Title: Use of nicotinic analogs for treatment of degenerative diseases of the nervous system
United States Patent: 6,630,491
Issued: October 7, 2003
Inventors: Zoltewicz; John A (Gainesville, FL); Kem; William R. (Gainesville, FL); Meyer; Edwin M. (Gainesville, FL)
Assignee: University of Florida
Appl. No.: 473667
Filed: June 7, 1995
Method of using anabaseine, DMAB-anabaseine, and anabaseine for stimulating brain cholinergic transmission and a method of making anabaseine.
SUMMARY OF THE INVENTION
The present invention arose out of the discovery that anabaseine, DMAB-anabaseine, and anabaseine could be used to improve overall brain neurocortical cholinergic activity. The interaction of these agents with nicotinic receptors has decreased levels of toxicity as compared to nicotine.
In the absence of long term studies for the clinical effectiveness of nicotine or its analogs for degenerative neural diseases, such as Alzheimer's or Parkinson's disease, the present invention has developed the nucleus basalis lesioned rat as a model for trans-synaptic neuronal degeneration caused by the loss of ascending neurons. Bilateral lesions of cholinergic neurons in the nucleus basalis were induced with ibotenic acid to cause long-term, essentially irreversible deficits in neocortical choline acetyltransferase activity, an enzyme selective for cholinergic neurons. However, passive avoidance behavior, a learning and memory paradigm particularly sensitive to nucleus basalis-lesions, reportedly recovers to normal levels sometime between 2-8 months post-lesioning.
Various other aspects and attendant advantages of the present invention will be more fully appreciated from an understanding of the following detailed description in combination with the accompanying examples.
DETAILED DESCRIPTION OF THE INVENTION
Anabaseine, 2-(3-pyridyl)-3,4,5,6-tetrahydropyridine, occurs in certain marine worms, which use the substance to paralyze prey and deter predators (Kem, et al., Toxicon, 9:23, 1971). Anabaseine is a potent activator of vertebrate neuromuscular nicotinic acetylcholine receptors (Kem, Amer.Zoologist, 25:99, 1985). Both nicotine and anabaseine possess a non-aromatic ring attached to the 3-position of a pyridyl ring. Anabaseine's non-aromatic tetrahydropyridine ring imine double bond is conjugated with .pi.-electrons of the 3-pyridyl ring. The imine nitrogen is a much weaker base than the pyrrolidinyl nitrogen of nicotine (Yamamoto, et al., Agr.Biol.Chem., 26:709, 1962). Considerable evidence (Barlow and Hamilton, BritJ.Pharmacol., 18:543, 1962) exists that the non-aromatic ring nitrogen of nicotine must be protonated (cationic) in order to avidly bind to the skeletal muscle nicotinic receptor and activate the opening of its channel. At physiological pH, anabaseine also exists in a hydrolyzed ammonium-ketone form as well as the cyclic imine (unionized) and cyclic iminium (monocationic) forms. The inventors have determined that anabaseine acts as a central nicotinic receptor agonist primarily through its cyclic iminium form.
The synthesis of anabaseine was first reported in 1936 (Spath, et a/., Chem. Ber., 69:1082, 1936). Unfortunately, this technique utilized an elaborate isolation scheme involving a distillation and preparation of a picrate. Medicinally, the picrate is of no useful value, in fact, since picrate is toxic and potentially explosive, its presence precludes the direct use of anabaseine in physiological systems when produced by this technique.
The first analog of anabaseine to be synthesized was 3-[p-(dimethylamino) benzylidene]-3,4,5,6-tetrahydro-2,3'-bipyridine, also termed DMAB-anabaseine (Kem, et al., Toxicon, 9:23, 1971). This compound, resulting from the electrophilic attack of Ehrlich's reagent upon anabaseine, is a stable orange-colored compound.
The present invention provides an improved method for synthesizing anabaseine which overcomes the problems associated with prior disclosed techniques for its synthesis.
The first part of the improved synthesis of anabaseine, the joining of an activated derive of nicotinic acid and a modified 2-piperidone, is performed. using a mixed Claisen condensation. The second pan of the synthesis involves the hydrolysis and decarboxylation at the condensed product The overall reaction sequence is shown below. ##STR1##
In the scheme presented herein, certain protecting and activating groups are specifically illustrated. However, one skilled in the art will recognize that other protecting and activating groups could have been used. For example, a variety of amino protecting group can be used to protect the nitrogen of 2-piperidone (1). Representative amino protecting groups are C1 -C4 alkanoyl, benzyl, and trialkylsilyl derivatives such as trimethylsilyl and butyldimethylsilyl. The preferred amino protecting group is trimethylsilyl (TMS). The TMS-protected 2-piperidone (2) is prepared by deprotonation and subsequent reaction with trimethylchlorosilane. Typical silylation conditions are the use of lithium diisopropylamide (LDA) in an inert solvent such as tetrahedrofuran (THF) at -70oC. For each one mole of 2-piperidone, at least one mole of LDA, preferably 11/2 moles, should be used to ensure complete silylation. While maintaining the temperature at -70oC., at least one molar equivalent of TMS is combined with the LDA-added reaction mixture. Normally, silylation is complete within a few hours by raising the reaction temperature to ambient temperature.
The protected 2-piperidone (2) is next enolyzed to an enolate by base. Conveniently, this enolization can be conducted by simply adding additional LDA to the reaction mixture containing compound (2). Although this is a preferred process, other suitable bases which can be employed include metal amides such as NaNH2 or KNH2, metal hydrides such as NaH or KH, and metals such as Na or K. In practice, the reaction mixture is cooled to -70oC., at which point at least one molar equivalent of LDA is added. Enolization is usually complete within an hour, and the resultant amide enolate (3) can be directly used in the next condensation reaction.
The key Claisen condensation between a 2-piperidone enolate and a nicotinic acid derivative can be carried out, e.g., by combining the lithium amide enolate (3) in an inert solvent such as THF with about one molar equivalent of ethyl nicotinate. Reaction temperature can be varied, but it is preferred to start the condensation at -70oC. and to allow the temperature to warm up to ambient temperature. Reaction requires a few hours to 24 hours until its completion.
Although an ethyl ester form of nicotinic acid (4) has been illustrated hereinabove, activation of the carboxylic group to expedite condensation can be achieved by other activating groups known in the art. Especially useful in the herein described condensation are anhydrides, particularly cyclic anhydrides, acid halides, and activated esters such as those derived from N-hydroxysuccimide and N-hydroxypthalimide. Alkyl esters of up to C5 other than ethyl ester can also be used.
The condensed product (5) is isolated after removal of TMS group by hydrolysis. The product (5) is normally subjected to hydrolysis and decarboxylation without further purification.
Conversion of compound (5) to the final anabaseine (6) is accomplished by first hydrolyzing compound (5) with a strong acid such as concentrated hydrochloric acid; and by second decarboxylating the intermediate .beta.-keto acid (not shown in the above scheme). Both hydrolysis and decarboxylation steps are conveniently conducted in one-pot in the presence of concentrated hydrochloric acid at an elevated temperature, e.g., under reflux. Anabaseine (6) is thus obtained as its dihydrochloride.
Anabaseine is commercially available from Aldrich Chemical Co. Alternative sources of anabaseine are reduction of anabaseine.
Reduction of anabaseine to anabaseine can be achieved by several ways: (1) Hydrogeneration with hydrogen over Pd/C, as described in E Spath et al., Chem. Ber. 69 1082 (1936); (2) Borohydride reduction with either NaBH3 CN or with NaBH4, as described in E. Leate, J. Org. Chem. 44 165 (1979); and (3) Reduction with hot formic acid.
Anabaseine can exist as an optically active form. The present invention embraces such optically pure anabaseine, the pure enantiomers thereof, and the racemate thereof.
Anabaseine and anabaseine in their free base form will form acid addition salts, and these acid addition salts are non-toxic and pharmaceutically acceptable for therapeutic use. The acid addition salts are prepared by standard methods, for example by combining a solution of anabaseine or anabaseine (base) in a sutable solvent (e.g., water, ethyl acetate, acetone, methanol, ethanol or butanol) with a solution containing a stoichiometric equivalent of the appropriate acid. If the salt precipitates, it is recovered by filtration. Alternatively, it can be recovered by evaporation of the solvent or, in the case of aqueous solutions, by dyophilization. Of particular value are the sulfate, hydrochloride, hydrobromide, nitrate, phosphate, citrate, tartrate, pamoate, perchlorate, sulfosalicylate, benzene sulfonate, toluene sulfonate and 2-napnthalene sulfonate salts. These acid addition salts are considered to be within the scope and ourview of this invention.
As a result of using the above method for the synthesis of anabaseine: (1) the chemistry is cleaner and simpler; (2) higher yields of anabaseine are obtained; and (3) picric acid is not present, such that a more directly pharmacologically useful form of anabaseine is produced.
The term "therapeutically effective" means that the amount of nicotinic receptor agent used is of sufficient quantity to increase brain cholinergic transmission. The dosage ranges for the administration of the agent of the invention are those large enough to produce the desired effect in which the nicotinic receptors show some degree of stimulation. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary from about 1 .mu.g/kg/dose to about 1000 .mu.g/kg/dose, preferably from about 10 .mu.g/kg/dose to about 500 .mu.g/kg/dose, most preferably from about 30 .mu.g/kg/dose to about 100 .mu.g/kg/dose in one or more dose administrations daily, for one or several days. Alternatively, the dosage can be administered indefinitely in order to prevent a recurrence of cognitive function loss, for example, by administration of the agent in a slow-release form.
The nicotinic receptor agent of the invention can be administered enterally, parenterally, or by gradual perfusion over time. The nicotinic receptor agent of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, or orally.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the nicotinic receptor agent, together with a suitable amount of a carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or adsorb the nicotinic receptor agent. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the nicotinic receptor agent into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating the nicotinic receptor agent into these polymeric particles, it is possible to entrap the nicotinic receptor agent in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such teachings are disclosed in Remington's Pharmaceutical Sciences (17th Ed., A. Oslo, ed., Mack, Easton, Pa., 1985).
The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the nicotinic receptor agent of the invention, the medicament being used for therapy to stimulate brain cholinergic transmission.
Claim 1 of 9 Claims
What is claimed is:
1. A pharmaceutical learning and memory improving composition comprising brain cholinergic neurocortical stimulating amounts of anabaseine or DMAB-anabaseine together with a pharmaceutically inert carrier.