125.3 (C-), 116.1 (C-3 & C-5), 106.2 (C-1), 103.1 (C-3), 91.6 (C-5), 78.3 (C-6), 55.9 (OCH.sub.3), 28.4 (C-7& C-8)

Example 3: Synthesis of 5-hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3,9,10-tetrahydro-8H-pyrano[2,3-f]chromen-4-one (Compound 3a; ENDF 2)

(28) The compound 3a (ENDF2) was prepared in accordance with the method of Example 1 but using isoxanthohumol instead of xanthohumol. Purification gave 86.4 mg (43.4%) of a yellow solid, M=352 g/mol (C.sub.21H.sub.20O.sub.5).

(29) .sup.1H-NMR (Acetone-d.sub.6):

(30) (ppm): 1.45 (s, 6H, 2CH.sub.3), 2.65 (d, 1H, J=13.65, H-3), 2.99 (t, 1H, H-3), 3.83 (s, 3H.sub.2OCH.sub.3), 5.44 (d, 1H, J=12.63 Hz, H-5), 5.58 (d, 1H, J=10.11 Hz, H-2), 6.11 (s, 1H, H-6), 6.55 (d, 1H, J=9.86 Hz, H-4), 6.91 (d, 2H, J=8.34 Hz, H-3 & H-5), 7.42 (d, 2H, J=8.08 Hz H-2 & H-6)

(31) .sup.13C-NMR (Acetone-d.sub.6):

(32) 188.25 (CO), 163.0 (C-9), 160.28 (C-7), 159.67 (C-5), 158.46 (C-4), 131.2 (C-1), 128.78 (C-2 & C-6), 127.18 (C-5), 116.7 (C3 & C5), 116.1 (C-4), 106.5 (C), 103.4 (C-8), 94.39 (C-6), 79.81 (C-3), 78.42 (C-6), 56.20 (OCH.sub.3), 46.08 (C-2), 28.5 (CH.sub.3), 28.2 (CH.sub.3)

Example 4: Synthesis of (E)-1-(5-hydroxy-7-methoxy-2,2-dimethylchroman-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (Compound 1b; ENDF 3)

(33) ##STR00020##

(34) A solution of xanthohumol (200 mg, 0.56 mmol) in formic acid (2 ml) was radiated in a 10 ml microwave vial with snap-on-cap at 50 C. with pressurized air cooling for 5 min. The resulting mixture was given to ice-water. The precipitate was filtrated, washed with cold water and was purified by silica gel chromatography (ethyl acetate/hexane: 1/3) to give 75 mg (37.5%) yellow solid, M=354 g/mol (C.sub.21H.sub.22O.sub.5).

(35) .sup.1H-NMR (CDCl.sub.3):

(36) (ppm): 1.35 (s, 6H, 2CH.sub.3), 1.79 (t, J=7.32 Hz, 2H, H-5), 2.62 (t, J=6.12 Hz, 2H, H-4), 3.86 (s, 3H, OCH.sub.3), 5.88 (s, 1H, H-5), 6.86 (d, J=8.56 Hz, 2H, H-3 & H-5), 7.47 (d, J=7.36 Hz, 2H, H-2 & H-6), 7.85 (dd, J=15.9 Hz, 2H, H- & H-), 14.80 (s, 1H, OH)

(37) .sup.13C-NMR (CDCl.sub.3):

(38) 192.39 (CO), 165.14 (C-2), 160.53 (C-4), 160.49 (C-4), 157.49 (C-6), 141.91 (C-), 129.93 (C-2 & C-6), 128.01 (C-1), 124.97 (C-), 115.60 (C-3 & C-5), 105.26 (C-1), 101.75 (C-5), 91.62 (C-3), 75.90 (C-6), 55.35 (OCH.sub.3), 31.83 (C-5) 26.38 (C-7 & C-8), 15.76 (C-4)

Example 5: Synthesis of 1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-ethanone (ENDF 4)

(39) ##STR00021##

(40) A solution of 24-Dihydroxy-6-methoxyacetophenone (100 mg, 0.55 mmol), 3-methyl-2-butenal (180.6 mg, 0.64 mmol) and ethylenediamine-N,N-diacetic acid (EDDA) (9.89 mg, 0.05 mmol) in xylene (5 ml) was radiated in a 10 ml microwave vial with snap-on cap at 170 C. for 60 min. The reaction mixture was given to water and was extracted with ethyl acetate, washed with brine and dried over sodium sulfate. The solution was concentrated in vacuo. The residue was purified by silica gel chromatography (ethyl acetate/hexane: 4/1) to give 74.0 mg (53.5%) of a yellow solid, M=248. g/mol (C.sub.14H.sub.16O.sub.4) (Lee, Xia. Synthesis-Stuttgart 2007, (20), 3240-3246).

(41) .sup.1H-NMR (acetone):

(42) (ppm): 1.29 (s, 6H, 2CH.sub.3), 2.42 (s, 3H, COCH.sub.3), 3.79 (s, 3H, OCH.sub.3) 5.42 (d, J=9.84, 1H, Hz H-4), 5.85 (s, 1H, H-5), 6.45 (d, J=9.88 Hz, 1H, H-5)

(43) .sup.13C-NMR (CDCl.sub.3):

(44) 204.01 (CO), 164.21 (C-4), 162.55 (C-5), 161.21 (C-2), 126.42 (C-4), 116.40 (C-5), 106.21 (C-3), 103.14 (C-1), 92.14 (C-2), 78.81 (?), 56.35 (CH.sub.3), 33.09 (OCCH.sub.3), 28.54 (2CH.sub.3)

Example 6: Synthesis of 1-(5-hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)ethanone

(45) This compound can be prepared in accordance with the method described in Example 5 using 3,7-dimethyl-2,6-octadienal instead of 3-methyl-2-butenal.

(46) ##STR00022##

(47) .sup.1H-NMR (d.sub.6-acetone)

(48) (ppm)=1.39 (s, 3H, 7-H), 1.55 (s, 3H, 12-H), 1.63 (s, 3H, 13-H), 1.65-1.77 (m, 2H, 8-H), 2.05-2.13 (m, 2H, 9-H), 2.55 (s, 3H, CH.sub.3), 3.92 (s, 3H, OCH.sub.3), 5.03-5.11 (m, 1H, 10-H), 5.51 (d, 1H, J=10.07 Hz, 5-H), 5.99 (s, 1H, 5-H), 6.63 (d, 1H, J=10.07 Hz, 4-H), 14.24 (s, 1H, OH).

(49) .sup.13C-NMR (d.sub.6-acetone)

(50) (ppm)=17.62 (C-12), 23.32 (C-9), 25.58 (C-13), 27.37 (C-8), 33.13 (CH.sub.3), 42.25 (C-7), 56.32 (OCH.sub.3), 81.30 (C-10), 91.91 (C-5), 102.91 (C-3), 106.10 (C-1), 116.90 (C-4), 124.77 (C-10), 125.26 (C-5), 132.08 (C-11), 161.54 (C-4), 162.46 (C-2), 164.22 (C-6), 203.88 (CO).

Example 7: Synthesis of 7-hydroxy-2-(4-hydroxy-phenyl)-5-methoxy-8-(3-methyl-but-2-enyl)-chroman-4-one (Isoxanthohumol)

(51) ##STR00023##

(52) 500 ml of 1% aqueous NaOH solution were cooled to 0 C. and xanthohumol (500 mg) was added. The solution was stirred for 2 h. H.sub.2SO.sub.4 was added to the mixture and the yellow precipitate was filtered off. After washing with cold water and crystallization from methanol the product is a light yellow solid (Wilhelm, Wessjohann. Tetrahedron 2006, 62(29), 6961-6966).

Example 8: Synthesis of 5-hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3-dihydro-8H-pyrano[3,2-g]chromen-4-one (Compound 2a; ENDF 5)

(53) The compound was prepared in accordance with the method of Example 5 but using naringenin instead of 24-Dihydroxy-6-methoxyacetophenone. Purification gave a yellow solid, M=338 g/mol (C.sub.20H.sub.18O.sub.5).

(54) ##STR00024##

(55) .sup.1H-NMR (acetone):

(56) (ppm): 1.41 (s, 6H, H-7 & H-8), 2.75 (dd, 1H, J=16.94 Hz, H-3a), 3.19 (dd, 1H, J=13.2, H-3b) 5.47 (dd, 1H, J=13.05 Hz H-2), 5.88 (s, 1H, H-8), 6.57 (d, 1H, J=10.07 Hz, H-4), 6.89 (d, 2H, J=8.24 Hz, H-3 & H-5), 7.38 (d, 2H, J=8.7 Hz, H-2 & H-6)

(57) .sup.13C-NMR (CDCl.sub.3):

(58) 197.70 (CO), 164.21 (C-4), 163.62 162.53 159.13 158.69 (C-8a/7/5/4)), (C-2), 130.53 (C-1), 129.02 (C-2 & C-6), 127.27 (C-5), 116.14 (C-3), 115.57 (C-5), 103.45 (C-8), 96.52 (C-6), 79.98 (C-2), 78.91 (C-6), 43.38 (C-3), 28.4 (2CH.sub.3)

Example 9: Synthesis of 5-hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3-dihydro-8H-pyrano[2,3-f]chromen-4-one (Compound 3b; ENDF 6)

(59) This compound can be prepared in accordance with the method described in Example 5, using naringenin instead of 24-dihydroxy-6-methoxyacetophenone and purifying the title compound.

(60) ##STR00025##

(61) .sup.1H-NMR (d.sub.6-acetone):

(62) (ppm)=1.39 (s, 3H, H-7), 1.42 (s, 3H, H-8), 2.77 (dd, 1H, J=17.4 Hz, H-3a), 3.20 (dd, 1H, J=16.94 Hz, H-3b), 5.50 (dd, 1H, J=13.05=Hz, H-2), 5.58 (d, 1H, J=10.07, H-5), 5.87 (s, 1H, H-6), 6.49 (d, 1H, J=10.07 Hz, H-4), 6.90 (dd, 2H, J=8.7 Hz, H-3 & H-5), 7.41 (d, 2H, J=8.7 Hz, H-2 & H-6), 8.59 (s, 1H, OH-4), 12.24 (s, 1H, OH-5)

(63) .sup.1313-NMR (d.sub.6-acetone):

(64) (ppm)=197.61 (C-4), 164.61 (C-5), 162.65 (C-7), 158.69 (C-4), 158.02 (C-8a), 130.50 (C-1), 128.92 (C-2 & C-6), 127.33 (C-5), 116.19 (C-3 & C-5), 116.17 (C-4), 103.51 (C-4a), 102.50 (C-8), 97.50 (C-6), 80.03 (C-2), 78.78 (C-6), 43.22 (C-3), 28.56 (C-7), 28.29 (C-8)

Example 10: Synthesis of (2E)-1-(5-Hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)-3-(4-(methoxymethoxy)phenyl)prop-2-en-1-one

(65) 1.5 ml of an aqueous solution of potassium hydroxide (50%) was poured to a solution of 1.00 mmol 1-(5-hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)ethanone (Example 6) and 1.20 mmol 4-(methoxymethoxy)benzaldehye in 13 ml methanol. The reaction mixture was heated to boiling point for 3 hours. After cooling to room temperature the reaction mixture was poured into 20 ml water, acidified with HCl (10%) and extracted three times with 15 ml ethyl acetate. The combined organic fractions were dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel chromatography (n-hexane/ethyl acetate 2/1).

(66) ##STR00026##

(67) The yellow product was directly deprotected in accordance with the method described in Example 14 below in order to give the title compound.

Example 11: Synthesis of (2E)-3-(3,4-bis(methoxymethoxy)phenyl)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one

(68) This compound can be prepared in accordance with the method described in Example 10 using ENDF4 and 3,4-bis(methoxymethoxy)benzaldehyde.

(69) ##STR00027##

(70) The yellow product was directly deprotected in accordance with the method described in Example 14 below in order to give the title compound.

Example 12: Synthesis of (2E)-3-(benzo[d][1,3]dioxol-6-yl)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (Compound 1f; ENDF 11)

(71) This compound can be prepared in accordance with the method described in Example 10 using ENDF4 and benzo[1,3]dioxole-5-carbaldehyde.

(72) ##STR00028##

(73) .sup.1H-NMR (d.sub.6-acetone)

(74) (ppm)=1.43 (s, 6H, 7-H & 8-H), 4.00 (s, 3H, OCH.sub.3), 5.56 (d, 1H, J=10.26 Hz, 5-H), 6.02 (s, 1H, 5-H), 6.08 (s, 2H, OCH.sub.2O), 6.61 (d, 1H, J=10.26 Hz, 4-H) 6.91 (d, 1H, J=8.30 Hz, 5-H), 7.23 (d, 1H, J=1.47 Hz, 2-H), 7.27 (dd, 2H, J=8.30 Hz, 6-H), 7.70 (d, 1H, J=15.63 Hz, -H), 7.85 (d, 1H, J=15.63 Hz, -H), 14.68 (s, 1H, OH).

(75) .sup.13C-NMR (d.sub.6-acetone)

(76) (ppm)=28.51 (C-7& C-8), 56.60 (OCH.sub.3), 78.66 (C-6), 92.45 (C-5), 102.68 (OCH.sub.2O), 103.37 (C-3), 106.54 (C-1), 107.38 (C-6), 109.35 (C-5), 116. 46 (C-4), 126.01 (C-6), 126.27 (C-), 126.40 (C-5), 130.80 (C-1), 143.29 (C-), 149.45 (C-4), 150.74 (C-3), 161.26 (C-4), 163.20 (C-2), 163.81 (C-6), 193.37 (CO).

Example 13: Synthesis of (2E)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (Compound 1d; ENDF 9)

(77) This compound can be prepared in accordance with the method described in Example 10 using ENDF4 and 4-methoxybenzaldehyde.

(78) ##STR00029##

(79) .sup.1H-NMR (d.sub.6-acetone)

(80) (ppm)=1.43 (s, 6H, 7-H & 8-H), 3.86 (s, 3H, OCH.sub.3), 4.00 (s, 3H, OCH.sub.3), 5.56 (d, 1H, J=10.26 Hz, 5-H), 6.02 (s, 1H, 5-H), 6.61 (d, 1H, J=10.26 Hz, 4-H), 7.00 (d, 2H, J=8.79 Hz, 3-H & 5-H), 7.69 (d, 2H, J=8.79 Hz, 2-H & 6-H), 7.77 (d, 1H, J=15.63 Hz, -H), 7.89 (d, 1H, J=15.63 Hz, -H), 14.75 (s, 1H, OH).

(81) .sup.13C-NMR (d.sub.6-acetone)

(82) (ppm)=28.51 (C-7& C-8), 55.76 (OCH.sub.3), 56.56 (C-4), 78.85 (C-6), 92.45 (C-5), 103.40 (C-3), 106.53 (C-1), 115.31 (C-3 & C-5), 116.48 (C-4), 125.75 (C-), 126.39 (C-5), 128.93 (C-1), 131.16 (C-2 & C-6), 143.35 (C-), 161.20 (C-4), 162.64 (C-6), 163.26 (C-4), 163.78 (C-2), 193.43 (CO).

Example 14: Synthesis of (2E)-1-(5-hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (Compound 1g; ENDF 7)

(83) Five drops of aqueous HCl (3M) were poured to 10 ml of a methanolic solution of (2E)-1-(5-Hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)-3-(4-(methoxymethoxy) phenyl)prop-2-en-1-one (Example 10) and was heated to 50 C. for 60 minutes. After cooling the reaction mixture was poured into 15 ml water and extracted three times with 15 ml ethyl acetate. The combined organic fractions were dried over sodium sulfate and concentrated in vacuo.

(84) ##STR00030##

(85) .sup.1H-NMR (d.sub.6-acetone)

(86) (ppm)=1.41 (s, 3H, 7-H), 1.60 (s, 3H, 12-H), 1.63 (s, 3H, 13-H), 1.65-1.79 (m, 2H, 8-H), 2.10-2.14 (m, 1H, 9-H), 4.00 (s, 3H, OCH.sub.3), 5.12 (t, 1H, J=6.84 Hz, 10-H), 5.53 (d, 1H, J=10.26 Hz, 5-H), 6.04 (s, 1H, 5-H), 6.67 (d, 1H, J=9.77 Hz, 4-H), 6.91 (d, 2H, J=8.79 Hz, 3-H & 5-H), 7.61 (d, 2H, J=8.30 Hz, 2-H & 6-H), 8.94 (s, 1H, 4-OH), 14.82 (s, 1H, 2-OH).

(87) .sup.13C-NMR (d.sub.6-acetone)

(88) (ppm)=17.62 (C-12), 23.34 (C-8), 25.76 (C-13), 27.39 (C-7), 42.26 (C-9), 56.54 (OCH.sub.3), 81.33 (C-10), 92.23 (C-5), 103.22 (C-3), 106.43 (C-1), 116.78 (C-3 & C-5), 117.07 (C-4), 124.79 (C-10), 125.00 (C-5), 125.14 (C-), 127.95 (C-1), 131.34 (C-2 & C-6), 132.06 (C-11), 143.75 (C-), 160.71 (C-4), 161.47 (C-4), 163.24 (C-2), 163.79 (C-6), 193.36 (CO).

Example 15: Synthesis of (2E)-1-(5-Hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (Compound 1e; ENDF10)

(89) This compound can be prepared in accordance with the method described in Example 14 using (2E)-3-(3,4-bis(methoxymethoxy)phenyl)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (Example 11) instead of the compound of Example 10.

(90) ##STR00031##

(91) .sup.1H-NMR (d.sub.6-acetone)

(92) (ppm)=1.42 (s, 6H, 7-H & 8-H), 3.99 (s, 3H, OCH.sub.3), 5.55 (d, 1H, J=10.26 Hz, 5-H), 6.02 (s, 1H, 5-H), 6.61 (d, 1H, J=10.26 Hz, 4-H), 6.88 (1H, d, J=7.82 Hz, 5-H), 7.11 (dd, 1H, J=8.30 Hz, 6-H), 7.24 (d, 1H, J=1.95 Hz, 2-H), 7.69 (d, 1H, J=15.63 Hz, -H), 7.81 (d, 1H, J=15.63 Hz, -H), 14.81 (s, 1H, OH).

(93) C-.sup.13C-NMR (d.sub.6-acetone)

(94) (ppm)=28.51 (C-7& C-8), 56.55 (OCH.sub.3), 78.81 (C-6), 92.44 (C-5), 103.42 (C-3), 106.53 (C-1), 115.33 (C-5), 116.50 (C-4), 123.20 (C-6), 125.70 (C-), 126.36 (C-5), 128.60 (C-1), 144.18 (C-), 146.35 (C-4), 149.00 (C-3), 161.08 (C-4), 163.27 (C-2), 163.74 (C-6), 193.41 (CO).

Example 16: Synthesis of 1-(5-hydroxy-7-methoxy-2,2-dimethylchroman-6-yl)-3-(4-hydroxyphenyl)propan-1-one (Compound 1c; ENDF8)

(95) 122.0 mg (E)-1-(5-hydroxy-7-methoxy-2,2-dimethylchroman-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (ENDF3) were diluted in 7 ml methanol. After addition of 15 mg Pd/C, hydrogen was passed through the reaction mixture. The residue was purified by silica gel chromatography (n-hexane/ethyl acetate 1/1) to give the product.

(96) ##STR00032##

(97) .sup.1H-NMR (d6-acetone)

(98) (ppm)=1.31 (s, 6H, 7-H & 8-H), 1.80 (t, 2H, J=6.84 Hz, 5-H), 2.54 (t, 2H, J=6.84 Hz, 4-H), 2.86 (t, 2H, J=8.30 Hz, former ), 3.25 (t, 2H, J=7.82 Hz, former ), 3.86 (s, 3H, OCH.sub.3), 5.90 (s, 1H, 5-H), 6.75 (d, 2H, J=8.79 Hz, 3-H & 5-H), 7.08 (d, 2H, J=8.30 Hz, 2-H & 6-H), 14.42 (s, 1H, OH).

(99) .sup.13C-NMR (d6-aceton)

(100) (ppm)=16.68 (C-4), 26.85 (C-7& C-8), 30.75 (former ), 32.61 (C-5), 46.94 (former ), 56.05 (OCH.sub.3), 76.64 (C-6), 92.39 (C-3), 102.31 (C-5), 105.45 (C-1), 115.99 (C-3 & C-5), 130.15 (C-2 & C-6), 133.33 (C-1), 156.42 (C-4), 161.66 (C-4), 161.99 (C-6), 165.57 (C-2), 205.46 (CO).

Biological Assays

(101) In the following examples, primary cells derived from developing mouse brain, primary chicken embryonic day 8 and day 16 dorsal root ganglion neurons, the mouse Neuro-2a neuroblastoma cell line and the rat phaeochromocytoma PC12 cell line were used to test the activity of chromane-like cyclic prenylflavonoids and other substances to enhance neuronal differentiation, survival and neuroregeneration.

(102) The following substances were tested:

(103) Compound 1a (ENDF1): (E)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one.

(104) ##STR00033##

(105) Compound 1b (ENDF3): (E)-1-(5-hydroxy-7-methoxy-2,2-dimethylchroman-6-yl-3-(4-hydroxyphenyl)prop-2-en-1 one.

(106) ##STR00034##

(107) Compound 1c (ENDF8): 1-(5-hydroxy-7-methoxy-2,2-dimethylchroman-6-yl)-3-(4-hydroxyphenyl)propan-1-one.

(108) ##STR00035##

(109) Compound 1d (ENDF9): (2E)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one.

(110) ##STR00036##

(111) Compound 1e (ENDF10): (2E)-1-(5-Hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one.

(112) ##STR00037##

(113) Compound 1f (ENDF11): (2E)-3-(benzo[d][1,3]dioxol-6-yl)-1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one.

(114) ##STR00038##

(115) Compound 1g (ENDF7): (2E)-1-(5-hydroxy-7-methoxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one.

(116) ##STR00039##

(117) Compound 2a (ENDF5): 5-Hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3-dihydro-8H-pyrano[3,2-g]chromen-4-one.

(118) ##STR00040##

(119) Compound 3a (ENDF2): 5-Hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3,9,10-tetrahydro-8H-pyrano[2,3 f]chromen-4-one.

(120) ##STR00041##

(121) Compound 3b (ENDF6): 5-Hydroxy-2-(4-hydroxy-phenyl)-8,8-dimethyl-2,3-dihydro-8H-pyrano[2,3-f]chromen-4-one.

(122) ##STR00042##

(123) ENDF4: 1-(5,7-dihydroxy-2,2-dimethyl-2H-chromen-6-yl)ethanone.

(124) ##STR00043##

(125) Xanthohumol: (E)-1-(2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one.

(126) ##STR00044##

(127) Isoxanthohumol: 7-hydroxy-2-(4-hydroxyphenyl)-5-methoxy-8-(3-methylbut-2-en-1-yl)chroman-4-one.

(128) ##STR00045##

(129) Osajin: 5-hydroxy-3-(4-hydroxyphenyl)-8,8-dimethyl-6-(3-methylbut-2-enyl)pyrano[2,3-h]chromen-4-one.

(130) ##STR00046##

(131) 6-Prenylnaringenin: 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-(3-methylbut2-en-1yl)chroman-4-one.

(132) ##STR00047##

(133) 8-Prenylnaringenin: 5,7-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut2-en-1yl)chroman-4-one.

(134) ##STR00048##

(135) Chromanol: 2,2,5,7,8-Pentamethyl-6-chromanol.

(136) ##STR00049##

(137) Tocopherol: 2,5,7,8-tetramethyl-2-(4,8,12 trimethyltridecyl)chroman-6-ol.

(138) ##STR00050##

(139) Retinoic acid: (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid.

(140) ##STR00051##

(141) Valproic acid: 2-propylpentanoic acid.

(142) ##STR00052##

(143) Stock solutions for the substances were 100 mM in DMSO. All trans-retinoic acid 10 M+valproic acid sodium salt 50 M (Sigma-Aldrich, Taufkirchen, Germany) diluted in DMEM-KO medium or Neurobasal medium+1% FCS were used as positive control. DMEM-KO medium or NB-B27+1% FCS was used as negative control.

(144) Readouts were the abilities i) to induce the promoter of the neuronal precursor specific gene doublecortin (DCX) in a firefly-luciferase assay using primary fetal mouse brain derived cells (Karl, 2005), ii) to promote neuronal differentiation/neurite extension in different neuronal cells, iii) to promote neurite extension in a fetal chicken dorsal root ganglion neurite outgrowth assay (Aigner, 1993; Aigner, 1995), and iv) to promote neuronal survival in a PC12 cobalt-chloride stress test (Wang, 2000).

(145) Experiments were performed in triplicates or tetraplicates and p-values of 0.05 to 0.001 were considered to be significant. Data are presented as mean+/SD. Statistical analysis was performed using PRISM5 (GraphPad, San Diego, Calif., USA) and significance was acquired by one-way ANOVA-Tukey post hoc.

Example 17: The Chromane-Like Cyclic Prenylflavonoids According to the Invention Activate a Neuronal Differentiation Program

(146) Chromane-like cyclic prenylflavonoids, including the compounds 1a to 1g, 2a, 3a and 3b according to the invention, were tested for their ability to induce the promoter of the neuronal precursor specific gene doublecortin (DCX) in a firefly-luciferase assay using primary fetal mouse brain derived cells (Karl, 2005) in Neurobasal plus 1% FCS.

(147) Neuronal Stem Cell (NSC) Isolation from Mouse Embryonic Forebrain (MEF E16) Cultures

(148) Primary mouse embryonic day 16 forebrain (MEF) cultures were prepared as follows. Pregnant NMRI mice (Charles River Laboratories, Sulzfeld, Germany) were sacrificed and the uteri promptly removed and immersed in ice-cold Dulbecco's phosphate buffered saline solutionDPBS (PAA, Pasching, Austria). Eight embryonic day (E) 16 embryos were released from the uteri; forebrains were taken out, separated from surrounding tissues and mechanically dissociated using a razor blade. Subsequently the dissociated cells were collected by 5 min centrifugation at 120g. The pellet was resuspended in 10 ml PPD-solution containing 0.01% papain (Worthington, England), 0.1% Dispasell (Roche, Basel, Switzerland), 0.01% DNasel (Worthington, England), 12.4 mM MgSO.sub.4 dissolved in HBSS w/o Ca/Mg (PAA, Pasching, Austria). The cell suspension was incubated at 37 C. and triturated every ten minutes for three times. The cell suspension was then centrifuged at 120g for 5 minutes and washed three times in Neurobasal medium (Gibco BRL, Germany) supplemented with 2% (v/v) B27 (Gibco BRL, Karlsruhe), 2 mM L-glutamine, 100 g/ml Penicillin/Streptomycin (PAN Biotech GmbH, Aidenbach, Germany). Finally, the cell preparation was resuspended in 12 ml Neurobasal medium supplemented with B27 (Gibco BRL, Karlsruhe), 2 mM L-glutamine, 100 g/ml Penicillin/Streptomycin (PAN Biotech GmbH, Aidenbach, Germany), 20 ng/ml Epidermal Growth FactorrhEGF, 20 ng/ml Fibroblast Growth FactorrhFGF (R&D Systems, Wiesbaden-Nordenstadt, Germany) and 2 g/ml Heparin (Sigma-Aldrich, Taufkirchen, Germany) (NB plus all), seeded in one T75 flask (TPP, Switzerland) and maintained in a humidified atmosphere at 37 C. and 5% CO.sub.2. Within the first two days in culture, MEF cells had formed neurospheres. On this day (day 2), half of the medium was refreshed and on day 4 cells were passaged. To passage the cells, the culture medium containing the floating neurospheres was collected in a 15 ml tube (Corning, Lowell, USA) and centrifuged at 120g for 5 min. For dissociation, the pellet was resuspended in 500 l Accutase (PAA, Pasching, Austria) and triturated using a 1 ml pipette. The cell suspension was incubated at 37 C. for 10 min, and then, 10 ml media were added. Cell number was determined by Trypan blue exclusion.

(149) Luciferase Assay

(150) Luciferase assays were performed using a noncommercial dual luciferase enzyme assay system (Dyer et al., 2000, Anal Biochem). In this system, a co-transfection with pDCX-Luci and a control vector driving Renilla-luciferase under the CMV promoter was performed. The two different luciferases have different specific substrates and, therefore, the DCX regulatory region driven firefly luciferase activity can be distinguished from CMV driven Renilla-luciferase. For analysis, the firefly luciferase activity was normalized against the CMV driven Renilla-luciferase. For each transfection reaction, a volume containing two million cells was transferred into a 15 ml tube (Corning, Lowell, USA), centrifuged at 120g for 5 min to pellet the cells and resuspended in 100 l Amaxa Mouse Neural Stem Cell Nucleofector Kit (Lonza, Kln, Germany). Then, pDCX-Luci (Karl, 2005) and pRL3 (Promega, Mannheim, Germany) (5 g each) were added and the entire reaction mix was transferred into an Amaxa transfection cuvette. Transfection was done using the A-033 program in the Amaxa Nucleofector II (Lonza, Kln, Germany). Transfected cells were then pipetted into 20 ml of DMEM knockout medium (Gibco BRL, Germany) containing 20% serum replacement supplement (PAN Biotech GmbH, Aidenbach, Germany), 2 mM L-glutamine (PAN Biotech GmbH, Aidenbach, Germany) and 100 g/ml Penicillin/Streptomycin (PAN Biotech GmbH, Aidenbach, Germany) (DMEM-KO plus supplements) in a 50 ml tube (Corning, Lowell, USA). From this resulting cell suspension, 200 l per well were plated in Poly-Ornithine/Laminin coated 96-well-luciferase plates (Wimmer & Macho, Wels, Austria) in a cell density of 20.000 cells per well, as described herein below. For some experiments, cells were taken up after transfection and further cultivated in Neurobasal medium (Gibco BRL, Germany) supplemented with 2% (v/v) B27 (Gibco BRL, Karlsruhe) and 1% fetal calf serum (PAN Biotech, Aidenbach, Germany), 2 mM L-glutamine, 100 g/ml Penicillin/Streptomycin (PAN Biotech, Aidenbach, Germany) (NB-B27, 1% FCS) instead of DMEM-KO plus supplement.

(151) For coating of the 96-well-luciferase plates, wells were incubated for one hour at 37 C. with Poly-Ornithine 100 g/ml in sterile H.sub.2O. After three times of washing with sterile H.sub.2O the wells were coated with Laminin (Sigma-Aldrich, Taufkirchen, Germany) (5 g/ml in DPBS) for two hours at 37 C. Immediately after removing Laminin, cells were plated.

(152) Substances were tested in 0.1 M, 1 M, 10 M and 100 M final concentrations in DMEM-KO plus supplement or NB-B27, 1% FCS. Cells were stimulated starting one day after seeding for a total of three days by replacing the medium with 200 l fresh medium containing the various substances. Stimulations were done in 4 wells/condition (tetraplicates) at 37 C. in 5% CO.sub.2 containing humidified atmosphere.

(153) Prior to measurement the stimulation medium was carefully taken off with a pipette. Cells were then lysed in 25 l lysis solution consisting of 25 mM Tris-phosphate pH 7.8, 2 mM DTT, 1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany), 2 mM EDTA, 10% Glycerol (Merck, Darmstadt, Germany) per well for ten minutes. For analysis of the firefly luciferase activity, 80 l of 25 mM glycylglycine (Acros, Geel, Belgium), 15 mM KH.sub.2PO.sub.4 pH 8.0, 4 mM EGTA, 15 mM MgSO.sub.4 (Merck, Darmstadt, Germany), 2 mM ATP, 1 mM DTT, 0.1 mM Coenzyme A, 75 mM luciferin (Sigma-Aldrich, Taufkirchen, Germany) were added to each well and the bioluminescent measurement is performed in a Victor X Multilable Plate Reader (Perkin

(154) Elmer, Calif., USA) according to the manufacturer's instructions. Then, 100 l of freshly prepared Renilla assay solution consisting of 1.1 M NaCl (VWR, Vienna, Austria), 2.2 mM Na.sub.2EDTA, 0.22 M KH.sub.2PO.sub.4 pH 5.1 (Merck, Darmstadt, Germany), 0.44 mg/mL BSA (Biomol, Hamburg, Germany), 1.3 mM NaN.sub.3 (Sigma-Aldrich, Taufkirchen, Germany), 1.43 mM coelenterazine (p.j.k., Kleinblittersdorf, Germany) per well were added and measurements were performed using the Victor X Multilable Plate Reader as described above. Data were normalized the following way: i) firefly (FF) luciferase activity is related to the renilla (R) luciferase activity; and ii) the FF/R values of the individual samples are related to the FF/R value obtained by stimulation with the control medium and expressed as x-fold change compared to control. The renilla luciferase activity values (R values) obtained for the test substances were furthermore related to the R value obtained by stimulation with control medium (i.e., R.sub.substance/R.sub.control) to give normalized renilla activity values which, supposing a constant number of transfected cells, provide an approximate measure of the test substances' effect on cell survival and, thus, their relative cytotoxicities.

(155) Results

(156) Among all substances tested, the compounds 1a (ENDF1), 1b (ENDF3), 1c (ENDF8), 1e (ENDF10) and 1f (ENDF11) according to the invention exerted a very strong and significant stimulatory effect on the DCX promoter activity, as also shown in FIGS. 1A and 1C, respectively. The effect was similar or even higher compared to the one exerted by the positive control retinoic acid plus valproic acid (denoted as RA10+VPA50 in FIG. 1A). A stimulatory effect on the DCX promoter activity was also observed for compounds 1d (ENDF9) and 2a (ENDF5) and, to a lesser extent, for compounds 1g (ENDF7), 3a (ENDF2) and 3b (ENDF6) according to the invention, as can be seen in FIGS. 1A, 1B and 1C. The FF/R values indicated in FIGS. 1A, 1B and 1C are relative to the respective control.

(157) The reference compounds ENDF4, tocopherol, osajin, chromanol and isoxanthohumol did not have any significant stimulatory effect on the DCX promoter activity. Actually, a 10 M concentration of osajin induced a down-regulation of the DCX promoter (FIG. 1A).

(158) The compounds according to the present invention, and particularly compounds 1a, 1b, 1c, 1e and 1f, as well as compounds 1d, 1g, 2a, 3a and 3b, have thus been shown to induce the activity of the neuronal precursor and neuronal differentiation specific promoter DCX, which demonstrates the suitability of the compounds according to the invention as neuroprotective agents and in promoting neuronal differentiation. Moreover, the compounds of the invention show a considerably improved efficacy in inducing the DCX promoter as compared to the reference compounds tested. The normalized renilla activity values of the substances tested (FIG. 1D) furthermore indicate that the compounds according to the invention exhibit a favorably low cytotoxicity, particularly in comparison to the reference compounds xanthohumol and osajin.

Example 18: The Chromane-Like Cyclic Prenylflavonoids According to the Invention Enhance the Percentage of Cells Expressing the Neuronal Specific Markers DCX and Map2ab in MEF Cultures

(159) Following the results from the Luciferase assay described in Example 17, the inventors examined the ability of compounds according to the invention and reference compounds to promote differentiation of MEF E16 cells, using immunocytochemical staining with DCX and Map2ab antibodies.

(160) Compound 1a at 10 and 100 M was analyzed for its effect on the expression of the neuronal specific markers DCX and Map2ab using primary fetal mouse brain derived cells stimulated in DMEM-KO media. Furthermore, compounds 1a (ENDF1), 1b (ENDF3) and 3a (ENDF2) according to the invention as well as the reference compounds xanthohumol, isoxanthohumol, ENDF4, 6-prenylnaringenin, 8-prenylnaringenin, chromanol and tocopherol were analyzed for their effect on the expression of the neuronal specific markers DCX and Map2ab using primary fetal mouse brain derived cells stimulated in Neurobasal plus 1% FCS media.

(161) Mouse embryonic day 16 forebrain cells were prepared as described in Example 17. Before seeding the cells, glass-coverslips were coated and prepared. Glass-coverslips (13 mm) (Menzel GmbH, Braunschweig, Germany) were incubated in 1 M HCl at 65 C. overnight and stored in isopropanol until further use. Coverslips were put into 24-well test plates and air-dried. Subsequently coverslips were incubated for 1 h with 100 g/ml Poly-L-ornithin solution at 37 C. After washing three times with sterile water coverslips were incubated with 5 g/ml Laminin solution for 2 h. Immediately after removal of the Laminin solution cells were seeded (40.000 cells/well) in DMEM-KO plus supplement or in NB-B27 plus 1% FCS medium. One day later, cells had attached to the substrate. Cells were stimulated with the above-mentioned substances (in DMEM-KO plus supplement medium: 10 M and 100 M; in NB-B27 plus 1% FCS medium: 10 M) for 3 or 7 days. In the 7 day group, medium including the substances was replaced after three days. Cultures were maintained at 37 C. in a humidified incubator with 5% CO.sub.2.

(162) Cells were fixed with phosphate-buffered 4% paraformaldehyde (Sigma-Aldrich, Taufkirchen, Germany). The fixed cells were washed three times in DPBS (PAA, Pasching, Austria) and subsequently blocked for a minimum of 1 hour at room temperature in fish skin gelatine buffer (FSGB) containing 0.1M Tris-HCl pH 7.5, 0.15 M NaCl, 1% bovine serum albumin, 0.2% Teleostean gelatin and 0.1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany) to make the cell membrane permeable for the antibody. The specimens were incubated overnight at 4 C. with the primary antibodies at the following dilutions in FSGB: rabbit anti Doublecortin (DCX) 1:500 (NEB, Frankfurt, Germany) and mouse anti-Map 2a+2b 1:400 (Sigma-Aldrich, Taufkirchen, Germany). After washing three times with FSGB, cells were incubated with the species-specific secondary antibodies, which were conjugated to fluorochromes, for 2 hours in the dark at room temperature. To remove unbound secondary antibodies, cells were washed again with PBS. Nuclear counterstaining was performed with 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) at 0.25 g/l (Sigma Aldrich, Taufkirchen, Germany). Finally, cells were washed again three times with PBS and mounted on microscope slides using Prolong Antifade reagent (Invitrogen, Oreg., USA).

(163) The immunostainings were examined using Olympus IX81 inverted research microscope and the software Volocity 5.3.1. To quantify the number of cells, five indiscriminately chosen visual fields per coverslip were selected under a focus of 40 times. The total number of cells (DAPI positive cells), the number of DCX positive, Map2ab positive, and DCX/Map2ab double positive cells was determined.

(164) The results of these experiments are shown in FIGS. 2 and 3. As can be seen, compound 1a according to the invention enhances the expression of the neuronal specific markers DCX and Map2ab in primary fetal mouse brain derived cells stimulated in DMEM-KO media (FIG. 2). When primary fetal mouse brain derived cells were stimulated in Neurobasal plus 1% FCS media, compound 1a showed the most vigorous effect on DCX and Map2ab expression (p<0.001), while compound 1b (p<0.01) and ENDF4 (p<0.01) also strongly enhanced the number of DCX positive cells (FIG. 3A). The effect of compound 3a (p<0.05) was similar to that of 8-prenylnaringenin (p<0.05), 6-prenylnaringenin (p<0.05) and tocopherol (p<0.05) whereas ENDF4 increased slightly more the number of DCX/Map2ab double positive cells (FIG. 3B). The highest number of DCX and Map2ab double positive cells showed compounds 1a (p<0.001) and 1b (p<0.001). It has thus been demonstrated that the compounds of the present invention enhance neuronal differentiation.

Example 19: The Chromane-Like Cyclic Prenylflavonoids According to the Invention Promote Neurite Growth and Branching

(165) Further to the effects on the activation of the DCX promoter and on the percentage of neuronal cells in MEF cultures as determined in Examples 17 and 18, the effects of chromane-like cyclic prenylflavonoids, including compound 1a according to the invention, on neuronal neurite extension and branching was tested in primary MEF cells, in the neuroblastoma cell line Neuro-2a and in fetal chicken dorsal root ganglion cells (RDGs).

(166) Effects of Chromane-Like Cyclic Prenylflavonoids on Neurite Length and Branching in MEF Cultures

(167) Mouse embryonic day 16 forebrain cells were prepared and treated as described in Example 17.

(168) The morphology of MEF cells after a 7 day treatment with retinoic acid/valproic acid or with compound 1a (ENDF1) is illustrated in FIG. 4. Cells were fixed and stained with DCX and Map2ab antibodies as described in Example 18. Three independent experiments were performed and evaluated. Data acquisition was done by using the software Volocity 5.3.1 for measurement and photographical documentation. The following parameters were analyzed: polarity, process, number of branches, number and length of sprouts, neurite-length and growth cone morphology. At the qualitative level, compound 1a strongly increases neurite length and neurite branching (FIG. 4).

(169) For quantitative analysis of neurite length, neurons with long neurites were taken into consideration. MEF cells stimulated with 10 M and 100 M compound 1a (ENDF1) displayed much longer neurites compared to control or retinoic acid/valproic acid treated MEF cells, as also shown in FIG. 4I. For quantitative analysis of neurite branching, the branch density (number of branches per 100 m of neurite length) was counted. For analysis, the DCX staining with DMEM-KO medium was used, since DCX, in contrast to Map2ab, is localized throughout the neurites. MEF cells stimulated with 10 M and 100 M compound 1a (ENDF1) displayed a dramatic increase in branching/sprouting compared to control or retinoic acid/valproic acid treated MEF cells, as shown in FIG. 4J.

(170) Effects of Chromane-Like Cyclic Prenylflavonoids on Neurite Length in Neuro2a Cells

(171) For more precise analysis of neurite length Neuro2a cells were selected and stimulated for 2 days with RA/VPA as positive control and ENDFs in MEM+10% FCS. Immunocytochemical analysis was performed using GAP43 antibody which is known as a strong neuronal plasticity marker.

(172) Neuro2a cells were grown in MEM with Earle's Salts (PAA, Pasching, Austria) containing 2 mM L-glutamine, 100 g/ml Penicillin/Streptomycin (PAN Biotech GmbH, Aidenbach, Germany), 100 mM sodium pyruvate (Sigma-Aldrich, Taufkirchen, Germany) and 10% FCS (Lonza, Wuppertal, Germany).

(173) The cells were passaged with 6 ml trypsin 0.5 mg/ml+EDTA 0.22 mg/ml (PAA, Pasching, Austria) two times per week. For analysis of neurite length Neuro2a cells were seeded on coverslips in a 24 well plate coated with Poly-L-ornithine and Laminin in a differentiation media MEM with Earle's Salts (PAA, Pasching, Austria) containing 2 mM L-glutamine, 100 g/ml Penicillin/Streptomycin (PAN Biotech GmbH, Aidenbach, Germany), 100 mM sodium pyruvate (Sigma-Aldrich, Taufkirchen, Germany) and 1% FCS (Lonza, Wuppertal, Germany) and treated for 2 days with retinoic acid plus valproic acid and compound 1a (ENDF1) 10 M, compound 1b (ENDF3) 10 M, compound 3a (ENDF2) 10 M, or ENDF4 10 M. Cells were fixed as described in Example 18. Immunostaining was performed using GAP-43 1:500 (AbD Serotec, Oxford, UK).

(174) Neuro2a cells stimulated with compound 1a (ENDF1) and compound 1b (ENDF3) 10 M display a strong and significant increase on neurite length and more elaborated branches as compared to control and RAA/PA treated cells, which is also shown in FIG. 5. Compound 3a (ENDF2) shows more sprouting than the other substances.

(175) Effects of Chromane-Like Cyclic Prenylflavonoids on Neurite Length in Primary Chicken DRG Neurons

(176) Dorsal root ganglion neurons comprise of an axon with two branches and are a good model for neuronal differentiation. Embryonic day 8 and 15 chicken dorsal root ganglions were examined for immunochemical staining with GAP43 after incubation with NGF 20 ng/ml and compound 1a (ENDF1) 10 M to analyze whether compound 1a alone or in combination with the nerve growth factor enhances neurite outgrowth and branching.

(177) Embryonic day (E) 8 and E15 chicken Dorsal Root Ganglions (DRGs) were prepared as described in: Aigner, 1993; Aigner, 1995. Briefly, foetuses were taken out of the egg, decapitated and the DRGs were targeted from the ventral side. The 10 lumbar region DRGs per foetus were dissected, and a total of 100 DRGs were collected in 10 ml DPBS in a 15 ml Falcon tube. DRGs were centrifuged 120g for 3 min and then enzymatically treated with 1 ml trypsin 0.5 mg/ml+EDTA 0.22 mg/ml (PAA, Pasching, Austria) for 15 min. Then, 9 ml of DMEM 10% FCS were added to inactivate the trypsin and DRGs were centrifuged 120g for 5 min. DRGs were resuspended and washed with 5 ml of DMEM 10% FCS, pipetted up and down to dissociate them, centrifuged, resuspended in 5 ml of DMEM 10% FCS and transferred into a 60 mm diameter cell culture dish. This cell culture dish with the DRGs were placed into a humidified incubator with 5% CO.sub.2 and 37 C. for three hours to allow non-neuronal cells to adhere to the bottom of the dish, while DRG neurons remained non-adherent. After three hours of incubation, the non-adherent fraction was collected, centrifuged 5 min at 120g and resuspended in 700 l of DMEM 10% FCS. 20 l of cell suspension were transferred into each well of a 24 well plate, in which a Poly-L-ornithine/Laminin coated glass coverslip and 400 l of DMEM 10% FCS with the different compounds (compound 1a and/or NGF) were prepared. Cultures were maintained at 37 C. in a humidified incubator (Heraeus, Germany) with 5% CO.sub.2 for 24 hours.

(178) Cells were fixed with phosphate-buffered 4% paraformaldehyde (Sigma-Aldrich, Taufkirchen, Germany). The fixed cells were washed three times in DPBS (PAA, Pasching, Austria) and subsequently blocked for a minimum of 1 hour at room temperature in fish skin gelatine buffer (FSGB) containing 0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 1% bovine serum albumin, 0.2% Teleostean gelatin and 0.1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany) to make the cell membrane permeable for the antibody. The specimens were incubated overnight at 4 C. with primary antibody rabbit anti GAP-43 1:500 (AbD Serotec, Oxford, UK). After washing three times with FSGB, cells were incubated with the species-specific secondary antibody, which was conjugated to fluorochromes, for 2 hours in the dark at room temperature. To remove unbound secondary antibodies, cells were washed again with PBS. Nuclear counterstaining was performed with 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) at 0.25 g/l (Sigma Aldrich, Taufkirchen, Germany). Finally, cells were washed again three times with PBS and mounted on microscope slides using Prolong Antifade reagent (Invitrogen, Oreg., USA). The immunostainings were examined using Olympus IX81 inverted research microscope and the software Volocity 5.3.1.

(179) The results are indicated in FIG. 6 and show NGF as a potent enhancer of neuronal differentiation by increasing neurite growth and branching. Interestingly, compound 1a (ENDF1) was almost in the range of NGF and in combination (ENDF1+NGF) there was a slight but significant increase compared to NGF alone.

(180) In view of the above, it has been demonstrated that the compounds of the present invention, including compound 1a, promote neuronal differentiation and enhance neurite outgrowth and sprouting, e.g., in MEF cultures, Neuro2a cells and chicken dorsal root ganglions. Moreover, it has been found that the compounds of formula (III) according to the invention, including compound 3a, are particularly advantageous with respect to the promotion of neurite sprouting.

Example 20: The Chromane-Like Cyclic Prenylflavonoids According to the Invention are Neuroprotective

(181) For the neuroprotection assay PC12 cells were stressed with cobalt chloride 300 M for 24 hours and the activity of Caspase 3/7 and LDH release were measured. NGF 50 ng/ml was used as a positive control for Caspase 3/7 activity and NGF 100 ng/ml was used as a positive control for LDH release.

(182) For the PC12-CoCl.sub.2-Assay PC12 cells were used (ATCC: CRL-1721 ). These cells were growing in RPMI supplemented with 10% Horse Serum (heat inactivated) (Sigma Aldrich, Taufkirchen, Germany), 5% Fetal Bovine Serum (Lonza, Wuppertal, Germany), 2.5% Penicillin/Streptomycin and Glutamine (PAA, Pasching, Austria) as adherent cultures when the plates are coated with Poly-L-ornithine (100 mg/ml in water) for at least one hour at 37 C. For passaging PC12-cells were resuspended with Accutase (PAA, Pasching, Austria) for 10 minutes at 37 C. once a week.

(183) For all experiments cells were plated (quadruplicates) on Poly-L-ornithine-coated 96-well plates at a density of 104 cells per well. For Caspase 3/7-measurement cells were seeded in a white 96-well-plate to detect luminescence. As positive-control for neuroprotection Nerve Growth Factor (NGF) (50 ng/ml and 100 ng/ml) was used. To induce hypoxia CoCl.sub.2 (300 M) was added to the cells after 20-24 hours. The control-treatment was compound 1a (ENDF1), compound 1b (ENDF3), compound 3a (ENDF2), ENDF4 or Osajin in the concentrations 1 M and 10 M in proliferation-media without CoCl.sub.2.

(184) For Caspase 3/7-activation Caspase 3/7 substrate was added to the cells in an equal amount 22 hours after stimulation. Plates are incubated for 2 hours at room temperature in the dark, luminescence was measured 24 hours after stimulation and the blank-values are subtracted.

(185) For LDH-release cells were centrifuged at 1050 rpm for 5 minutes. After 24 hours of stimulation, the supernatant was mixed in an equal amount with LDH-substrate and incubated in the dark for 30 minutes. Afterwards stop solution was added and absorbance was measured at 490 nm, the blank-values are subtracted.

(186) The results shown in FIG. 7A confirm that CoCl.sub.2 induces stress in PC12 cells whereas NGF inhibits CoCl.sub.2 induced Caspase 3/7 activity. Compounds 1a (ENDF1) and 1b (ENDF3) 10 M display a significant neuroprotective effect similar to NGF.

(187) The results of the LDH release assay (FIG. 7B) show that NGF 100 ng/ml as well as compound 1a (ENDF1), compound 1b (ENDF3) and compound 3a (ENDF2) 10 M slightly decrease the LDH release whereas Osajin does not significantly diminish cell death.

(188) These results indicate that the compounds of the present invention, including compounds 1a, 1 b and 3a, are neuroprotective, as has been demonstrated in PC12 cells.

LITERATURE

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