This invention is directed to compounds of the following formula:
##STR00001##
wherein when a is 0, b is 1 or 2; when a is 1, b is 1, m is from 0-3, X is O or S, Y is halogeno, R1 is hydrogen C1-4 alkyl, R2 is hydrogen, C1-4 alkyl, or optionally substituted propargyl and R3 and R4 are each independently hydrogen, C1-6 alkyl, C6-12 aryl, C6-12 aralkyl each optionally substituted.
This invention is also directed to the use of these compounds for treating depression, Attention Deficit Disorder (ADD), Attention Deficit and Hyperactivity Disorder (ADHD), Tourette's Syndrome, Alzheimer's Disease and other dementia's such as senile dementia, dementia of the Parkinson's type, vascular dementia and Lewy body dementia.
This invention is further directed to a pharmaceutical composition comprising a therapeutically effective amount of the above-defined compounds and a pharmaceutically acceptable carrier.
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0. 29. A compound having the structure:
##STR00027##
wherein when a is 0, b is 1 or 2, and when a is 1, b is 1;
m is 0-3; X is O or S; Y is halogeno; R1 is hydrogen or C1-4 alkyl; R2 is hydrogen, C1-4 alkyl, propargyl or substituted propargyl; and R3 and R4 are each independently C1-8 alkyl, C6-12 aryl, C6-12 aralkyl or C6-12 cycloalkyl, each of which may be optionally substituted, or hydrogen,
such compound being a racemic mixture, an enantiomer, or a salt thereof.
1. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of a compound having the structure:
##STR00019##
wherein when a is 0, b is 1 or 2, and when a is 1, b is 1;
wherein when a is 1, b is 1, m is 0-3, ; X is O or S, ; Y is halogeno, ; R1 is hydrogen or C1-4 alkyl, ; R2 is hydrogen, C1-4 alkyl, propargyl, or optionally substituted propargyl; and R3 and R4 are each independently hydrogen, C1-8 alkyl, C6-12 aryl, C6-12 aryl aralkyl or C6-12 cycloalkyl, each of which may be optionally substituted, or is hydrogen, such compound being a racemic mixture, an enantiomer, or a salt thereof;
a racemic mixture, an enantiomer, or salt thereof, so as to thereby treat the subject's depression.
19. A method of treating a subject suffering from depression comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound having the structure:
##STR00025##
wherein when a is 0, b is 1 or 2, and when a is 1, b is 1;
wherein when a is 1, b is 1, m is 0-3, ; X is O or S, ; Y is halogeno, ; R1 is hydrogen or C1-4 alkyl, ; R2 is hydrogen, C1-4 alkyl, propargyl, or optionally substituted propargyl; and R3 and R4 are each independently hydrogen, C1-8 alkyl, C6-12 aryl, C6-12 aryl aralkyl or C6-12 cycloalkyl, each of which may be optionally substituted, or is hydrogen, such compound being a racemic mixture, an enantiomer, or a salt thereof a racemic mixture, an enantiomer, or salt thereof , and
a pharmaceutically acceptable carrier, ; so as to thereby treat the subject's depression.
10. A method of selectively inhibiting monoamine oxidase-B (MAO-B) activity in the brain of a subject in need of such inhibition comprising administering to the subject a therapeutically effective amount of a compound having the structure:
##STR00022##
wherein when a is 0, b is 1 or 2, and when a is 1, b is 1;
wherein when a is 1, b is 1, m is 0-3, ; X is O or S, ; Y is halogeno, ; R1 is hydrogen or C1-4 alkyl, ; R2 is hydrogen, C1-4 alkyl, propargyl, or optionally substituted propargyl; and R3 and R4 are each independently hydrogen, C1-8 alkyl, C6-12 aryl, C6-12 aryl aralkyl or C6-12 cycloalkyl, each of which may be optionally substituted, or is hydrogen, such compound being a racemic mixture, an enantiomer, or a salt thereof;
a racemic mixture, an enantiomer, or salts thereof, so as to thereby selectively inhibit MAO-B activity in the brain of the subject.
24. A method of selectively inhibiting monoamine oxidase-B (MAO-B) activity in the brain of a subject in need of such inhibition comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound having the structure:
##STR00026##
wherein when a is 0, b is 1 or 2, and when a is 1, b is 1;
wherein when a is 1, b is 1, m is 0-3, ; X is O or S, ; Y is halogeno, ; R1 is hydrogen or C1-4 alkyl, ; R2 is hydrogen, C1-4 alkyl, propargyl or optionally substituted propargyl; and R3 and R4 are each independently hydrogen, C1-8 alkyl, C6-12 aryl, C6-12 aryl aralkyl or C6-12 cycloalkyl, each of which may be optionally substituted, or is hydrogen, such compound being a racemic mixture, an enantiomer, or a salt thereof a racemic mixture, an enantiomer, or salt thereof , and
a pharmaceutically acceptable carrier, ; so as to thereby selectively inhibit MAO-B activity in the brain of the subject.
2. The method of
3. The method of
5. The method of
6. The method of
8. The method of
9. The method of
11. The method of
12. The method of
14. The method of
15. The method of
17. The method of
18. The method of
20. The method of
21. The method of
22. The method of
23. The method of
25. The method of
26. The method of
27. The method of
28. The method of
0. 30. The compound of
wherein the group OC(O)NR3R4 is on the 6 position of the indan ring counting from the amino substituted carbon atom; m is 0; R1 is H; R2 is H; R3 is methyl; and R4 is ethyl.
0. 31. The compound of
0. 32. The compound of
wherein the group OC(O)NR3R4 is on the 6 position of the indan ring counting from the amino substituted carbon atom; m is 0; R1 is H; R2 is H; R3 is H; and R4 is ethyl.
0. 33. The compound of
wherein the group OC(O)NR3R4 is on the 6 position of the indan ring counting from the amino substituted carbon atom; m is 0; R1 is H; R3 is H; and R4 is ethyl.
0. 34. The compound of
0. 35. A pharmaceutical composition comprising the compound of
0. 36. A pharmaceutical composition comprising the compound of
0. 37. A pharmaceutical composition comprising the compound of
0. 38. A pharmaceutical composition comprising the compound of
0. 39. A pharmaceutical composition comprising the compound of
0. 40. A pharmaceutical composition comprising the compound of
0. 41. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 42. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 43. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 44. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 45. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 46. A method of treating a subject suffering from Alzheimer's disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 47. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 48. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 49. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 50. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 51. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 52. A method of treating a subject suffering from depression comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 53. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 54. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 55. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 56. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 57. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
0. 58. A method of treating a subject suffering from Attention Deficit Disorder, Attention Deficit and Hyperactivity Disorder, Tourette's syndrome, dementia, neurotraumatic disorder or memory disorder which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition of
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This application is a continuation of U.S. Ser. No. 09/336,493, filed Jun. 18, 1999, a continuation of PCT International Application No. PCT/US97/24155, filed Dec. 18, 1997, designating the United States of America and claiming priority of Israeli Patent Application Nos. 119853, filed Dec. 18, 1996 and 120510, filed Mar. 24, 1997, the contents of which are hereby incorporated by reference.
The present invention relates to novel compounds, pharmaceutical compositions containing said compounds and their use in the treatment of various CNS disorders.
Dementia exists in several forms including static dementia, Alzheimer's-type dementia, senile dementia, pre-senile dementia and progressive dementia. One of the common pathological features of several types of dementia is the lack of the neurotransmitter acetylcholine. This has led to the development of acetylcholine esterase inhibitors for use in the treatment of dementias such as the compound tacrine. A summary of the different approaches to and progress made in the treatment of Alzheimer's Disease may be found in Drugs of the Future (1995) 20(11): 1145-1162.
Recently, compounds that in addition to inhibiting acetylcholine esterase, possess inhibitory activity against monoamine oxidase type A (MAO-A) have been developed. The perceived benefit of having the anti-MAO-A activity is stated to be an anti-depressant effect (European Patent Publication Nos. 614,888 and 664,291).
U.S. Pat. Nos. 5,387,133, 5,453,446, 5,457,133 and 5,519,061 all disclose that the compound (R)-N-propargyl-1-aminoindan, a highly selective monoamine oxidase type B (MAO-B) inhibitor is effective in the treatment of dementias of the Alzheimer type and memory disorders. There is no indication given therein that the compound might have acetylcholine esterase inhibitory activity. Furthermore, the compound is only very weakly active as a MAO-A inhibitor.
PCT International Publication No. WO95/18617 discloses various aminoindan derivatives that are active in a variety of CNS disorders including dementias of the Alzheimer type. There is no indication given therein that any of the compounds disclosed might have acetylcholine esterase inhibitory activity.
The present invention relates to compounds of formula I
##STR00002##
wherein when a is 0; b is 1 or 2; when a is 1, b is 1; m is from 0 to 3; X is C or S; Y is halogeno; R1 is hydrogen or C1-4 alkyl; R2 is hydrogen, C1-4 alkyl or optionally substituted propargyl; and R3 and R4 are each independently hydrogen, C1-8 alkyl, C6-12 aryl, C6-12 aralkyl or C6-12 cycloalkyl optionally substituted.
The invention relates to the compounds themselves, pharmaceutical compositions containing said compounds and their use in the treatment of depression, Attention Deficit Disorder (ADD), Attention Deficit and Hyperactivity Disorder (ADHD), Tourette's Syndrome, Alzheimer's Disease and other dementias such as senile dementia, presenile dementia, progressive dementia, dementia of the Parkinson's type, vascular dementia and Lewy body dementia.
A further aspect of the present invention relates to the use of the compounds of formula I in the treatment of neurotraumatic disorder. As used herein the term “neurotraumatic disorder” is meant to include damage caused to the nervous system (both central and peripheral) by virtue of ischemic damage such as that which occurs in stroke, hypoxia or anoxia, neurodegenerative diseases, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, neurotoxic injury, head trauma injury, spinal trauma injury, peripheral neuropathy or any form of nerve damage.
An additional aspect of the present invention relates to the use of the compounds of formula I in the treatment of memory disorder or depression.
The present invention relates to the racemic compounds themselves and optically active enantiomers thereof.
The present invention is directed to compound of Formula I:
##STR00003##
wherein when a is 0, b is 1 or 2; when a is 1, b is :, m is from 0-3, X is O or S; Y is halogen; R1 is hydrogen or C1-4 alkyl; R2 is hydrogen, C1-4 alkyl, or optionally substituted propargyl and R3 and R4 are each independently hydrogen, C1-6 alkyl, C6-11 aryl, C6-11; aralkyl or C6-11 cycloalkyl each optionally substituted.
In an embodiment of the present invention, a is 0 and b is 1. In another embodiment of the present invention, a is 0, b is 1, and X is O.
In an embodiment of the present invention, X is O. In an additional embodiment of the present invention, X is S.
In an embodiment of the present invention, R1 is selected from the group consisting of hydrogen, methyl, ethyl or optionally substituted propargyl.
In another embodiment of the present invention, R1 is propargyl.
In a further embodiment of the present invention, the compound is selected from the group consisting of: (rac) 6-(N-methyl, N-ethyl-carbanyloxy)-N′-propargyl-1-aminoindan HCl; (rac) 6-(N,N-dimethyl, carbanyloxy)-N′-methyl-N′-propargyl-1-aminoindan HCl; (rac) 6-(N-methyl, N-ethyl-carbamyloxy)-N′-propargyl-1-aminotetralin HCl; (rac)6-(N,N-dimethyl-thiocarbamyloxy)-1-aminoindan HCl; (rac)6-(N-propyl-carbamyloxy)-N′-propargyl-1-aminoindan HCl; (rac)5-chloro-6-(N-methyl, N-propyl-carbamyloxy)-N′-propargyl-1-aminoindan HCl; (S)-6-(N-methyl, N-propyl-carbamyloxy)-N′-propargyl-1-aminoindan HCl; and (R)-6-(N-methyl, N-ethyl-carbamyloxy)-N′-propargyl-1-aminoindan hemi-(L)-tartrate.
In a further embodiment of the present invention, R1 is hydrogen, methyl or ethyl and R2 is hydrogen, methyl, ethyl or optionally substituted propargyl. In a further embodiment of the present invention, the propargyl group is substituted with a C1-4 alkyl group on the methylene group (R6 in Scheme I)
According to the present invention, the term “halogeno” is used to refer to fluoro, chloro, bromo, or iodo.
In an embodiment of the present invention, when m is greater than 1 each Y may be the same or different.
In an additional embodiment of the present invention, the group OC(X)NR3R4 is on the 4, 6 or 7 position of the indan ring counting from the amino substituted carbon.
In another embodiment of the present invention, at least one of R3 and R4 is methyl and the other is hydrogen, methyl, ethyl, propyl, butyl, hexyl, phenyl, benzyl or cyclohexyl.
In the practice of this invention, pharmaceutically acceptable salts include, but are not limited to, the esylate, mesylate, maleate, fumarate, tartrate, hemi-tartarate, hydrochloride, hydrobromide, p-toluenesulfonate, benzoate, acetate, phosphate and sulfate salts.
The subject invention further provides a pharmaceutical composition which comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The “therapeutically effective amount” of a compound of formula I or a pharmaceutically acceptable salt thereof may be determined according to methods well known to those skilled in the art, indications of such amounts are given below.
These compositions may be prepared as medicaments go be administered orally, parenterally, rectally, or transdermally.
Suitable forms for oral administration include tablets, compressed or coated pills, dragees, sachets, hard or soft gelatin capsules, sublingual tablets, syrups and suspensions. In one embodiment, the pharmaceutically acceptable carrier is a solid and the pharmaceutical composition is a tablet. The therapeutically effective amount may be an amount from about 0.5 mg to about 2000 mg, preferably from about 1 mg to about 1000 mg.
In an alternative embodiment, the pharmaceutically acceptable carrier is a liquid and the pharmaceutical composition is an injectable solution. The therapeutically effective amount may be an amount from about 0.5 mg to about 2000 mg, preferably from about 1 mg to about 1000 mg. The volume administered may be an amount between 0.5 and 10 ml.
In a further alternative embodiment, the carrier is a gel and the pharmaceutical composition is a suppository. For parenteral administration the invention provides ampoules or vials that include an aqueous or non-aqueous solution or emulsion. For rectal administration there are provided suppositories with hydrophilic or hydrophobic vehicles. For topical application as ointments and transdermal delivery there are provided suitable delivery systems as known in the art. For oral or suppository formulations, 0.5-2000 mg per dosage unit and preferably 1-1000 mg per dosage unit.
These compositions may be used alone to treat the above-listed disorders, or alternatively, for example, in the case of Alzheimer's Disease, they may be used as an adjunct to the conventional treatments such as haloperidol, tacrine or deprenyl.
The invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
Compounds of general formula I may be prepared, as shown in Scheme I, from the corresponding carbamoyl derivatives of aminoindan III by reacting the latter with propargyl compounds bearing an appropriate leaving group at the 3-position, e.g. a halide group, mesylate, tosylate, etc., under basic cenditions provided by an inorganic base, e.g. K2CO3, NaOH, or an organic base e.g. a tertiary amine, in a polar organic solvent, e.g. CH3CN, DMF, etc., at 15-40° C., preferably at 20-25° C., for a period of time in the range of 5-48 hours, preferably 20-30 hours. The products, obtained after a suitable work-up and purification, are in the form of free bases. Preferably these are converted into their pharmaceutically acceptable salts, e.g. HCl, mesylate, hemi-tartarate, etc.
As shown in Scheme I, compounds of general formula III may be prepared by Boc deprotection of compounds of general formula IV. In turn, compounds of general formula IV may be prepared by carbamylating a compound of general formula V in a conventional manner, e.g. by reacting the compound of formula V with an appropriate carbamoyl halogenide or by an alkylisocyanate. Finally, compounds of general formula V may be prepared by Boc protection of the appropriate hydroxy amines, by methods known to those skilled in the art. N,N-dialkyl aminoindan derivatives may be prepared as shown on in Scheme I by the direct carbamylation of the corresponding N,N-dialkyl-hydroxy-aminoindan or by alkylation of a compound of formula III.
Although Scheme I shows the preparation of carbamoyl derivatives the same process and description above is relevant to the preparation of the thiocarbamates of the present invention.
Starting Materials
6- and 7-Hydroxy-1-aminoindans may be prepared by demethylation of the respective 6- and 7-methoxy-1-aminoindans. The latter may be obtained from the corresponding 1-indanones, either by their conversion to the oximes, followed by reduction, or by their reductive amination (NaCNBH3 and NH4OAc)2.
6-Hydroxy aminoindan may also be prepared from aminoindan via a regioselective Friedel—Crafts acylation of a suitably N-protected aminoindan, followed by a Baeyer—Williger oxidation and finally hydrolysis5. 6-hydroxy-(R)-1-aminoindan may thus be prepared by the method described in the Example below and Scheme II, wherein “R” is optionally substituted alkyl.
N-Methyl-6-hydroxy-1-aminoindan was prepared by demethylation of 6-methoxy-N-methyl-1-aminoindan, which was prepared from 6-methoxy-1-aminoindan by reductive alkylation (e.g. ethyl formate, followed by LiAlH4 reduction), or alternatively, by reductive amination (MeNH2, HCl, NaCNBH3) of 6-methoxy-1-indanone2. N-ethyl-6-hydroxy-1-aminoindan was obtained by acetylation of 6-hydroxy-1-aminoindan (Ac2O, KOH), followed by reduction (LiAH4). N,N-Dimethyl-6-hydroxy-1-aminoindan was prepared by demethylation of the corresponding 6-methoxy analogue, which was prepared by reductive alkylation (formaldehyde, formic acid) of 6-methoxy-1-aminoindan. 4-Hydroxy-1-aminoindan may be prepared from 4-hydroxy indanone by converting the latter to the oxime, followed by reduction1. 4-Hydroxy indanone may be prepared from dihydrocoumarin.3
7-Hydroxy-1-aminotetralin and 7-hydroxy-2-aminotetralin were prepared by demethylation of the corresponding 7-methoxy analogues. The latter were prepared by reductive amination (as above) of the corresponding 7-methoxy-1- and 2-tetralones.
7-Methoxy-2-tetralone was prepared from 2,7-dimethoxytetralin according to Copinga,et al4.
Preparation of 6-Hydroxy-(R)-1-aminoindan (As Shown in Scheme II)
N-Trifluoroacetyl-(R)-1-aminoindan
To a cooled (0-5° C.) solution of trifluoroacetic anhydride (194.6 g, 0.926 mol) in toluene (680 ml) was added dropwise a solution of (R)-1-aminoindan (base) (113.32 g 0.85 mol) in toluene (50 ml) and stirred under ice-cooling for 3½ hours. A solution of KOH (67.25 g, 1.2 mol) in water (1000 ml) was then added, under cooling. The reaction mixture was stirred for further 2 hours at room temperature and filtered. The solid was collected by filtration, washed with water (680 ml) and dried in vacuo at 60° C. to give 152 g (78%) of a white solid, mp:153-154° C. The solution was evaporated in vacuum and the crystals were filtered and washed with water. The solid was dried in vacuo at 60° C. The second crop (25 g) was crystallized from a mixture of hexane and ethyl acetate to give 189 (9%) of a white solid, mp:153-154° C. The total yield was 170 g (87%).
6-Chloroacetyl-N-trifluoroacetyl-(R)-1-aminoindan
To a suspension of AlCl3 (89.2 g, 0.67 mol) in 1,2-dichloroethane (600 ml) was added chloroacetyl chloride (55.7 ml, 78.9 g, 0.7 mol) dropwise at 0-5° C. under nitrogen for 20 minutes and it was then left to warm up to 20-25° C. To this mixture was added N-trifluoroacetyl-(R)-1-aminoindan (34.4 g, 0.15 mol) for 3 hours at 20-25° C. The resulting mixture was then stirred for an additional 30 minutes and poured into a mixture of ice-cold water (1.5 l) and 1,2-dichloroethane (1l). The mixture was stirred for 5 minutes and the layers were separated. The aqueous layer was extracted with 1,2-dichloroethane (2×750 ml). The combined organic layers were washed with water (2×900 ml) and 5% aqueous NaHCO3 solution (3×900 ml). The organic layer was dried (Na2SO4) and the solvent was removed under reduced pressure to give a solid, which was recrystallized from ethanol to give 15 g (48%) of a white solid mp: 166-167° C.
6-Chloroacetoxyl-N-trifluoroacetyl-(R)-1-aminoindan
6-Choroacetyl-N-trifluoroacetyl-(R)-1-aminoindan (30.57 g, 0.1 mol) was dissolved in anhydrous dichloromethane (210 ml) and 3-chloroperoxybenzoic acid (70%, 44.87 g, 0.26 mol) was added all at once. The suspension was cooled to 0° C. and trifluoroacetic acid (11.4 g, 0.1 mol) was added dropwise for 5-10 minutes. The reaction flask was protected from light and the mixture was stirred for 3-5 days at room temperature. The reaction mixture was poured into water (300 ml.). The mixture was neutralized with ammonium hydroxide solution. The layers were separated. The aqueous layer was extracted with dichloromethane (200 ml). The combined organic layers were dried (Na2SO4) and the solvent was removed under reduced pressure to give a solid, which was recrystallized from ethanol to give 15 g (48%) of a white solid mp: 169-170° C.
6-Hydroxy-(R)-1-aminoindan
A suspension of 6-chloroacetoxy-N-trifluoroacetyl-(R)-1-aminoindan (25.4, 0.11 mol) and K2CO3 (38.0 g, 0.275 mol) in a mixture of methanol (275 ml) and water (175 ml) was stirred at 70° C. for 1.5 hours. Methanol was removed in vacuo, and the aqueous phase was neutralized with 10% hydrochloric acid. The mixture was filtered and the solid was washed with water. The mother liquor was evaporated under reduced pressure to a small volume. The suspension was neutralized, filtered and the brown solids were crystallized from methanol (twice) to give 7.0 g (43%) of a white solid mp:200-203° C.
Preparation of the corresponding S-enantiomer may be carried out in the same manner using (S)-1-aminoindan as the starting material.
Resolution of Enantiomers
The R- and S-enantiomers of each compound may be obtained by optical resolution of the corresponding racemic mixtures. Such a resolution can be accomplished by any conventional resolution method well known to a person skilled in the art, such as those described in U.S. Pat. No. 4,833,273, issued May 23, 1989 (Goel) and in J. Jacques, A. Collet and S. Wilen, “Enantiomers, Racemates and Resolutions,” Wiley, N.Y. (1981). For example, the resolution may be carried out by preparative chromatography on a chiral column. Another example of a suitable resolution method is the formation of diastereomeric salts with a chiral acid such as tartaric, malic, mandelic acid or N-acetyl derivatives of amino acids, such as N-acetyl leucine, followed by recrystallization to isolate the diastereomeric salt of the desired enantiomer.
Alternatively, selected starting materials, intermediates or end products may be resolved into their respective enantiomers by the method described in PCT International Application Publication No. WO/96US/21640, wherein the compound to be resolved is first converted into its N-benzyl derivative. The N-benzyl derivative is then resolved using either R or S-mandelic acid. The resolved product is converted to its base and reduced under acidic conditions to provide the desired enantiomer. Preferably, the starting material is resolved prior to Boc protection and carbamylation.
The R and S enantiomers of the starting materials may also be prepared from R and S enantiomers c: aminoindan via a regioselective Friedel—Crafts acylation so a suitably N-protected optical isomer of aminoindan, followed by a Baeyer-Williger oxidation and finally hydrolysis5, thus obviating the need for optical resolution.
A: Boc—protection and carbamylation
1. Boc Protection
6-hydroxy N-Boc aminoindan
A solution of 6-hydroxy aminoindan (16 g, 107 mmol), di-t-butyl dicarbonate (23.8 g, 109.2 mmol) and Et3N (16.74 ml, 120 mmol) in THF (375 ml) was stirred at room temperature (RT) for 20 hrs. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was dissolved in CH2Cl2 (200 ml), washed with water (200 ml), dried over Na2SO4 and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (hexane/EtOAc 2:1) to give 23 g of a solid (86%).
2. Carbamylation
6-(N-Me, N-Et carbamyloxy) N-Boc aminoindan
To a stirred and ice-cooled solution of N-Boc 6-hydroxy aminoindan (7.5 g, 30 mmol) in acetonitrile (75 ml) was added N-Me,N-Et carbamoyl chloride (6.3 g, 51.8 mmol), followed by a dropwise addition of NaH (60% in oil, 1.56 g, 39 mmol). The reaction mixture was stirred for 2 hrs at RT under argon. After evaporation of the solvent in-vacuo, water (100 ml) was added, and extracted with ether (3×100 ml). The organic phase was washed with dilute NaOH (pH 10-11), dried and evaporated to dryness in-vacuo. Purification by column chromatography (hexane:EtOAc 2:1) afforded 7.8 g (77%) of an oil.
In this manner the intermediates in Tables 1 and 2 were prepared. In Table 1 and all further Tables the heading “position” refers to the ring position of the carbamyl group unless otherwise indicate
TABLE 1
N-Boc protected carbomyloxy aminoindans
##STR00004##
position
Y
R1
R3
R4
yield (%)
6-
H
H
Me
Me
92
6-
H
H
Me
Pr
95
6-
H
H
Me
Et
77
7-
H
H
Me
Me
92
7-
H
H
Me
Et
83
7-
H
H
Me
Pr
95
6-
H
Et
Me
Me
76
6-
H
Me
Me
Me
92
7-
H
Me
Me
Me
78
6-
H
Me
Me
Pr
80
6-
H
H
Me
n-hexyl
98
4-
H
H
Me
Me
85
4-
H
H
Me
Et
87
6-
H
H
Me
Et
89
6-
H
H
Me
cyclohexyl
98
6-
H
H
Me
p-OMe-phenyl
97
6-
H
H
Me
phenyl
93
6-
H
H
Me
CH2-phenyl
83
6-
5-Cl
H
Me
Et
88
6-
5-Cl
H
Me
Pr
97
6-
H
H
Me
Bu
99
6-
H
H
Et
Bu
93
6-
H
H
Et
cyclohexyl
94
TABLE 2
N-Boc protected carbomyloxy tetralins
##STR00005##
position of
amine
R1
R3
R4
yield (%)
2-
H
Me
Me
85
2-
H
Me
Et
79
1-
H
Me
Me
85
1-
H
Me
Et
98
B: Boc—Deprotection
6-(N-Me,N-Et Carbamyloxy) aminoindan HCl (Compound 3)
6-(N-Me,N-Et Carbamyloxy) N-Boc aminoindan (7.8 g, 23.3 mmol) was dissolved in dioxane (80 ml), and a 20% solution of gas. HCl in dioxane (80 ml) was added. After 2 hr stirring at RT the solvent was evaporated in-vacuo and the residue was treated with dry ether (200 ml) and the mixture stirred at RT for 4 hrs and filtered, to give 6.15 g (0.7 mmol, 97%) of 6-(N-Me, N-Et carbamyloxy) aminoindan hydrochloride.
In this manner the following compounds of general formula I as shown in Tables 3, 3a and 4 were prepared. Spectral data relating to these compounds is given in Tables 7, 7a and 8.
TABLE 3
Carbamyloxy aminoindan HCl salts
##STR00006##
cryst/
slurry
yield
#
position
R1, R2
R3
R4
solvent
mp(° C.)
(%)
1
6-
H, H
Me
Me
Et2O
156-8
93
2
6-
H, H
Me
Pr
Et2O
165-7
27
3
6-
H, H
Me
Et
Et2O
150-2
50
4
7-
H, H
Me
Me
Et2O
156-60
93
5
7-
H, H
Me
Et
Et2O
185-7
55
6
7-
H, H
Me
Pr
Et2O
153-5
33
7
6-
H, Et
Me
Me
Et2O
172-4
91
8
6-
H, Me
Me
Me
Et2O
178-80
88
9
7-
H, Me
Me
Me
dioxane
169-71
98
10
6-
H, Me
Me
Et
Et2O
172-4
87
11
6-
H, Me
Me
Pr
Et2O
165-7
98
12
6-
Me, Me
Me
Me
Et2O
164-6
62
13
4-
H, H
Me
Me
Et2O
198-200
90
14
4-
H, H
Me
Et
Et2O
183-5
92
15
6-
H, H
Me
n-
dioxane
111-12
78
hexyl
16*
6-
H, H
Me
Et
Et2O
197-8
89
17
6-
H, H
Me
cyclo
Et2O
207-8
86
hexyl
18**
6-
H, H
Me
Et
Et2O
202-4
84
48
6-
H, H
H
Et
MeOH/
191-2
74
EtOAc
49
6-
H, H
H
Pr
MeOH/
171-3
67
EtOAc
50
6-
H, H
Me
p-OMe-
iPrOH
225-7
92
Phenyl
51
6-
H, H
Me
CH2—
Et2O
78
Ph
52*
6-
H, H
Me
Me
Et2O
83
53**
6-
H, H
Me
Me
Et2O
81
88
6-
H, H
Me
Ph
Et2O
96
66***
6-
H, H
Me
Et
Et2O
116-9
92
67***
6-
H, H
Me
Pr
Et2O
181-3
86
80
6-
H, H
Me
Bu
Et2O
54
84
6-
H, H
Et
cyclo-
Et2O
196-8
89
hexyl
*R < nantiomer
**S-cnantiomer
***5-chloro
TABLE 3a
Thiocarbamyloxy aminoindan HCl salts
##STR00007##
cryst/slurry
yield
#
position
R1, R2
R3
R4
solvent
mp(° C.)
(%)
44
6-
H, H
Me
Me
MeOH/EtO
244-5
55
45
6-
H, H
Me
Et
MeOH/EtOAc
236-8
58
TABLE 4
Carbamyloxy aminotetralin HCl salts
##STR00008##
position
of
cryst/slurry
yield
#
amine
R1
R3
R4
solvent
mp(° C.)
(%)
19
2-
H
Me
Me
ether
a)
96
20
2-
H
Me
Et
ether
a)
98
21
1-
H
Me
Me
ether
196-8
99
22
1-
H
Me
Et
ether
166-8
85
a) wide melting range; compound is a hemi-hydrate
C: Propargylation and salt formation
The compounds prepared in Step B may be optionally propargylated to provide further compounds of general formula I.
6-(N-Me, N-Et carbamyloxy) N-propargyl aminoindan, HCl (Compound 25)
To a stirred mixture of 6-(N-Me, N-Et carbamyloxy) aminoindan. HCl (5.2 g, 19.2 mmol), potassium carbonate (5.31 g, 38.4 mmol) in acetonitrile (250 ml), was added a solution of propargyl bromide (2.06 g, 17.28 mmol) in acetonitrile (10 ml). The reaction mixture was stirred at RT under nitrogen for 25 hrs, and filtered. The filtrate was evaporated to dryness in-vacuo and the residue was purified by column chromatography (EtOAc) to give 3.6 g (13.2 mmol, 69%) of the free base as a yellow oil.
The free base was dissolved in dry ether (150 ml) and HCl/ether (15 ml) was added. The mixture was stirred at RT for 1 hr, filtered and the solid was recrystallized from iPrOH/ether to give 3.5 g (11.3 mmol, 59%) of the title compound as a white solid.
6-(N,N-Dimethylcarbamyloxy)-N-propargyl aminoindan mesylate (Compound 24)
To a stirred mixture of 6-(N,N-dimethylcarbamyloxy) aminoindan HCl (1.88 g, 7.33 mmol), K2CO3 (2.03 g, 14.66 mmol) and acetonitrile (70 ml) was added a solution of propargyl bromide (0.79 g, 6.6 mmol) in CH3CN (5 ml) dropwise over 5 min, under nitrogen. The mixture was stirred under N2 for 24 hrs, filtered and the solvent was removed at reduced pressure. The residue was taken up into water (150 ml) and toluene (150 ml). This mixture was stirred while adjusting the pH of the aqueous layer to 3.75 by the addition of 20% aq. HCl. The aqueous layer was separated and extracted with toluene (2×100 ml) and brought carefully to pH 7.5 by the addition of 10% aq. NaOH solution. It was then extracted with toluene (100 ml+4×70 ml). The combined toluene layers were dried (Na2SO4), filtered and the solvent was removed under reduced pressure to give 1.06 g (62%) of a yellow oil.
To a stirred solution of the free base (1.65 g, 6.4 mmol) in anh. ether (60 ml) was added dropwise a solution of methanesulfonic acid (0.7 g, 7.29 mmol) in ether (10 ml). The resulting suspension was stirred at 25° C. for 30 man and then allowed to settle for an additional 30 min. The ether was then decanted off, and the residue was dried under vacuum. It was then recrystallized from iPrOH/ether to give 2.05 g of a white solid (90.3%).
In this manner the following compounds of general formula I as shown in Tables 5, 5a and 6 were prepared. Analytical data relating to these compounds is given in Tables 9, 9a and 10.
TABLE 5
Carbamyloxy-N-propargyl aminoindans
##STR00009##
cryst/slurry
mp
yield
#
X
position
R1
R3
R4
solvent
(° C.)
(%)
23
Cl
6-
H
Me
Me
iPrOH/Et2O
180-2
52
24
mesylate
6-
H
Me
Me
iPrOH/Et2O
147-9
60
25
Cl
6-
H
Me
Et
iPrOH/Et2O
194-6
59
26
Cl
6-
H
Me
Pr
iPrOH/Et2O
183-5
46
27
Cl
7-
H
Me
Me
iPrOH/Et2O
219-20
65
28
Cl
7-
H
Me
Pr
iPrOH/Et2O
185-6
53
29
Cl
6-
Me
Me
Me
iPrOH/Et2O
199-201
55
30
Cl
6-
Me
Me
Et
Et2O
196-8
47
31
Cl
6-
Et
Me
Me
iPrOH/Et2O
212-3
71
32
Cl
7-
Me
Me
Me
iPrOH/Et2O
169-71
63
33
Cl
7-
H
Me
Et
iPrOH/Et2O
208-9
64
34
Cl
4-
H
Me
Me
Et2O
196-8
85
35
Cl
4-
H
Me
Et
Et2O
183-5
85
36
Cl
6-
H
Me
n-hexyl
iPrOH/Et2O
106-8
53
37*
Cl
6-
H
Me
Et
Et2O
159-6
88
38
Cl
6-
H
Me
cyclohexyl
Et2O
174-5
55
39**
Cl
6-
H
Me
Et
Et2O
160-2
61
54*
mesylate
6-
H
Me
Me
Et2O
139-41
54
55**
mesylate
6-
H
Me
Me
Et2O
138-40
52
56
Cl
6-
H
H
Et
iPrOH/Et2O
175-7
38
57
Cl
6-
H
H
Pr
iPrOH/Et2O
165-7
48
58
mesylate
6-
H
Me
Et
Et2O
92-4
64
59**
mesylate
6-
H
Me
Et
iPrOH/Et2O
72
60
mesylate
6-
H
Me
Et
Et2O
121-3
87
61
Cl
6-
H
Me
p-OMe-Ph
Et2O
172-4
84
62
Cl
6-
H
Me
Ph
Et2O
182-4
61
63
Cl
6-
H
Me
CH2Ph
Et2O
188-90
58
64***
Cl
6-
H
Me
Me
iPrOH/Et2O
195-7
55
65***
Cl
6-
H
Me
Et
iPrOH/Et2O
188-90
51
68****
fumarate
6-
H
Me
Et
iPrOH
146-8
48
69*
fumarate
6-
H
Me
Et
iPrOH
115-7
35
70
crylate
6-
H
Me
Et
EtOAc
109-11
60
71****
Cl
6-
H
Me
Et
Et2O
161-3
55
72****
Cl
6-
H
Me
Pr
Et2O
164-6
58
73**
fumarate
6-
H
Me
Et
iPrOH
114-6
81
74**
crylate
6-
H
Me
Et
EtOAc
95-7
82
75**
½D-tartrale ½D-tartrate
6-
H
Me
Et
iPrOH
143-5
44
76*
½L-tarate ½L-tartrate
6-
H
Me
Et
iPrOH
143-5
41
77*
crylate
6-
H
Me
Et
EtOAc
106-8
93
78*
Cl
6-
H
Me
Pr
Et2O
126-8
89
79*
Cl
6-
H
Me
Pr
Et2O
135-7
33
81
Cl
6-
H
Me
Bu
Et2O
168-70
63
83
Cl
6-
H
Et
Bu
Et2O
148-50
42
85
Cl
6-
H
Et
cyclohexyl
Et2O
178-80
56
86*
Cl
6-
H
Me
Bu
Et2O
86-8
51
87**
Cl
6-
H
Me
Bu
Et2O
88-9
52
*R-enantiomer
**S-enantiomer
***substituted propargyl derivatives, Rn in Scheme 1 is methyl
****Y: 5-Cl
TABLE 5a
Thiocarbamyloxy-N-propargyl aminoindans
##STR00010##
cryst/slurry
mp
yield
#
X
position
R1
R3
R4
solvent
(° C.)
(%)
46
Cl
6-
H
Me
Me
Et2O
152-4
53
47
Cl
6-
H
Me
Et
Et2O
193-5
54
TABLE 6
N-Propargyl aminotetralins
##STR00011##
position
of
cryst/slurry
mp
yield
#
amine
R1
R3
R4
solvent
(° C.)
(%)
40
2-
H
Me
Me
MeOH/Et2O
206-8
66
41
2-
H
Me
Et
iPrOH/Et2O
208-9
65
42
1-
H
Me
Me
ether
207-9
57
43
1-
H
Me
Et
ether
201-3
42
TABLE 7
Analytical Data of Compounds of the Invention shown in Table 3
##STR00012##
NMR1
MS
elem. anal.
#
aryl
index
R1, R2
R3, R4
IR
(MH*)
(C, H, N)
1
7.38, 7.20
4.85, 3.10
3.10, 2.96
3446, 2943
221
calc: 56.14, 6.62, 10.90
7.10
2.96, 2.63
1711, 1487
found: 55.90, 6.67, 10.89
2.14
1393, 1240
2
7.40, 7.21
4.80, 3.10
3.43, 3.27
2970, 2863
249
calc: 59.05, 7.38, 9.84
7.10
2.95, 2.65
3.10, 2.95
1735, 1608
found: 58.75, 7.33, 9.86
2.15
1.70, 1.63
1396, 1241
0.94, 0.90
2a
7.40, 7.21
4.80, 3.10
3.43, 3.27
2970, 2863
249
calc: 57.23, 7.55, 9.54
(½
7.10
2.95, 2.65
3.10, 2.95
1735, 1608
found: 57.54, 7.29, 9.45
H2O
2.15
1.70, 1.63
1396, 1241
0.94, 0.90
4
7.47, 7.36
4.91, 3.25
3.18, 3.03
2950, 1701
7.09
3.07, 2.60
1504, 1396
2.25
1234, 1177
5
7.44, 7.29
4.88, 3.20
3.55, 3.39
3446, 2920
235
calc: 57.70, 7.25, 10.35
7.02
3.14, 2.55
3.14, 2.99
1710, 1472
found: 57.38, 6.97, 10.32
2.23
1.26, 1.18
1403, 1235
6
7.45, 7.30
4.86, 3.20
3.50, 3.32
3448, 2923
249
calc: 59.05, 7.43, 9.84
7.02
3.04, 2.55
3.13, 2.98
1710, 1485
found: 58.78, 7.47, 9.91
2.23
1.70, 1.63
1226, 1154
0.94, 0.90
7
7.45, 7.29
4.83, 3.17
3.20, 1.33
3.15, 3.0
2948, 2766
249
calc: 59.05, 7.38, 9.84
7.17
3.02, 2.65
2680, 1725
found: 57.75, 7.40, 9.65
1485, 1386
8
7.43, 7.27
4.75, 3.14
2.73
3.13, 2.97
2950, 2722
235
calc: 57.70, 7.02, 10.35
7.17
3.03, 2.60
1720, 1390
found: 56.83, 7.09, 10.27
2.30
1160
9
7.52, 7.37
4.83, 3.27
2.74
3.19, 3.04
2963, 2710
235
calc: 57.70, 7.02, 10.35
7.10
3.10, 2.55
1715, 1579
found: 57.46, 6.73, 10.36
2.38
1472, 1389
10
7.44, 7.29
4.80, 3.15
2.74
3.55, 3.35
2950, 2705
calc: 59.08, 7.38, 9.84
7.15
3.03, 2.62
3.12, 2.98
1720, 1450
found: 58.74, 7.51, 9.72
2.30
1.25, 1.18
1402
11
7.42, 7.25
4.75, 3.15
2.72
3.45, 3.30
2963, 2723
calc: 60.33, 7.70, 9.38
7.14
3.10, 2.60
3.10, 2.95
1715, 1465
found: 60.32, 7.75, 9.42
2.28
1.65, 0.94
1404, 1234
0.88
12
7.43, 7.27
4.96, 3.12
2.75
3.10, 2.96
3480, 1718
249
calc: 59.05, 7.38, 9.84
7.17
3.05, 2.55
1475, 1390
found: 58.75, 7.41, 9.84
2.42
1237, 1174
1311
7.53, 7.29
4.71, 2.95,
8.75
3.04, 2.9
221
7.08
2.74, 2.45,
2.0
1411
7.53, 7.3,
4.71, 2.95,
8.7
3.41, 3.3,
235
7.08
2.73, 2.48,
3/01, 2.89,
2.0
1.18, 1.07
15
7.35, 7.23
4.83, 3.3
3.1, 3.06
2930, 1720
291
calc: 62.47, 8.33, 8.57
7.01
2.6, 2.16
2.95, 2.91
1471, 1405
found: 62.54, 8.30, 8.61
1.6, 1.29
1248
0.85
16
7.42, 7.22
4.87, 3.16
3.53, 3.39
235
7.12
3.01, 2.65
3.92, 2.99
2.17
1.26, 1.17
17
7.42, 7.22
4.87, 3.15
4.10, 3.85
289
calc: 62.85, 7.76, 8.63
7.11
2.95, 2.65
3.00, 2.85
found: 62.55, 7.81, 8.33
2.17
1.90- 1.40
1.34, 1.13
3
7.43, 7.20
4.86, 3.15
3.51, 3.38
235
calc: 55.70, 7.25, 10.35
7.12
3.02, 2.64
3.10, 2.95
found: 57.44, 7.06, 10.38
2.18
1.25, 1.15
18
7.43, 7.20
4.86, 3.15
3.51, 3.38
235
calc: 55.70, 7.25, 10.35
7.12
3.02, 2.64
3.10, 2.95
found: 57.44, 7.06, 10.38
2.18
1.25, 1.35
48
7.41, 7.24
4.87, 3.13
3.23, 1.17
221
calc: 56.13, 6.68, 10.91
7.13
3 0, 2.65
found: 56.00, 6.66, 10.81
2.17
49
7.41, 7.24
4.87, 3.12
3.17, 1.56
235
calc: 57.67, 7.07, 10.35
7.13
2.98, 2.65
0.94
found: 57.32, 7.13, 10.31
2.17
50
7.37, 7.16
4.80, 3.10
7.40- 7.0
calc: 61.98, 6.02, 8.03
7.03
2.96, 2.61
3.82, 3.43
found: 61.16, 6.07, 7.77
2.15
3.29
66
7.57, 7.39
4.91, 3.18
3.61, 3.43
269
calc: 50.41, 6.02, 9.05
3.05, 2.71,
3.20, 3.03
271
found: 50.46, 6.11, 8.77
2.25
1.33, 1.23
67
7.55, 7.36
4.89, 3.14
3.52, 3.36
283
calc: 52.67, 6.32, 8.78
3.02, 2.68
3.18, 3.02
285
found: 52.67, 6.28, 8.48
2.20
1.77, 1.67
0.99, 0.93
1D2O, unless otherwise specified
11DMSO-d6
TABLE 7a
Analytical Data of Compounds of the Invention shown in Table 3a
##STR00013##
NMR(D3O)
MS
elem. anal.
#
aryl
indan
R1, R2
R3, R4
IR
(MH+)
(C, H, N, S)
44
7.45, 7.20,
4.87, 3.15,
3.44, 3.36
2933, 1714,
calc: 52.83, 628, 10.27, 11.75
7.11
3.05, 2.65,
1599, 1536,
found: 51.11, 6.48, 10.23, 12.16
2.20
1488, 1392
45
7.45, 7.20,
4.75, 3.10,
3.88, 3.79
2934, 1719,
calc: 51.22, 6.94, 9.19, 10.52
7.11
2.97, 2.65,
3.39, 3.32,
1594, 1522,
found: 51.04, 7.30, 9.31, 11.24
2.20
1.28, 1.25
1497, 1402
TABLE 8
Analytical Data of Compounds of the Invention shown in Table 4
##STR00014##
NMR2
MS
elem. anal.
#
aryl
cyclohex.
R1, R2
R3, R4
IR
(MH+
(C, H, N)
19
7.22, 6.95
3.69, 3.22
3.12, 2.97
3484, 2930
235
calc: 55.81, 7.20, 10.02
(½H2O)
2.93, 2.87
2362, 1699
found: 55.29, 6.93, 9.71
2.22, 1.92
1612, 1500
1391
20
7.20, 6.94
3.70, 3.19
3.48, 3.35
249
calc: 57.23, 7.55, 9.54
(½H2O)
2.90, 2.23
3.08, 2.94
found: 57.50, 7.53, 9.54
1.90
1.20, 1.12
21
7.28, 7.11,
3.10, 2.96
3.10, 2.96
235
calc: 57.70, 7.02, 10.35
7.06
2.77, 2.16
found: 56.97, 6.93, 10.06
2.05, 1.88
22
7.29, 7.13,
4.57, 2.88
3.52, 3.37
249
calc: 59.05, 7.38, 9.84
7.07
2.79, 2.15
3.10, 2.97
found: 58.91, 7.18, 9.99
2.05, 1.90
1.25, 1.17
2D2O, unless otherwise specified
TABLE 9
Analytical Data of Compounds of the Invention shown in Table 5
##STR00015##
NMR3
MS
elem. anal.
#
aryl
indan
R1
proparg
R3, R4
IR
(MH+)
(C, H, N)
23
7.46, 7.30
5.01, 3.20
4.0, 3.16
3.15, 3.0
259
calc: 61.12, 6.50, 9.51
7.18
3.15, 2.65
found: 60.93, 6.38, 9.47
2.36
24
7.46, 7.30
5.01, 3.20
4.0, 3.16
3.15, 3.02
1711, 1482,
259
calc: 54.22, 6.26, 7.91
7.18
3.15, 2.65
1439, 1394,
found: 53.92, 6.28, 7.84
2.36
1192, 1170
25
7.42, 7.27
4.97, 3.16
3.97, 3.02
3.52, 3.36
1728, 1435,
273
calc: 62.23, 6.86, 9.57
7.15
3.0, 2.62
3.10, 2.97,
1403, 1242,
found: 62.42, 6.84, 8.94
2.32
1.24, 1.15
1166
25a
7.50, 7.32
4.78, 3.10
3.91, 3.74
3.43, 3.32
1728, 1435,
273
calc: 62.23, 6.86, 9.57
7.10
2.85, 2.45
3.03, 2.90
1403, 1242,
found: 62.42, 6.84, 8.94
2.28
1.20, 1.10
1166
26
7.45, 7.30
5.0, 3.16
4.0, 3.03
3.48, 3.32
1725, 1465,
287
calc: 63.25, 7.18, 8.68
7.17
3.04, 2.65
3.12, 2.98
1429, 1403,
found: 63.13, 7.28, 8.93
2.33
1.72, 1.63
1232, 1165
0.96, 0.92
27
7.52, 7.38
5.05, 3.26
3.90, 3.21
3.12, 3.03
3200, 1722,
259
calc: 61.12, 6.50, 9.51
7.10
3.07, 2.56
1567, 1434,
found: 61.01, 6.46, 9.64
2.40
1408, 1238
28
7.52, 7.37
5.02, 3.27
3.98, 3.10
3.65, 3.42
3200, 1727,
287
calc: 63.25, 7.18, 8.68
7.07
3.09, 2.55
3.18, 3.02
1566, 1468,
found: 63.06, 7.30, 8.37
2.38
1.75, 0.98
1438, 1406,
0.93
1222
29
7.44, 7.30
5.20, 3.15
2.80
4.01, 3.13
3.12, 2.97
1729, 1388,
273
calc: 62.33, 6.80, 9.07
7.19
3.03, 2.57,
1234, 1165
found: 61.97, 6.80, 8.78
2.44
31
7.48, 7.30
5.34, 3.20
3.36,
4.05, 3.12
3.16, 3.01
3180, 1723,
287
calc: 63.25, 7.18, 8.68
7.23
3.08, 2.65
1.37
1490, 1440,
found: 63.42, 7.09, 8.71
2.50
1389, 1230,
1160
32
7.56, 7.39
5.30, 3.28
2.78
4.12, 3.23
3.20, 3.02
1712, 1472,
273
calc: 62.23, 6.86, 9.07
7.15
3.09, 2.55
1392, 1238,
found: 62.05, 6.81, 8.87
1171
33
7.46, 7.32
4.96, 2.50
3.92, 3.04
3.13, 2.96
1719, 1426,
273
calc: 62.23, 6.86, 9.07
7.03
2.33
1.24, 1.15
1404, 1233,
found: 62.19, 6.77, 9.08
1154
34
7.48, 7.23
5.07, 3.08
4.05, 3.07
3.29, 3.03
3238, 2907
259
calc:
2.95, 2.65
2769, 2635
found:
2.35
1714, 1470
1392, 1240
35
7.48, 7.23
5.07, 3.08
4.05, 3.07
3.56, 3.41
3197, 2934
273
calc:
2.95, 2.65
3.15, 3.01
2565, 2431
found:
2.35
1.29, 1.21
1707, 1445
1403, 1236
36
7.45, 7.28
4.98, 3.16
3.98, 3.04
3.49, 3.35
calc: 65.83, 8.01, 7.68
7.15
3.03, 2.63
3.11, 2.97
found: 65.65, 8.11, 7.82
2.33
1.66, 1.33
0.88
37
7.44, 7.29
4.98, 3.15
3.98, 3.03
3.53, 3.38
3275, 2754
273
calc: 62.23, 6.86, 9.07
7.18
3.01, 2.63
3.12, 2.98
1719, 1445
found: 62.30, 6.94, 9.09
2.31
1.25, 1.16
1395, 1303
38
7.44, 7.27
4.98, 3.14
3.98, 3.04
4.09, 3.85
3227, 2936
327
calc: 66.19, 7.50, 7.72
7.16
3.00, 2.64
3.01, 2.88
2612, 2128
found: 65.90, 7.63, 7.55
2.33
1.90-1.45
1713, 1584
1.35, 1.14
1440, 1401
39
7.46, 7.30
4.97, 3.17
3.97, 3.03
3.54, 3.39
3275, 2933
273
calc: 62.23, 6.86, 9.07
7.19
3.04, 2.64
3.13, 3.0
2758, 1720
found: 62.27, 6.95, 9.03
2.32
1.27, 1.19
1442, 1396
1303
54
7.46, 7.30
5.00, 3.17
3.99, 3.05
3.15, 3.0
1711, 1482
259
calc: 54.17, 6.20, 7.90
7.19
3.05, 2.64
1438, 1395
found: 54.18, 6.27, 7.78
2.33
1192, 1169
55
7.46, 7.30
5.00, 3.17
3.99, 3.05
3.15, 3.0
1711, 1482
259
calc: 54.17, 6.20, 7.90
7.19
3.05, 264
1438, 1395
found: 54.07, 6.25, 7.88
2.33
1192, 1169
56
7.46, 7.32
4.99, 3.17
3.99, 3.05
3.27, 1.20
259
calc: 61.12, 6.50, 9.51
7.20
3.04, 2.65
found: 60.87, 6.47, 9.34
2.33
57
7.47, 7.32
4.99, 3.18
3.99, 3.06
3.20, 1.61
273
calc: 62.23, 6.86, 9.07
7.20
3.05, 2.65
0.98
found: 61.60, 6.93, 9.04
2.34
58
7.47, 7.32
5.01, 3.20
4.01, 3.07
3.56, 3.41
273
calc: 55.43, 6.52, 7.60
7.22
3.08, 267
3.14, 3.01
found: 55.08, 6.52, 7.31
2.36
1.29, 1.21
59
7.47, 7.32
5.01, 3.20
4.01, 3.07
3.56, 3.41
273
calc:
7.22
3.08, 2.67
3.14, 3.01
found:
2.36
1.29, 1.21
60
7.47, 7.32
5.01, 3.20
4.01, 3.07
3.56, 3.41
273
calc: 55.43, 6.52, 7.60
7.22
3.08, 2.67
3.14, 3.01
found: 55.21, 6.64, 7.40
2.36
1.29, 1.21
61
7.40-7.0
4.96, 3.10
3.96, 3.90
7.40-7.0
351
calc: 65.20, 5.95, 7.24
2.97, 2.57
3.03
3.81
found: 64.72, 6.04, 6.81
2.30
62
7.60-7.10
4.96, 3.15
3.98, 3.07
7.60-7.10
321
calc: 67.32, 5.89, 7.85
3.00, 2.61
3.42
found: 67.22, 6.00, 7.54
2.34
63
7.55-7.10
4.97, 3.17,
3.99, 3.07
7.55-7.10
335
calc: 67.47, 6.20, 7.55
3.00, 2.64,
4.73, 4.59
found: 67.75, 6.32, 7.47
2.36, 2.36
3.14, 3.05
64
7.48, 7.35
5.16, 5.12
4.44, 4.27
3.17, 3.03
273
calc: 62.23, 6.86, 9.07
7.21
3.20, 3.05
3.17, 1.68
found: 62.22, 6.86, 8.96
2.70, 2.35
1.63
65
7.44, 7.36,
5.15, 5.09
4.43, 4.25
3.55, 3.39
calc: 63.25, 7.18, 8.68
7.27, 7.19
3.20, 3.02
3.25, 3.17
3.13, 3.00
found: 63.15, 7.15, 8.31
2.65, 2.32
1.67, 1.61
1.27, 1.19
71
7.60, 7.44
5.02, 3.20,
4.02, 3.07
3.60, 3.43,
307
calc: 55.98, 5.87, 8.16
3.06, 2.68,
3.20, 3.02,
309
found: 55.72, 5.88, 8.11
2.36
1.33, 1.23
72
7.59, 7.44
5.01, 3.20,
4.03, 3.07
3.53, 3.36,
321
calc: 57.15, 6.21, 7.84
3.06, 2.68,
3.20, 3.02,
323
found: 57.05, 6.21, 7.81
2.38
1.79, 1.68,
1.01, 0.95
76
7.47, 7.31,
5.00, 3.20,
4.00, 3.07
3.56, 3.40,
3286, 2972,
273
calc: 62.17, 6.62, 8.05
7.20
3.06, 2.66,
3.16, 3.00,
1724, 1637,
found: 62.31, 6.66, 7.94
2.35
1.28, 1.20
1400, 1308,
1233
81
7.48, 7.31,
5.00, 3.20,
4.01, 3.07
3.53, 3.38,
calc: 64.19, 7.42, 8.32
7.20
3.07, 2.66,
3.14, 3.01,
found: 63.99, 7.42, 8.04
2.35
1.65, 1.39,
0.97
83
7.47, 7.31,
5.00, 3.19,
4.01, 3.07
3.52, 3.38,
315
calc: 65.04, 7.70, 7.98
7.19
3.04, 2.66,
1.68, 1.40,
found: 64.75, 7.72, 7.94
2.34
1.29, 1.22,
0.98
85
7.47, 7.31,
5.00, 3.19,
4.01, 3.07
3.84, 3.42
341
calc: 66.33, 7.70, 7.43
7.19
3.02, 2.63,
1.85, 1.66,
found: 66.75, 7.69, 7.36
2.34
1.23
2D2O, unless otherwise specified
aDMSO-dd
TABLE 9a
Analytical Data of Compounds of the Invention shown in Table 5a
##STR00016##
NMR (D2O)
MS
elem. anal.
#
aryl
indan
propargyl
R3, R4
IR
(MH+)
(C, H, N, S)
46
7.48, 7.29,
5.02, 3.19,
4.0, 3.07
3.46, 3.41
calc: 57.97, 6.11, 9.01, 10.30
7.16
3.05, 2.67,
found: 58.07, 6.06, 8.85, 10.23
2.37
47
7.50, 7.31
5.04, 3.21,
4.20, 3.09
3.95, 3.87
calc: 59.16, 6.47, 8.62, 9.86
7.19
3.07, 2.70,
3.45, 3.38
found: 59.23, 6.39, 8.52, 9.76
2.38
1.35, 1.32
TABLE 10
Analytical Data of Compounds of the Invention shown in Table 6
##STR00017##
NMR4
MS
elem. anal.
#
aryl
cyclohex.
R1
proparg.
R3, R4
IR
(MH+)
(C, H, N)
40
calc:
found:
41
7.22, 6.95
3.79, 3.26
4.06, 3.01
3.50, 3.36
3227, 2938
287
calc: 63.25, 7.18, 8.68
2.95, 2.32
3.09, 2.96
2768, 1718
found: 63.16, 6.93, 8.69
1.91
1.24, 1.16
1587, 1494
1394, 1301
42
7.21, 7.03
4.60, 2.81
3.88, 3.95
3.01, 2.87
3234, 2936
273
calc: 62.23, 6.80, 9.07
2.72, 2.15
2774, 2130
found: 62.20, 7.01, 9.3
2.02, 1.84
1732, 1499
1.80
1390
43
7.32, 7.12
4.65, 2.88
3.99, 3.04
3.51, 3.37
3216, 2933
287
calc: 63.06, 7.41, 8.65
2.80, 2.20
3.10, 2.96
2768, 2663
found: 63.2, 7.14, 8.81
2.12, 1.94
1.23, 1.16
2129, 1723
1.85
1425, 1399
4D2O, unless specified otherwise
Acetylcholinesterase Inhibition in Mice
1.1 In vitro measurement of Acetylcholinesterase (AChE) Inhibition
Human erythrocyte acetylcholinesterase (type XIII, Sigma Israel), was prepared in a stock solution of 1 U/ml, containing Triton (1%) and bovine serum albumin (0.05%) in phosphate buffer (pH 8). The enzyme (0.05U) was incubated with 3-5 different concentrations of test compound (in triplicate) for periods of from 15 to 60 minutes at 37° C. The substrate acetylthiocholine (0.075M) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, 0.01M) were then added and the rate of hydrolysis of the substrate which yields a yellow product monitored spectrophotomerically at 412 nM (Ellman et al., Biochem Pharmacol. (1961) 7: 88-95). The percentage inhibition of AChE by each concentration of drug is calculated by comparison with that of enzyme in the absence of drug. The concentration of each drug that inhibits AChE by 50% (IC50) at the time of peak activity was calculated and is given in Table 11 below.
1.2 Ex vivo measurement of Acetylcholinesterase (AChE) Inhibition
Test drugs or saline were administered sub-cutaneously to male mice (Sabra strain, 28-35 g). At least 4-5 mice were used per dose and a minimum of 3 doses per drug were tested. The mice were sacrificed 15, 30, 50, 70, 90, 120 or 180 minutes after drug administration, the brains rapidly removed (minus cerebellum), weighed and homogenized in 0.1 M phosphate buffer, pH 8.0, containing Triton (1 mg/100 g tissue) and centrifuged to remove cell debris. Aliquots (25 μl) of the supernatant were then incubated with acetylthiocholine and DTNB. AChE activity was measured as described above. The % inhibition of whole brain AChE by each dose of drug was calculated by comparison with enzyme activity from 3 saline treated control mice run at the same time. The dose of each drug that inhibits AChE by 50% at the peak of activity (ED50) was calculated and is given in Table 11.
1.3 Acute Toxicity in Mice
Drugs were administered sub-cutaneously in at least 3 doses, to a minimum of 10 mice per dose. The dose that was lethal to 50% of the mice (LD50) within 6 hours after administration was calculated for each drug and is given in Table 11. Therapeutic Ratio was calculated as LD50 divided by ED50 of ex vivo acetylcholine esterase inhibition.
2.1 Inhibition of MAC activity in vitro
The MAO enzyme source was a homogenate of rat brain in 0.3M sucrose, which was centrifuged at 600 g for 15 minutes. The supernatant was diluted appropriately in 0.05M phosphate buffer, and pre-incubated with serial dilutions of test compounds for 20 minutes at 37° C. 14C-Labeled substrates (2-phenylethylamine, hereinafter PEA; 5-hydroxytryptamine, hereinafter 5-HT) were then added, and the incubation continued for a further 20 minutes (PEA), or 30-45 minutes (5-HT). Substrate concentrations used were 50 μM (PEA) and 1 mM (5-HT). In the case of PEA, enzyme concentration was chosen so that not more than 10% of the substrate was metabolized during the course of the reaction. Deaminated products were extracted into toluene-ethyl acetate (1:1 v/v) containing 0.6% (w/v) 2,5-diphenyloxazole (ppo) prior to determination by liquid scintillation counting. Radioactivity n the eluate indicates the production of neutral and acidic metabolites formed as a result of MAO activity. Activity of MAO in the sample was expressed as a percentage of control activity in the absence of inhibitors after subtraction of appropriate blank values. The activity determined using PEA as substrate is referred to as MAO-B, and that determined using 5-HT as MAO-A.
Concentrations of inhibitor producing 50% inhibition of substrate metabolism (IC50) were calculated from the inhibition curves, and are shown in Table 11.
2.2 Inhibition of MAO activity ex vivo
Male Sabra mice, weighing 45-50 g were injected with test compound solutions (prepared in 0.9% saline). Each dose was administered to two or three mice. The mice were sacrificed two hours after drug administration or at a time corresponding to the peak AChE inhibition time (see Table 11). The brain and liver were rapidly dissected and stored in appropriate vials on ice. The tissues were weighed, diluted to ½ in sucrose 0.3M and stored at −20° C. before performance of the MAO assay described above. The results given in Table 11 relate to measurements made on brain tissue only.
2.3 Inhibition of MAO activity following sub-acute administration to rats
Experiments were done in Sprague Dawley male rats. Procedures were repeated as described in Examples 2.1 and 2.2 but drug administration was continued daily for 14 days. At the end of this period animals were sacrificed and MAO levels determined in the brain, liver and intestines. Compounds 24, 25, 37 and 39 were administered subcutaneously and/or pet os at a dose of 6 mg/kg(sc) and 10 mg/kg(po) (compound 24), 25 and 50 mg/kg (compound 25), 45 mg/kg (compound 37) and 40 mg/kg (compound 39). The results are shown in Table 11a from which it can be seen that these compounds displayed selectivity in inhibiting MAO enzyme sub-types in the brain in preference to the periphery.
TABLE 11
AChE Inhibition
Time
Ex vivo
to return to
MAO-B Inhibition
MAO-A Inhibition
Acute Toxicity
ED50
to peak
50% of
Ex vivo
Ex vivo
LD50
Therapeutic
In vitro
μmoles/kg
activity
peak
In vitro
ED50
In vitro
ED50
μmoles/kg
Ratio
#
IC50 μm
(AC)
t (min)
t (min)
IC50 μm
μmoles/kg
IC50 μm
μmoles/kg
(LD)
LD/AC
1
0.6
5.0
30
>120
>1000
>>80
75
>>80
83.8
16.8
23
3.5
22.4
15
70
600
100
800
>120
255
11.4
2
7.3
NT
>1000
32
3
20.0
46.3
60-90
>180
>1000
12.6
950
20.6
25
53.0
140.0
60
>180
>1000
200
270
>>350
1400
10.8
26
17.0
120
30-60
264
333
114
>>440
1200
9
27
5.72
30
15
>60
>1000
>>160
>1000
>>160
300
10
28
100.0
NT
5
11.5
85.0
60
>120
>>277
>>277
840
9.9
7
32.0
NT
>1000
600
8
1.0
10.0
15-30
>60
>1000
>>50
50
>>50
87
8.7
9
0.18
19
15
93
4.9
29
8.5
53.7
15
>60
40
30
40
50
500
9.3
10
38.0
34.7
60-90
>180
>1000
>175
22
>175
740
21.3
30
1300.0
NT
31
10.0
110
>1000
>100
>1000
>100
32
3.7
7.8
15
500
>>20
190
>>20
68
9.0
12
2.0
8.0
15
>1000
130
<20
<2.5
33
540.0
NT
>1000
1000
>1000
>>1200
34
0.046
0.65
30
100
0.5
3.7
5.7
35
2.2
10
60
100
<1
33
3.3
37
51
125
500
200
750
>200
1700
13.6
39
36
80
30-60
>180
1000
>>200
550
>>200
1150
14.4
24
3
16.6
15
750
100
850
>120
179
10.8
60
42
58
51
>1000
300
54
1.8
>100
>100
55
2
>100
>100
56
11.5
180
57
2.4
70
25
89
48
10
49
2
17
4
16
9
50
0.26
61
0.75
47
500
>100
700
>100
64
1.9
13.2
>1000
>120
1000
>120
150
11.4
38
33
>1000
10
>400
170
>400
36
15
>400
>1000
>100
>1000
>100
>1000
62
0.57
290
60
100
>>200
80
>>200
63
2.5
140
60-90
120
>300
40
>300
1300
9.3
71
29
>100
130
>100
72
38
>200
>100
>100
78
10
101
60-90
>120
450
>>450
1300
12.9
79
9.4
94
90
>180
>>450
>>450
1000
10.6
81
11.5
40
90
>120
>>100
>>100
920
23
83
80
86
10.5
87
9.1
85
17
>100
TABLE 11a
Effect of Compounds 24, 25, 37 and 39 on MAO
activity after chronic sub-acutetreatment to rats
% MAO-A inhibition
24
% MAO-B inhibition
Compound
6 (sc)
25
37
39
24
25
37
39
Dose (mg/kg)
10 (po)
25
50
45
40
25
25
50
45
40
Brain
sc
30
53
75
78
17
50
61
85
87
27
po
0
70
67
20
80
82
Intestine
sc
0
0
30
0
0
0
29
45
26
40
po
30
25
0
20
30
21
Liver
sc
0
0
10
0
0
0
14
40
29
0
po
10
25
28
0
35
28
Effect of Drug Treatment Following Closed Head Injury (CHI) in Mice
The procedure for closed head injury followed was as described for rats in Shohami, et al. (J Neurotrauma (1993) 10 (2): 109-119) with changes as described.
Animals: Male Sabra mice (Hebrew University strain) weighing 34-40 g were used. They were housed in groups of 10 per cage, in a 12 hr:12 hr light:dark cycle. Food and water were provided ad libitium.
Trauma was induced under ether anesthesia. A longitudinal incision was performed in the skin covering the skull and the skin retracted to expose the skull. The head was fixed manually at the lower plane of the impact apparatus. A weight of 333 g was delivered by an electric device from a distance of 3 cm to the left hemisphere, 1-2 mm lateral to the midline in the midcoronal plane. Test compounds were injected sub-cutaneously at a dosage corresponding to the ED50 acetylcholinesterase, once 15 min. after CHI.
3.1 Assessment of Motor Function
Motor function and reflexes were evaluated in the injured mice at different times after closed head injury (CHI) using a neurological severity score (NSS) as shown in Table 12 below, which is modified from that described for rats (Shohami, et al. supra.). One point was awarded for the lack of a tested reflex or for the inability to perform the tasks outline in the Table. The maximal score that can be reached at 1 hour post-CHI is 25 points and 21 at later times. The difference in NSS at 1 hr and at any other time reflects the recovery, and is referred to as ΔNSS. An NSS score of 15-19 at 1 hr denotes severe injury, 11-14 moderate injury and less than 10 mild injury. The NSS recorded after treatment with test compound or control is shown in Table 13.
TABLE 12
Neurological Severity Score for mice after Closed Head Injury
Points at
Points at any
Parameter
1 hour
other time
Inability to exit from a circle (30
cm diameter when left in its center
for 30 min
1
for 60 min
1
for >60 min
1
1
Loss of righting reflex
for 10 second
1
for 20 seconds
1
for >30 seconds
1
1
Hemiplegia - inability of mouse to
1
1
resist forced changes in position
Flexion of hind limb when
1
1
lifted by tail
Inability to walk straight when
1
1
placed on the floor
Reflexes
Pinna reflex
1
1
Corneal reflex
1
1
Startle reflex
1
1
Clinical grade
Loss of seeking behaviour
1
1
Prostration
1
1
Loss of reflexes
Left forelimb
1
1
Right forelimb
1
Left hindlimb
1
1
Right hindlimb
1
1
Functional test
Failure in beam balancing task
1
1
(0.5 cm wide)
for 20 seconds
1
1
for 40 seconds
1
1
for >60 seconds
Failure in round stick balancing
task (0.5 cm is diameter
for 10 seconds
1
1
Failure in beam walking task
3 cm wide
1
1
2 cm wide
1
1
1 cm wide
1
1
Maximum Points
25
21
TABLE 13
Change in Neurological Severity Score
after Closed Head Injury in Mice
ΔNSS, 24 hr
ΔNSS, 7 days
ΔNSS, 14 days
Drug/dose
N
post-CHl
post-CHl
post-CHl
Saline, 1 ml/kg
51
4.75 ± 0.17
5.83 ± 0.36
5.96 ± 0.4
1 (1.3 mg/kg)
10
5.50 ± 0.34m
7.31 ± 0.45m
9.21 ± 0.47
24 (6.5 mg/kg)
12
6.31 ± 0.23m
8.67 ± 0.41m
9.67 ± 0.66m
25 (46 mg/kg)
10
5.00 ± 0.42
7.42 ± 0.62m
9.01 ± 0.69m
251 (46 mg/kg)
10
4.90 ± 0.43
7.70 ± 0.33m
8.80 ± 0.33m
10 (15 mg/kg)
11
5.36 ± 0.39
6.64 ± 0.41m
6.73 ± 0.52
37 (30 mg/kg)
12
5.50 ± 0.26
6.92 ± 0.38
8.25 ± 0.62
39 (30 mg/kg)
14
5.36 ± 0.25
6.71 ± 0.45
7.64 ± 0.48
1administered 60 min before CHl
msignificantly different from saline control (p < 0.05)
3.2 Assessment of Reference Memory
Morris Water Maze Test: the water maze consists of a circular aluminium pool, im in diameter and 60 cm in depth, filled with water to a depth of 17.5 cm. The hidden goal platform is a glass vessel (15 cm diameter×16.5 cm height) placed upside down at a fixed location in the pool, 1 cm below the surface of the water. The water temperature is maintained at 24° C. and the pool is always placed in the same position in the room to provide the same extra-maze cues. Prior to CHI (as described in Example 3 above), mice were given 3 trials per day for 5 consecutive days to establish a baseline performance—measured as the latency to find the platform from the same start location. Commencing 24 hr after CHI, mice were retested daily for 2 weeks in 3 trials per day.
Effect On Mice Having Experienced A Hypobaric Hypoxic Episode
The hypobaric hypoxic model is a well accepted model for assessing the activity of compounds believed to possess neuroprotective activity. The model is based on that described in Nakanishi, M., et al. Life Sci. (1973) 13: 467, Oshiro, et al., J. Med. Chem. (1991) 34: 2004-2013 and U.S. Pat. No. 4,788,130.
A 12 liter desiccator (desiccator A) and a 2.5 liter desiccator (desiccator B) were separately connected to a vacuum pump. Desiccator B was disconnected and allowed to equilibrate with room air whilst desiccator A was evacuated to a pressure of 100 mmHg. Four male ICR albino mice (22-28 g) were placed in desiccator B. Desiccator B was then closed to room air and connected to desiccator A. The pressure inside desiccator B was monitored using a mercury manometer and at the point were the pressure in desiccator B reached 200 mmpg (usually within 14 seconds), the two desiccators were disconnected from the vacuum pump and the pump switched off. The survival time from the moment of induction of hypoxia to the time of cessation of respiration was recorded for each mouse for a maximum of 15 minutes after which time room air was reintroduced to desiccator B. Survivors were monitored for signs of lethargy or vitality.
Effect of drug treatment was assessed as the percent of the survival time of the drug treated group with respect to the saline injected or vehicle injected control group. Control groups were run twice, before and after each experimental group and consisted of 8 mice in groups of 4 mice to ensure a constant residual volume of oxygen in all tests. The effect of each dose of test drug was determined in duplicate i.e. two groups of 4 mice. The range of survival times of control mice was from 108-180 seconds.
Positive reference drugs were sodium pentobarbital at a dose of 40 mg/kg, and diazepam 10 mg/kg given 0.5 h prior to hypoxia, physostigmine 0.2 and 0.4 mg/kg and neostigmine 0.2 mg/kg given sc 30 min before hypoxia. Methyl atropine 1 mg/kg was given sc. 10 min. before physostigmine.
Test drugs were dissolved in 0.9% saline, and injected sc. in the nip of the neck at a dose in accordance with body weight, 60-90 min. before hypoxia. The volume of injection was 0.2-0.3 mL per mouse (10 mL/kg). The initial dose was about one third of the reported LD50 for acetylcholine esterase inhibition. If no protection could be obtained, the dose was further increased to the nearest non-toxic dose. In case of protection, the dose was further reduced in an attempt to locate the “protective” dose range.
Per cent survival times as compared to saline treated control is shown in Table 14.
TABLE 14
Survival Time of Mice Having Experienced a Hypobaric Episode
Time of dose
Dose
(min before
Protection
Compound
mg/kg
hypoxin)
(% of control)
p
Control
100
(saline)
Nembutal
40
30
253 ± 200
<0.005
Diazepam
10
30
316 ± 78
<0.003
Neostigmine
0.2
30
141 ± 32
<0.01
Physostigmine
0.2
30
453 ± 222
<0.001
0.4
30
552 ± 210
<0.001
Physostigmine
0.4
30
296 ± 193
<0.05
and Atropine
1.0
40
methyl nitrate
1
8
60
637 ± 116
0.007
4
60
470 ± 200
0.001
2
60
120 ± 51
NS
24
50
60
738 ± 00
<0.001
21
60
269 ± 166
<0.02
25
100
60
761 ± 91
0.001
75
60
559 ± 225
0.001
50
60
380 ± 231
0.01
25
60
84 ± 35
NS
27
50
60
455 ± 23
<0.001
3
60
287 ± 319
<0.001
15
60
143 ± 56
<0.05
8
60
119 ± 45
NS
29
77
60
508 ± 206
<0.001
53
60
638 ± 10
<0.001
25
60
131 ± 56
NS
25
30
273 ± 183
<0.02
10
50
90
705 ± 101
0.001
25
90
700 ± 201
0.001
10
90
304 ± 129
0.001
12
20
60
725 ± 128
<0.001
15
60
649 ± 221
<0.001
10
60
386 ± 238
<0.01
7
60
248 ± 97
<0.001
Neurological Score and Brain Infarct Size in Male Wistar Rats After Middle Cerebral Artery Occlusion (MCA-O)
A modification of the procedure described by Tamura, et al was used (Tamura A, Graham D1, McCulloch J, Teasdale G H (1981) J. Cereb. Blood Flow and Metab. 1: 53-60). Male Wistar rats (Olac England-Jerusalem) 300-400 g each were anesthetized with a solution of Equitesine administered i.p. at a dose of 3 ml/kg. Equitesine consists of 13.5 ml sodium pentothal solution (60 mg/ml), 3.5 g chloral hydrate, 1.75 g MgSO4, 33 ml propylene glycol, 8.3 ml absolute alcohol, made up to 83 ml with distilled water.
Surgery was performed with the use of a high magnification operating microscope, model SMZ-2B, type 102 (Nikon, Japan) In order to expose the left middle cerebral artery, a cut was made in the temporal muscle. The tip of the coronoid process of mandible was excised as well and removed with a fine rongeur. Craniectomy was made with a dental drill at the junction between the median wall and the roof of the inferotemporal fossa.
The dura matter was opened carefully using a 27 gauge needle The MCA was permanently occluded by microbipolar coagulation at low power setting, beginning 2-3 mm medial to the olfactory tract between its cortical branch to the rhinal cortex and the laterate striate arteries. After coagulation, the MCA was severed with microscissors and divided to ensure complete occlusion. Following this, the temporalis muscle was sutured and laid over the craniectomy site. The skin was closed with a running 3-0 silk suture. A sham craniectomy operation was performed on a parallel group of rats, but without cauterization of the MCA.
During the entire surgical operation (20-25 min) in either group, body temperature was maintained at 37 to 38° C. by means of a body-temperature regulator (Kyoristsu, Japan) consisting of a self-regulating heating pad connected to a rectal thermistor. At 24 and 48 hours post surgery a neurological score was taken in order to assess the severity of the injury in the drug-treated rats with respect to their untreated controls.
Drugs were administered as an s.c. injection, according to the following schedule:
Compound 24: 7.8 mg/kg 15 minutes prior to MCA-O and 6.5 mg/kg 2 hours post MCA-O.
Compound 25: 43 mg/kg 90 minutes prior to MCA-O and 30 mg/kg 3 hours post MCA-O.
After 48 hours of ischemia induced by permanent occlusion morphometric, the animals-were anesthetized with Equitesine and measurement of infarct volume was performed as follows by TTC (2,3,5-triphenyl tetrazolium chloride) staining. TTC 1% in saline was prepared immediately before use and protected from exposure to light by aluminum foil wrap. MCA-O rats were deeply anesthetized and a 23-gauge butterfly needle with an extended tubing and a 20 ml syringe was inserted into the ventricle via thoracotomy. The right atrium was incised to allow outflow of saline. Heparine 50 i.u. in saline was delivered until the perfusate was bloodless. A 30-ml TTC-filled syringe was exchanged for the saline syringe and TTC was injected into the left ventricle at a rate of 5 ml/min. Both perfusate solutions were administered at 37.5° C. The brains were removed and immersed into 20 ml of 1% TTC contained in tightly closed glass vials. These were further placed for 2 hours in a water bath maintained at 37° C. The TTC solution was decanted, the brains removed, wiped dry and placed into 10% buffered formalin solution for 3 days. Six coronal slices each 2 mm thick, 3, 5, 7, 9, 11 and 13 mm distal from the frontal pole were obtained with a brain matrix (Harvard Apparatus, South Natick, Mass.). Infarction areas were measured with a video imaging and analyzer from both sides of the coronal slices and expressed in mm2. The volume of the infarcted region in mm was calculated by taking the sum of the ischemic areas in all six slices. The volume of infarcted region for the saline control and compounds 24 or 25 are given in Table 15a.
Neurological Score
The neurological score was measured in a manner slightly different from that given in Example 3. This method consists of the sum total of a series of ratings assigned to the performance of specific locomotor activities in a given rat. The scale runs from 0 (fully normal rats) to 13 (fully incapacitated rats). Most parameters are rated as either 0 (normal), or 1 (incapacitated) others are graded. The following tests were used in the present study:
General observation tests: hypoactivity, sedation, piloerection.
Motor reflex. Rats were lifted by the tail about 15 cm above the floor. Normal rats assume a posture in which they extend both forelimbs towards the floor and spread that hind limbs to the sides in a trapeze-like manner. MCAO, when severe, causes consistent flexion of the contralateral limb.
Motor ability. This is seen as the ability to grasp a rod 1 cm in diameter by the contralateral limb for 5-15 sec when the rat is left hanging on the rod through the arm pit.
Motor coordination. Normal rats are able to walk up and down a beam, 5 cm wide placed at a moderate slant. Failure to walk the beam in either direction reveals some motor incoordination, lack of balance and limb weakness.
Gait. Ability to restore normal position to either hand contralateral or fore contralateral limb when intentionally displaced while on a narrow beam.
Balance. Ability to grasp and balance on a narrow beam 2 cm wide.
Locomotor activity. Total movements over a period of 15 min in an automated activity cage.
Ratings assigned to each of the above parameters are given in Table 15.
TABLE 15
Neurological scores assigned to each of 10
parameters of posture and locomotion
Parameter
Score
a.
Activity in home cage
normal = 0
hypoactive = 1
b.
Sedation
none = 0
marked = 1
c.
Piloerection
none = 0
marked = 1
d.
Extenstion of contra-
good = 0
flexed limb = 1
lateral forelimb to-
wards floor when
lifted by tail
e.
Spread of contralateral
good = 0
flexed limb = 1
hind limb when lifted by
tails (trapezoid posture)
f.
Grasp rod with contra-
good = 0
poor = 1
lateral limb for 5-15 sec.
when suspended by
armpit
g.
Walk on beam 5 cm wide
good = 0
poor = 1
h.
Restoration of contra-
good = 0
poor =
lateral hind and/or
1 (one limb)
forelimb to original
2 (two limbs)
position when
intentionally displaced
i.
Grasping & balance on
good = 0
poor = 1
beam 2 cm wide
j.
Motor activity with
0-25% of control = 3
respect to control
26-50% of control = 2
(15 min in activity cage)
51-75% of control = 1
76-100% of control = 0
k.
Tendency to lean on
1
contralateral side
l.
Contralateral circling
1
when pulled by tail
m.
Contralateral circling
1
spontaneous.
Table 15a shows the effect of compounds 24 and 25 in this model, comparing the change in NSS measured in 24 and 48 hours post injury.
TABLE 15a
Volume infarction
Compound
ΔNSS*
Mean ± SD mm*
Saline
0.745
211 ± 75
24
1.625
152 ± 45
25
1.78
189 ± 54
*Difference is ΔNSS measured at 24 hours and 48 hours. From this it can be seen that compounds 24 and 25 have a longer lasting effect than the saline treated control.
##STR00018##
Youdim, Moussa B. H., Sterling, Jeffrey, Chorev, Michael, Weinstock-Rosin, Marta, Goren, Tamar, Herzig, Yacov
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