Compounds for Treatment of Neurodegenerative Diseases

The present invention relates to certain novel compounds for treating patients having neurodegenerative diseases, and in particular, to compounds useful for treating patients suffering from those diseases which cause a cholinergic deficit.

Senile dementia of the Alzheimer's type (SDAT) is a debilitating neurodegenerative disease, mainly afflicting the elderly; characterized by a progressive intellectual and personality decline, as well as a loss of memory, perception, reasoning, orientation and judgment. One feature of the disease is an observed decline in the function of cholinergic systems, and specifically, a severe depletion of cholinergic neurons (i.e., neurons, that release acetylcholine, which is believed to be a neurotransmitter involved in learning and memory mechanisms). See, Jones, et al., Intern. J. Neurosci., Vol. 50, p. 147 (1990); Perry, Br. Med. Bull., Vol. 42, p. 63 (1986) and Sitaram, et al., Science, Vol. 201, p. 274 (1978). It has been observed that nicotinic acetylcholine receptors, which bind nicotine and other nicotinic agonists with high affinity, are depleted during the progression of SDAT. See, Giacobini, J. Neurosci. Res., Vol. 27, p. 548 (1990); and Baron, Neurology, Vol. 36, p. 1490 (1986). As such, it would seem desirable to provide therapeutic compounds which either directly activate nicotinic receptors in place of acetylcholine or act to minimize the loss of those nicotinic receptors.

Parkinson's disease (PD) is a debilitating neurodegenerative disease, presently of unknown etiology, characterized by tremors and muscular rigidity. A feature of the disease appears to involve the degeneration of dopaminergic neurons (i.e., which secrete dopamine). One symptom of the disease has been observed to be a concomitant loss of nicotinic receptors which are associated with such dopaminergic neurons, and which are believed to modulate the process of dopamine secretion. See, Rinne, et al., Brain Res., Vol. 54, pp. 167-170 (1991) and Clark, et al., Br. J. Pharm., Vol. 85, pp. 827-835 (1985).

Certain attempts have been made to treat SDAT. For example, nicotine has been suggested to possess an ability to activate nicotinic cholinergic receptors upon acute administration, and to elicit an increase in the number of such receptors upon chronic administration to animals. See, Rowell, Adv. Behav. Biol., Vol. 31, p. 191 (1987); and Marks, J. Pharmacol. Exp. Ther., Vol. 226, p. 817 (1983). Other studies indicate that nicotine can act directly to elicit the release of acetylcholine in brain tissue, to improve cognitive functions, and to enhance attention. See, Rowell, et al., J. Neurochem., Vol. 43, p. 1593 (1984); Hodges, et al., Bio. of Nic., Edit, by Lippiello, et al., p. 157 (1991); Sahakian, et al., Br. J. Psych., Vol. 154, p. 797 (1989); and U.S. Pat. No. 4,965,074 to Leeson.

It would be desirable to provide a method for treating neurodegenerative diseases, such as SDAT and PD, by administering a nicotinic compound to the patient suffering from such disease.

The present invention relates to compounds for the treatment of a neurodegenerative disease. The treatment involves treating a patient suffering from such disease (e.g., SDAT or PD) with an effective amount of a compound having a tropane functionality, such as a ferruginine compound or an anhydroecgonine compound.

The use of the compounds of the present invention provide benefits to the patient in that the compounds have the potential to (i) increase the number of nicotinic cholinergic receptors of the brain of the patient, (ii) exhibit neuroprotective effects, (iii) act as a pharmacological agonist to activate nicotinic receptors, and (iv) elicit neurotransmitter secretion.

The present invention relates to compounds for the treatment of neurodegenerative diseases, such as SDAT and PD. The method involves treating a patient with an effective amount of a new compound having the general formula: ##STR1## where Z represents --R or --OR; R and R' independently or individually represent H, or straight chain or branched alkyl (e.g., C.sub.1 to about C.sub.7, or other lower alkyl); and R" represents H, straight chain or branched alkyl (e.g., C.sub.1 to about C.sub.7, or other lower alkyl), or aryl (e.g., benzyl, phenyl and pyridyl; and substituted benzyl, phenyl and pyridyl). Although not preferred, the other carbon atoms of the tropane ring of the compound can include substituent groups other than hydrogen (e.g., one or more of the other carbon atoms of the tropane ring can include a lower alkyl substituent group).

Compounds useful according to the method of the present invention include ferruginine (R'=H, R"=CH.sub.3, Z=CH.sub.3), norferruginine (R'=H, R"=H, Z=CH.sub.3), anhydroecgonine (R'=H, R"=CH.sub.3, Z=OCH.sub.3), and noranhydroecgonine (R'=H, R"=H, Z=OCH.sub.3). See, Bick, Aust. J. Chem., Vol. 32, P. 2537 (1979); Davies, et al., J. Org. Chem., Vol. 56, p. 5696 (1991); Spivak, et al., Mol. Pharm., Vol. 23, p. 337 (1983); El-Imam, et al., Phytochem, Vol. 27, p. 2181 (1988); and Al-Yahya, et al , J. Chem. Soc., Vol. 1, p. 2130 (1979); which are incorporated herein by reference. Other compounds, for example, those were (i) R'=CH.sub.3, R"=CH.sub.3, Z=CH.sub.3 and (ii) R'=CH.sub.3, R"=H, Z=CH.sub.3, can be employed. Other derivatives of ferruginine and noranhydroecgonine can be employed and as such, other ferruginine and noranhydroecgonine compounds can be employed. The compounds can be employed as racemic mixtures or as enantiomers.

The manner in which the compounds are administered can vary. The compounds can be administered by inhalation; in the form of an aerosol either nasally or using delivery articles of the type set forth in U.S. Pat. No. 4,922,901 to Brooks, et al. and U.S. Pat. application Ser. No. 486,025, filed Feb. 27, 1990; orally (e.g., in liquid form within a solvent such as an aqueous liquid, or within a solid carrier); intravenously (e.g., within a saline solution); or transdermally (e.g., using a transdermal patch). Exemplary methods for administering such compounds will be apparent to the skilled artisan. Certain methods suitable for administering compounds useful according to the present invention are set forth in U.S. Pat. No. 4,965,074 to Leeson. The administration can be intermittent, or at a gradual, continuous, constant or controlled rate to a warm-blooded animal, such as a human being or other mammal.

The dose of the compound is that amount effective to treat the neurodegenerative disease from which the patient suffers. By "effective amount" or "effective dose" is meant that amount sufficient to pass across the blood-brain barrier of the patient, to bind to relevant receptor sites in the brain of the patient, and to elicit neuropharmacological effects (e.g., elicit neurotransmitter secretion, thus resulting in effective treatment of the disease). Treatment of a neurodegenerative disease involves a decrease of symptoms of the particular disease.

For human patients, the effective dose of typical compounds generally does not exceed about 150 .mu.g, often does not exceed about 100 .mu.ug, and frequently does not exceed about 50 .mu.g, per kg patient weight. For human patients, the effective dose of typical compounds generally is at least about 5 .mu.g, often is at least about 10 .mu.g, and frequently is at least about 25 .mu.g, per kg of patient weight. For human patients, the effective dose of typical compounds generally requires administering the compound in an amount of at least about 2.0, often at least about 1.0, and frequently at least about 0.1 mg/hr./patient. For human patients, the effective dose of typical compounds requires administering the compound in an amount which generally does not exceed about 10, often does not exceed about 5, and frequently does not exceed about 2.5 mg/hr./patient.

The compounds of the present invention useful according to the method of use have the ability to pass across the blood-brain barrier of the patient. As such, such compounds have the ability to enter the central nervous system of the patient. The log P values of typical compounds useful in carrying out the present invention generally are greater than 0, often are greater than about 0.1, and frequently are greater than about 0.5. The log P values of such typical compounds generally are less than about 3.0, often are less than about 2.5, and frequently are less than about 2.0. Log P values provide a measure of the ability of a compound to pass across a diffusion barrier, such as a biological membrane. See, Hansch, et al., J. Med. Chem., Vol. 11, p. 1 (1968).

The compounds useful according to the present invention have the ability to bind to, and hence cause activation of, nicotinic cholinergic receptors of the brain of the patient. As such, such compounds have the ability to act as nicotinic agonists. The receptor binding constants of typical compounds useful in carrying out the present invention generally exceed about 1 nM, often exceed about 200 nM, and frequently exceed about 500 nM. The receptor binding constants of such typical compounds of such typical compounds generally are less than about 10 .mu.M, often are less than about 7 .mu.M, and frequently are less than about 2 .mu.M. Receptor binding constants provide a measure of the ability of the compound to bind to half of the relevant receptor sites of brain cells of the patient. See, Cheng, et al., Biochem. Pharmacol., Vol. 22, pp. 2099-3108 (1973).

The compounds also have the ability to demonstrate a nicotinic function by effectively eliciting neurotransmitter secretion from nerve ending preparations (i.e., synaptosomes). As such, such compounds have the ability to cause relevant neurons to release or secrete acetylcholine, dopamine, and other neurotransmitters. Generally, typical compounds useful in carrying out the present invention provide for the secretion of dopamine in amounts of at least about 5 percent, often at least about 25 percent, and frequently at least about 50 percent, of that elicited by an equal molar amount of S(-)nicotine.

The following examples are provided in order to further illustrate various embodiments of the invention but should not be construed as limiting the scope thereof. Unless otherwise noted, all parts and percentages are by weight.

Mice (DBA strain) were maintained on a 12 hour light/dark cycle and were allowed free access to water and food supplied by Wayne Lab Blox, Madison, Wis. Animals used in the present studies were 60 to 90 days of age and weighed 20 to 25 g. Brain membrane preparations were obtained from pooled brain tissue of both males and females.

Mice were killed by cervical dislocation. Brains were removed and placed on an ice-cold platform. The cerebellum was removed and the remaining tissue was placed in 10 volumes (weight:volume) of ice-cold buffer (Krebs-Ringers HEPES:NaCl, 118 mM: KCl, 4.8 mM; CaCl.sub.2, 2.5 mM; MgSO.sub.4, 1.2 mM; HEPES, 20 mM; pH to 7.5 with NaOH) and homogenized with a glass-Teflon tissue grinder. The resulting homogenate was centrifuged at 18000.times.g for 20 min. and the resulting pellet was resuspended in 20 volumes of water. After 60 min. incubation at 4.degree. C., a new pellet was collected by centrifugation at 18000.times.g for 20 min. After resuspension in 10 volumes of buffer, a new final pellet was again collected by centrifugation at 18000.times.g for 20 min. Prior to each centrifugation step, the suspension was incubated at 37.degree. C. for 5 min. to promote hydrolysis of endogenous acetylcholine. The final pellet was overlayered with buffer and stored at - 70.degree. C. On the day of the assay, that pellet was thawed, resuspended in buffer and centrifuged at 18000.times.g for 20 min. The resulting pellet obtained was resuspended in buffer to a final concentration of approximately 5 mg protein/ml. Protein was determined by the method of Lowry, et al., J. Biol. Chem., Vol. 193, pp. 265-275 (1951), using bovine serum albumin as the standard.

The binding of L-[.sup.3 H] nicotine was measured using a modification of the method of Romano, et al., Science, Vol. 210, pp. 647-650 (1980) as described previously by Marks, et al., Mol. Pharmacol., Vol. 30, pp. 427-436 (1986). The binding of L-[.sup.3 H] nicotine was measured using a 2 hr. incubation at 4.degree. C. Incubations contained about 500 .mu.g of protein and were conducted in 12 mm.times.75 mm polypropylene test tubes in a final incubation volume of 250 .mu.l. The incubation buffer was Krebs-Ringers HEPES containing 200 mM TRIS buffer, pH 7.5. The binding reaction was terminated by filtration of the protein containing bound ligand onto glass fiber filter (Micro Filtration Systems) that had been soaked in buffer containing 0.5 percent polyethyleneimine. Filtration vacuum was -50 to -100 torr. Each filter was washed five times with 3 ml of ice-cold buffer. The filtration apparatus was cooled to 2.degree. C. before use and was kept cold through the filtration process. Nonspecific binding was determined by inclusion of 10 .mu.M nonradioactive nicotine in the incubations. The inhibition of L-[.sup.3 H] nicotine binding by test compounds was determined by including one of eight different concentrations of the test compound in the incubation. Inhibition profiles were measured using 10 nM L-[.sup.3 H] nicotine and IC.sub.50 values were estimated as the concentration of compound that inhibited 50 percent of specific L-[.sup.3 H] nicotine binding. Inhibition constants (Ki values) were calculated from the IC.sub.50 values using the method of Cheng, et al., Biochem. Pharmacol., Vol. 22, pp. 3099-3108 (1973). The Ki values for all compounds for which an inhibition constant less than 100 .mu.M was determined from the inhibition curves described above were also calculated independently using Dixon plots for inhibition measured using 2 nM, 8 nM and 20 nM concentrations of L-[.sup.3 H] nicotine used in all experiments was purified chromatographically by the method of Romm, et al., Life Sci., Vol. 46, pp. 935-943 (1990).

Lop P values (log octanol/water partition coefficient), which have been used to assess the relative abilities of compounds to pass across the blood-brain barrier, were calculated according to the methods described by Hansch, et al., J. Med. Chem., Vol. 11, p. 1 (1968).

Dopamine release was measured by preparing synaptosomes from the striatal area of rat brain generally obtained from Sprague-Dawley rats according to the procedures set forth by Nagy, et al., J. Neurochem., Vol. 43, pp. 1114-1123 (1984). Striata from 4 rats were homogenized in 2 ml of 0.32M sucrose buffered with 5 mM HEPES (pH 7.5), using a glass-teflon tissue grinder. The homogenate was diluted to 5 ml with additional homogenization solution and centrifuged at 1000.times.g for 10 min. This procedure was repeated on the new pellet and the resulting supernatant was centrifuged at 12,000.times.g for 20 min. A 3 layer discontinuous Percoll gradient consisting of 16 percent, 10 percent and 7.5 percent Percoll in HEPES-buffered sucrose was made with the final pellet dispersed in the top layer. After centrifugation at 15,000.times.g for 20 min., the synaptosomes were recovered above the 16 percent layer with a pasteur pipette, diluted with 8 ml of perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl.sub.2, 1.2 mM KH.sub.2 PO.sub.4, 1.2 mM MgSO.sub.4, 25 mM HEPES pH 7.4, 10 mM dextrose, 1 mM ascorbate, 0.01 mM pargyline), and centrifuged at 15,000.times.g for min. The new pellet was collected and re-suspended in perfusion buffer. The synaptosome suspension was incubated for 10 min. at 37.degree. C. Then [.sup.3 H]-dopamine (Amersham, 40-60 ci/mmol) was added to the suspension to give a final concentration of 0.1 .mu.M in suspension, and the suspension was incubated for another 5 min. Using this method, 30 to 90 percent of the dopamine was taken up into the synaptosomes, as determined by scintillation counting following filtration through glass fiber filters soaked with 0.5 percent polyethyleneimine. A continuous perfusion system was used to monitor release following exposure to each ligand (i.e., ferruginine, norferruginine, anhydroecgonine and noranhydroecgonine). Synaptosomes were loaded onto glass fiber filters (Gelman type A/E). Perfusion buffer was dripped onto the filters (0.2-0.3 ml/min.) and pulled through the filters with a peristaltic pump. Synaptosomes were washed with perfusion buffer for a minimum of 20 min. before addition of the ligand. After the addition of a 0.2 ml of a 20 .mu.M solution of ligand, the perfusate was collected into scintillation vials at 1 min. intervals and the dopamine released was quantified by scintillation counting. Peaks of radioactivity released above background were summed and the average basal release during that time was subtracted from the total. Release was expressed as a percentage of release obtained with an equal concentration of S(-)nicotine.

Date regarding octanol-water partition coefficients, binding constants and neurotransmitter secretion capability for the ligands evaluated are set forth in Table I.

The date in Table I indicate that the compounds have the capability of passing the blood-brain barrier, binding to high affinity nicotinic receptors, and eliciting neurotransmitter secretion. Thus, the data indicate that such compounds have the capability of being useful in treating neurodegenerative diseases.

1,8-diazabicyclo[5.4.0] undec-7-ene (6.09 g, 40.0 mmol) was added to a stirred solution of mesityl oxide (1.96 g, 20 mmol) and p-acetamidobenzylsulfonyl azide (9.6 g, 40 mmol) in 50 ml acetonitrile at 0.degree. C. After stirring for about 12 hours, about 50 ml of a saturated aqueous ammonium chloride solution was added to the mixture and the resulting mixture was extracted five times using pentane. Purification on silica gel column chromatography (using 5 parts diethyl ether and 95 parts petroleum ether) afforded an orange liquid, which is 3-diazo-4-methyl pent-4-en-2-one.

4-dimethylaminopyridine (2.38 g, 0.019 mol) and ditert-butyl dicarbonate (51.07 g, 0.23 mol) were added to a stirred solution of pyrrole (16.77 g, 0.25 mol) in 25 ml dry acetonitrile at room temperature for about 24 hours. The mixture was concentrated using a rotary evaporator under reduced pressure and the residue were purified on silica gel column chromatography (using petroleum ether) to give a colorless liquid, which is N-tert-butyloxycarbonyl pyrrole.

A solution of 3-dizao-4-methyl pent-4-ene-2-one (2.30 g, 18.5 mmol) in hexane (25 mL) was added over 3 hours by means of a syringe pump to a stirred solution of N-tert-butyloxycarbonyl pyrrole (9.18 g, 55 mmol) and rhodium (II) octanoate (0.07 g, 0.09 mmol) in refluxing hexane (10 mL) under an argon atmosphere. The mixture was refluxed for a further 1 hour period, and solvent was then removed using a rotary evaporator under reduced pressure. Purification of the product by silica gel column chromatography (using 2 parts diethyl ether and 8 parts petroleum ether, and 4 parts diethyl ether and 6 parts petroleum ether) yielded 2-acetyl-3-methyl-8-(tert-butyloxycarbonyl)-8-azabicyclo [3.2.1]oct-2,6-diene, which is an orange liquid.

A solution of 2-acetyl-3-methyl-8-(tert-butyloxycarbonyl)-8-azabicyclo [3.2.1]oct-2,6-diene (0.74 g, 2.81 mmol) and RhCl(PPh.sub.3).sub.3 (0.026 g, 0.028 mmol) in 50 ml ethanol was pressurized with 45 psi hydrogen gas and shaken for about 12 hours. The solvent was then evaporated and the residue was purified by silica gel column chromatography (using 3 parts diethyl ether and 7 parts petroleum ether) to give 2-acetyl-3- methyl-8-(tert-butyloxycarbonyl)-8-azabicyclo [3.2.1]oct-2-en.

A solution of trifluoroacetic acid (18.8 mmol, 2.13 mL) and 2-Acetyl-3-methyl-8-(tert-butyloxycarbonyl)-8-azabicyclo [3.2.1]oct-2-en (0.49 g, 1.88 mmol) was stirred at room temperature for about 0.5 hour. The solution was concentrated three times under reduced pressure after the addition of hexane to remove excess trifluoroacetic acid. A concentrated solution of sodium carbonate was added to the residue to form a basic solution. The solution was extracted with methylene chloride, dried using sodium sulfate and purified by bulb-to-bulb distillation (80.degree. C. (1.0 mmHg)) to give 2-acetyl-3-methyl-8-azabicyclo [3.2.1]oct-2-ene.

The compound was evaluated as described in Example 1, and was found to provide dopamine release.

To a stirred mixture of 2-acetyl-3-methyl-8-azabicyclo [3.2.1]oct-2-ene (1,2083 g, 0.0073 mol) and aqueous formaldehyde (2.7 ml, 0.036 mol, 37 percent) in 15 ml acetonitrile was added sodium cyanoborohydride (0.7352 g, 0.0117 mol). After stirring for 15 minutes, the reaction was made acidic by addition of 15 ml glacial acetic acid over a 45 minute period. The solution was made basic by the addition of an aqueous solution of ammonium hydroxide. The basic solution was extracted 3 times with ethyl acetate, the organic layer was then dried using sodium sulfate and concentrated under reduced pressure. Purification on silica gel column chromatography using 9 parts diethyl ether and 1 part triethylamine followed by bulb-to-bulb distillation (80.degree. C. 0.4 mmHg)) to give 2-acetyl-3,8-dimethyl-8-azabicyclo[3.2.1]oct-2-ene as a brownish liquid.