NO304522B1 - Anology method for the preparation of therapeutically active heterocyclic compounds - Google Patents

Description

The present invention relates to analog methods for the production of therapeutically active compounds with general formulas I and II

including enantiomers, racemates and acid addition and quaternary salts thereof.

These compounds are potentially useful for the treatment of diseases in the central and peripheral nervous systems using such spiro compounds or pharmaceutical compositions.

New spiro-quinuclidine compounds, in which the oxathiolane rings were spiro-connected with quinuclidine rings, were described e.g. in European Patent Application No. 0205247 A2, published December 17, 1986, and in US Patent Nos. 4,855,290 (issued August 8, 1989), 4,981,858 (issued January 1, 1991), 4,900,830 (issued February 13, 1990) and 4,876,260 (issued Oct. 24, 1989). These new compounds were found to possess central nervous system activity. The biological activity of the compound 2-methylspiro(1,3-oxathiolane-5',3)quinuclidine, which exists as geometric cis- and trans-isomers, depends on whether the 2-methyl group is located on the same side of the oxatiolane ring as the quinuclidine ring nitrogen atom (cis) or on the other side of the quinuclidine ring nitrogen atom (trans), was particularly extensively investigated, and it was found on the basis of preclinical tests that the cis compound (code no. AF102B) was particularly promising for the control of senile dementia of the Alzheimer type (SDAT). It is also of interest that each of the cis and trans isomers can be optically resolved, and the biological activity of the optical isomers was also investigated in a number of cases.

A main purpose according to the invention is to prepare new 4- and 5-spiro-1,3-oxazoline and -1,3-thiazoline compounds, which are different in relation to the above-mentioned spirooxathiolane/quinuclidine compounds.

The present invention relates to analog methods for the production of therapeutically active compounds with general formulas (I) and (II)

including enantiomers, racemates and acid addition and quaternary salts thereof, wherein formula (I) Q is (CH2)or C(CH3)2 attached to one of the 1',4' or 1',5' positions of the six-membered the ring, where m is 1, 2 or 3, and in formula (II) Q is two hydrogen atoms, (CH 2 ) or C(CH 3 ) 2 wherein m is 1, 2 or 3 and n and p are each independently 0, 1, 2 or 3, with the assumption that n + p = 1-3, R° is hydrogen, methyl or hydroxyl,

the part

denotes any of the following: segment with a 4-spiro substituted 1,3-oxazoline, segment of a 5-spiro substituted 1,3-oxazoline, segment of a 4-spiro substituted 1,3-thiazoline or segment of a 5-spiro substituted 1,3-thiazoline; R is selected from hydrogen, NH2, NH-C1-6-alkyl, N(C1-6-alkyl)2, <C>1-6-alkyl, <C>2-6-alkenyl, C2-6_alkynyl, C3-7-cycloalkyl, C1-6-alkyl substituted with 1-6 halogen atoms, hydroxy-C 1-4 alkyl, C 1-4 alkoxy, C 1-6 thio, C 1-6 alkoxy-C 1-4 alkyl, carboxy-C 1-4 . 3-alkyl, (C 1-3 ,-Alkoxy )-carbonyl-<C>1-6-alkyl, amino-C 1-3-Alkyl, mono-(C 1-3-alkyl )-amino-C 1-6-alkyl), di- (C 1 -alkyl )amino-C 1 -alkyl, 2-oxo-pyrrolidin-1-yl-methyl, aryl, diarylmethylol and C 1 -alkyl substituted with one or two aryl groups, R' being independently selected from the group from which R is selected and C 1 -6 alkanoyl and arylcarbonyl; and aryl denotes unsubstituted phenyl or phenyl substituted with 1-3 substituents selected from halogen, C 1-6 alkyl, C 1-6 alkoxy and CF 3 , subject to the conditions (i), (ii), (iii), (iv) and ( v), i.e.: (i) in formula II, when Q is two hydrogen atoms, n=p=l, R^ is hydrogen, R is NH2 and R' is methyl, then the moiety is different from

the segment of a 5-spiro substituted 1-3-oxazoline,

(ii) in formula II, when Q is two hydrogen atoms, n=p=l, Ro is hydrogen and R is phenyl, R' is not tertiary butyl, (iii) in formula II, when 0 is two hydrogen atoms, n=p =l, R° is hydrogen, R is p-chlorophenyl and R' is tertbutyl then the moiety

neither the segment of a 4-spiro substituted 1,3-oxazoline nor

segment of a 5-spiro substituted

1,3-oxazoline,

(iv) in formula II, when Q is two hydrogen atoms, n = p = 1, R° is hydrogen, R is 3,5-dichlorophenyl and R' is tertiary butyl, then the moiety

different from

the segment of a 5-spiro substituted 1,3-oxazoline, and

(v) in formula I, Q is not CH 2 in the 1',4' position, characterized in that to prepare a compound of structural formula I a compound of general formula

wherein R° and Q are as defined above, subjected to oxazoline or thiazoline cyclization reaction at the carbonyl group or epoxide group of general formula (Ia) or (Ib) to produce a compound of general formula (I) wherein X, R and Y are as defined above; and wherein, to produce a compound having the structural formula II, a compound having the general formula wherein R°, R<1>, 0, n and p are as defined above is subjected to the oxazoline or thiazoline cyclization reaction at the carbonyl group or the epoxide group of general formula (Ila) or (Ilb) to prepare a compound of formula (II) wherein X, R and Y are as defined above. The compounds of formulas (I) and (II) as defined above are novel. The compound of formula (II) where 0 is two hydrogen atoms, n = p = 1, R" is hydrogen, R is NH 2 and R' is methyl, and is one of a series of compounds tested by Harnden and Rasmussen (J. Med. Chem. 1970, 23: 305-308) for CNS stimulant activity, but was not one of the five compounds selected to be the most active of the series.The compounds of formula (II) where Q is two hydrogen atoms, n = p = 1, R " is hydrogen, R is phenyl and R' is tertiary butyl are known, and so are those where Q, n, p, R" and R' have these values and either R is p-chlorophenyl, while

or

R is 3,5-dichlorophenyl, while

are known (see Jones et al, J. Chem. Soc. (B) 1308-1315, 1971), but no biological activity was reported for these compounds. European Patent Application No. 0337547A1 corresponding to NO A 891471, published October 18, 1989, describes compounds useful for the treatment of psychotic disorders, presenile and senile dementia and other physiological conditions, having the following formula: where the dashed line represents an optical bond in one of the two positions, A represents a group of formula (wherein R<*>and R^ independently are hydrogen or specified substituents, V is N, or

and W is 0, S or NH which can be

substituted with specified substituents), Q is the residue of an azacyclic or azabicyclic system, and of X, Y and Z two are independently selected from 0, S and N and the other is C, or Y can be C=0. This reference does not describe the results of any biological testing, but claims that certain of the disclosed compounds (not specified) demonstrate an affinity for 5-HT3 receptors. This reference does not describe biological activity for selecting any particular of the many possible structures represented by permutations of the moieties represented by the symbols, Q, X, Y and Z set forth above, in combination with the essentially bicyclic residue

A.

Examples of the bridged ring in formula (I) are: 1-azabicyclo[2,2,1]neptane, 7,7-dimethyl-l-azabicyclo[2,2,1]-heptane, l-azabicyclo[2,2 ,2]octane (quinuclidine), 1-azabi-cyclo[3,3,2]-nonane, l-azabicyclo[3,1,l]heptane, 7,7-dimethyl-l-azabicyclo- [3 , 1 , l]heptane, l-azabicyclo[3,2,l]octane, and 1-azabicyclo[3,3,l]nonane.

Examples of the ring spiro-linked to the oxazoline or thiazoline ring in formula (II) are, when Q is two hydrogen atoms, pyrrolidine, piperidine and hexamethyleneimine; and when Q is different from two hydrogen atoms, examples are: 5-azabicyclo[2,1,l]hexane, 6,6-dimethyl-5-azabicyclo[2,1,l]hexane, 7-azabickly[2, 2,1]heptane, 8-azabicyclo[3,2,1]octane, 6-azabicyclo[3,1,1]heptane, 7,7,-dimethyl-6-azabicyclo[3,1,1]-heptane, 8-azabicyclo[3,2,1]octane, 9-azabicyclo[3,3,1]nonane,

7-azabicyclo[4,1,1]octane, 8,8-dimethyl-7-azabicyclo[4,1,1]-5 octane, 9-azabicyclo[4,2,1]nonane, and 10-azabicyclo[4 ,3,1] dean.

As indicated by the symbol E" in formulas (I) and (II) which can

any of these ring systems be ring-substituted with i"methyl or OH.

The spiro compounds produced in the present invention have central and peripheral nervous system activity.

!5 In a currently preferred group of compounds of formula (I), R can be hydrogen, NH2, NH-C-j-alkyl, N(C-j-6-alkyl)2,

C-1-6 alkyl or aryl, while R' can be hydrogen or methyl. In a currently preferred group of compounds of formula (II), R can be hydrogen, NH-C1-6-alkyl, N(C-j-6-alkyl )2,<20>C]-6-alkyl or aryl, while R' can be hydrogen or methyl. Examples of the present compound are: 2-aminospiro(1,3-oxazoline-5,3')quinuclidine,

2-methylspiro(1,3-oxazoline-5,3')quinuclidine,

25 2-ethylspiro(1,3-oxazoline-5,3<*>)quinuclidine,

2-phenylspiro(1,3-oxazoline-5,3')quinuclidine,

1-methylpiperidine-4-spiro-4'-(2'-methyl-1',3'-oxazoline),

1- methylpiperidine-4-spiro-4'-(2'-ethyl-1',3'-oxazoline),

2- methylspiro(1,3-oxazoline-5,4')-1'-methylpiperidine,

50 2-methylspiro(1,3-thiazoline-5,4•)-1'-methylpiperidine,

2-ethylspiro(1,3-oxazoline-5,4')-1'-methylpiperidine,

including enantiomers, racemates and acid addition and quaternary salts thereof.

J5 Preparation of the oxazoline compounds according to formula (I) The above-mentioned spiro compounds of formula (I) can be prepared by a reaction which brings about the formation of the oxazoline or the tlazolin ring. To give an example, when Q = (CH 2 ) and n = 2, suitable starting materials for such cyclization reactions can be prepared according to reaction schemes A1 and B1 below.

It is noted that in Scheme B1 the reaction between sodium azide and the epoxide of 3-methylenequinuclidine in an aqueous solution results in the formation of a single product, 3-azidomethyl-quinuclidin-3-ol; addition to the latter of wet active Raney Nickel until the evolution of nitrogen ceased resulted in the formation of the desired compound. The main advantages of the approximation according to form Bl are: a) It is not necessary to isolate any intermediate products and b) there is no need for a hydrogenation apparatus because the hydrogen content of the catalyst is sufficient.

It has now surprisingly been discovered that the 3-aminomethyl product of any of these reaction schemes can readily be condensed with a carboxylic acid RCOOH to provide the spiro products, whereas previously see EP A2 350118 and US-PS 3,792,053 it was believed that the formation of the oxazoline ring by condensation of the amino alcohol and the carboxylic acid would only proceed when the amino alcohol is completely substituted on the carbon atom where the NH2~ group is attached (see "Oxazolines, their Preparation, Reactions and Applications", J.A. Frunp, Chem. Rev. 71, 483- 505 (1971)]. The corresponding imidate RC(:NH)-O-alkyl may alternatively be used in place of the carboxylic acid RCOOH. The reactions providing compounds of formula I when the bridged ring is, for example, quinuclidine, may be represented as follows:

In order to obtain the spiro-quinuclidine compound, for example when n = 2 and R = NH2, it is, on the other hand, preferred to react cyanogen bromide with 3-aminomethylquinuclidin-3-ol.

It is to be noted that while the foregoing methods are effective for preparing the compounds where X is 0 and Y is N, it is to use similar methods for the preparation of corresponding sub-group (a) compounds prepared according to the invention where X is N and Y is 0, a suitable starting material would be (where, for example, the ring spiro-linked to oxazoline is quinuclidine) 3-amino-3-hydroxy-methylquinuclidine. This starting material can e.g. be prepared according to form Dl:

Preparation of the oxazoline compounds of formula (II) The above-mentioned spiro compounds of formula (II) can be prepared by reacting substantially substituted compounds containing the saturated nitrogen-only ring system with a reactant which will achieve the formation of the oxazoline ring. A suitable starting material is thus, for example, 4-aminomethyl-1-methylpiperidin-4-ol, which can be prepared either from 4-nitromethyl-1-methylpiperidin-4-ol, e.g. as the HCl salt, as in Scheme A2, or from 4-azidomethyl-1-methylpiperidin-4-ol as in Scheme B2.

It is noted that in Scheme B2 the reaction of sodium azide with the epoxide of 4-methylene-l-methylpiperidine in aqueous solution results in the formation of a single product, 4-azidomethyl-l-methylpiperidin-4-ol, while addition to the latter of wet active Raney Nickel until the evolution of nitrogen ceases results in the formation of the desired compound. The main advantages of the form B2 approach have been described in connection with form Bl above.

It has now surprisingly been discovered that the 3-aminomethyl product of any of these reaction schemes can readily be condensed with a carboxylic acid RCOOH to provide the spiro products, whereas it was previously believed that the formation of the oxazoline ring by condensation of amino alcohol and carboxylic acid only would proceed smoothly when the amino alcohol is fully substituted on the carbon atom to which the NH2~ group is attached (see "Oxazolines, their Preparation, Reactions and Applications", J.A. Frunp, Chem. Rev. 71, 483-505 (1971)). The corresponding imidate RC(:NH)-O-alkyl can alternatively be used instead of the carboxylic acid RCOOH.

Reactions affording compounds of formula II may be illustrated (eg in the case of spirooxazoline/piperidines) as follows:

In order to obtain the spiro compounds when R = NH2, on the other hand, it is preferred to react cyanogen bromide with e.g. 4-aminomethyl-1-methylpiperidin-4-ol.

It is noted that while the foregoing methods will be effective in preparing the compounds prepared according to

the invention there

will to obtain similar methods for the preparation of the compounds there

a suitable starting material being (illustratively, when the ring spiro-linked to the oxazoline is piperidine) 4-amino-4-hydroxy-methyl-1-methylpiperidine. This starting material and the final product can, for example, be produced according to form D2 or

E:

Preparation of oxazoline and the thiazoline compounds (I) & (II) these compounds and the oxazoline analogues can be prepared by the following schemes F and G, e.g. when the ring spiro connected to thiazoline or oxazolines is piperidine or quinuclidine:

Further methods include preparing the corresponding epoxide or thioepoxide and reacting it with an appropriate nitrile, as in Scheme H, where the ring spiro connected to the oxazoline or the thiazoline ring is, for example, piperidine in quinuclidine.

The compounds of formula (I) and (II), whether they in themselves form an embodiment according to the invention, or included in the pharmaceutical compositions according to the invention, or used in the methods according to the invention, include addition and quaternary salts of these compounds. By way of example, acid addition salts for pharmaceutical use include those formed from such acids as hydrochloric acid, hydrobromic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, malic acid, acetic acid, citric acid, tartaric acid, dibenzoyl D- and L-tartaric acid, ditoluene D- and L-tartaric acid , salicylic acid, carbonic acid, aspartic acid and glutamic acid. In those cases where the spiro compounds according to the invention are optically active, the racemates can be dissolved by using optically active acids such as dibenzoyl-L- or D-tartaric acid, ditolyl-L- or D-tartaric acid.

At least the spiro compounds according to the present invention where R is methyl are centrally active muscarinic agonists. Due to their pharmacological properties, these compounds can activate central cholinergic functions under conditions where the cholinergic system is hypofunctional.

The spiro compounds prepared according to the invention are generally potentially useful for the treatment of presenile and senile dementia, senile dementia of the Alzheimer type (SDAT), atypical Alzheimer's disease (Perry et al, Advances in Neurology, eds. R.J. Wurtman et al., 51:41 , 1990), combined multi-infarct dementia and Alzheimer's disease, age-associated memory impairment (AAMI), acute confusional disorders, emotional and attention disorders, mania, tardive dyskinesia, hyperkinesia, mixed Alzheimer's and Parkinson's disease, aphasia, hallucinatory states, post encephalitic amnesic syndrome, alcohol withdrawal symptoms, Huntington's chorea, Pick's disease, Friedrick's ataxia, Gilles de la Tourette's disease and Down syndrome, because all of these disease states are disorders in which a central cholinergic hypofunction has been implicated at least to some extent . The spiro compounds prepared according to this invention are also potential analgesic agents and can therefore be useful for the treatment of several painful conditions such as rheumatism, arthritis and terminal diseases. The spiro compounds prepared according to the invention where R is methyl are of particular potential value for the treatment of SDAT and related disorders.

The spiro compounds prepared according to the present invention can be used in combination with the acetylcholinesterase inhibitors such as physostigmine or tetrahydroaminoacridine; in combination with acetylcholine precursors such as choline or lecithin; in addition to "neurotrophic" drugs such as piracetam, aniracetam, oxiracetam, or pramiracetam; in addition to compounds that react with Ca<2+>^ channels such as 4-aminopyridine or 3,4-diaminopyridine; or in addition to peptides that may have modulating effects on acetylcholine release, such as somatostatin; in combination with a peripheral antimuscarinic agent (such as pirenzepine, N-methylatropine, N-butylscopolamine, propantheline methantheline, glycopyrrolate or tropenzilium) to counteract peripheral adverse effects that may be expected at high doses, such as salivation, diarrhoea, gastric secretion or vomiting , or in combination with transdermal scopolamine such as Scopoderm(R) to counteract nausea and/or vomiting; in combination with antidepressants such as nortriptyline, amitriptyline, imipramine, minaprine to alleviate both cognitive disturbances and depressive symptoms sometimes associated with SDAT, AAMI, mixed SDAT/Parkinson's disease (PD); in combination with M2-antimuscarinic drugs such as secoverin, AFDX-116 (cf. Hammer et al, Life Sci. 38: 1653, 1986) to counteract peripheral negative side effects that may be expected at high doses of the compounds, to counteract inhibitory actions of such agonists at central inhibitory M2-type presynaptic and postsynaptic receptors and to potentiate the release of acetylcholine via inhibition of M2-type inhibitory autoreceptors at intact terminals; in combination with nicotinic agonists such as nicotine to stimulate both nicotinic and muscarinic receptors in the brain; in combination with an adrenergic agonist (clonidine or quanfamicin) to alleviate both cognitive and other disturbances associated with a mixed cholinergic-noradr-energetic deficiency in SDAT; in combination with inhibitors of the neuronal zerotin reuptake such as alaproclat, zimelidine to alleviate both cognitive and other emotional functions in SDAT; in combination with monoamine oxidase B inhibitors such as deprenyl to alleviate both cognitive and other motor disturbances associated with mixed conditions such as SDAT/PD; in combination with nerve growth factor (NGF, which is either administered using a nasal spray or intracerebroventricular).

The spiro compounds prepared according to the present invention, with or without the above-mentioned other active compounds, can for example be administered by injection in a suitable diluent or carrier, per os, rectally in the form of suppositories, by insufflation or nasal spray, by infusion or transdermally in a suitable carrier with or without physostigmine or tetrahydroaminoacridine, for example using the device disclosed in US Patent No. 2163347 (issued November 29, 1988).

The present spiro compounds, especially where R = methyl, are also potentially useful for the treatment of disorders requiring the use of a long-lasting cholinergic agent with weak local activity. Such an agent is needed in disorders such as glaucoma, due to the fact that the compound is not destroyed by the enzyme that activates choline, i.e. acetyl- and butyryl-cholinesterase, and may also be used for the treatment of peripheral cholinergic disorders such as myasthenia gravis , urinary bladder disorders, Addis disease and Eaton-Lambert disease. These compounds can also be used in disorders where cholinergic underactivity is induced by the drugs.

It appears that the present spiro-compounds, especially where R is C 3-4 -alkyl or aryl, are anticholinergic agents and may potentially be used for the treatment of disorders caused by cholinergic hyperfunction, whether spontaneous or drug-induced. These compounds can be used for the treatment of various diseases, such as PD, pseudo-PD, mixed AD/PD, primary dystonia, spasmodic torticollis, cranial dystonia, depression, motion sickness, akathisia (after neuroleptic withdrawal), central hypertension, human head injury , mixed tardive dyskinesia and PD, manic-depression, as adjuncts in surgery instead of atropine, scopolamine, etc., in intoxication caused by an excess of acetylcholine as inhibition of acetylcholinesterase. These can also be used in ophthalmology where neither prolonged nor short-term mydriasis is necessary.

Present spiro-compounds can also potentially be used for the treatment of the disease characterized by an excess of peripheral-like activity such as asthma, chronic obstructive pulmonary disease, peptic ulcer. For these peripheral disorders, it is recommended to use quaternary salts according to the formulas (Ia), (Ib) and (Ila)

wherein the tertiary nitrogen is quaternized by R", where this is, for example, lower (C^_^)alkyl, aryl such as phenyl, or aryl-substituted C]_(, alkyl such as benzyl.

The invention will now be illustrated by the following examples.

EXAMPLE 1:

2- metvl- splro( 1. 3- oxazoline- 5. 3') quinuclidine.

(a) 3-nitromethylquinuclidin-3-ol

Quiniclidin-3-one (125 g. , 1 mol) was dried by azeotrophic distillation (40$ w/w in toluene) and placed in a 3 L flask fitted with a mechanical stirrer, 1 L of methanolic sodium ethoxide was added and the clear solution was stirred at 15-20°C. Nitromethane, 61 g, (1.0 mol), dissolved in 500 ml of absolute ethanol was then added over 0.5 hour while maintaining the temperature below 20°C. The solution was acidified to pH=l using HCl-isopropanol, filtered and dried to provide 160 g of crude product which was used in the next step.

1-H NMR (D 2 O, DSS) (HCl salt): S 3.79 (d as part of an AB-type spectrum, a H of CH 2 NO 2 ), 3.38-3.33 (m, 7H), 2 .36 (m, 1H), 2.36-1.95 (m, 4H)

(b) 3-aminomethylquinuclidin-3-ol

(i) In the reduction of 3-nitromethylquinuclidin-3-ol.

A solution of 3-nitromethylquinuclidin-3-ol hydrochloride in methanol was reduced by catalytic hydrogenation using 10# Pd on activated carbon to provide the HCl salt of 3-aminomethylquinuclidin-3-ol, which was recrystallized from methanol-isopropanol for to provide the product as white crystals.

(ii) Via the azide.

3-Methylenequinuclidine epoxide (25 g, 0.18 mol) and sodium azide (20 g, 0.3 mol) were dissolved in 50 mL of water. The mixture was stirred overnight at room temperature, extracted with chloroform (2 x 200 mL) and the extracts were concentrated by evaporation. The oily residue containing 3-azidomethylquinuclidin-3-ol was dissolved in water and wet active Raney nickel was added in portions with stirring until the evolution of nitrogen ceased (35 g). The mixture was filtered, the filtrate was concentrated by evaporation and the residue was recrystallized from isopropanol to provide 9 g of pure 3-aminomethylquinuclidin-3-ol.

m/z M<+>156, base peak m/e 139 (M-NH3), and m/z 96 which is typical of the quinucllidine skeleton. 1 H NMR (D 2 O, DSS) (free base) : S 1.3-2.0(5H); 2,3-3(8H). (di-HCl salt): S 1.8-2.4(m,5H); 3.2-3.5(m, 8H). ■^H NMR of the dihydrochloride salt compared to the free base shows a downfield shift of 6 hydrogen bonded to the carbons adjacent to the nitrogens (e.g. 5 2.5-2.9 to 3.3-3.5 ppm) according to to the expected structure.

(c) 2- metvl- splro( 1. 3- oxazoline- 5. 3') qulnuclidine (AF125).

(i) The ethylacetamidate method.

3-Aminomethylquinuclidin-3-ol (9.1 g, 0.058 mol) was dissolved in 1500 ml of dichloromethane; The ethyl acetamidate HCl salt (14 g, 0.11 mol) was added and the mixture was stirred at 5°C for six hours. The solution was made alkaline with Dowex 1, filtered and the solvent evaporated to provide an oily residue which was distilled at 60-65°C/0.5 mm Hg to provide AF125 as a colorless liquid.

m/z (M<+>) 181 base peak, 139 (M-CH 3 CN).

1-H-NMR (CDCl 3 -TMS): S 1.97 (dd, 3H)(J=1.3Hz); 2.8-2.9(m,1H); 3.14(d,1H)(J=12Hz); 3.5(dd,1H)(J=13Hz).

3.96(dd,lH)(J=13Hz, 1.3Hz).

^H-NMR including spin decoupling experiments enables assignment of the hydrogens adjacent to C2, Cg and the methyl group. Each of the hydrogens next to Cg shows a double doublet (J=13Hz geminal coupling) and a homo-allylic coupling with the methyl group (J=1.3Hz). Irradiation at 1.97 (methyl group) brings about the disappearance of homo-allylic coupling at 3.5 and 3.9 ppm and vice versa. Irradiation at 3.5 provides the signal 3.96 and vice versa which must be caused by geminal coupling of Hga and Hg^ (table 1).

The <13>C-NMR spectrum (in CDCI3) is very informative and enables the assignment of most resonances to appropriate carbons.

<13>C-NMR (CDCl3-TMS): S 13.2(CH3); 20.5 (CH₂CH); 21.5(CH 2 CH ); 30.0 (CHCH 2 ); 45.3, 45.7 (CH 2 CH 2 N); 62.2, (64.2(CH2N); 84.0(COCH2); 165(CH3).

GC (one peak): column, 25 m, diameter, 0.2 mm, packing, 556 phenylmethylsilicone; detector type, FID; temperature: column, 125°C, injection port, 220°C, detector, 220"C, carrier gas: N2, 0.7 ml/min.; retention time: 6.2 min.

(ii) The acetic acid method.

3-Aminomethylquinuclidin-3-ol (10 g) was dissolved in acetic acid (50 ml), azeotrophically distilled in xylene for 30 hours and the solution was cooled and made alkaline with cold aqueous potassium carbonate.

The organic phase was dried and evaporated to provide 5.2 g of crude product which was distilled under reduced pressure (60-65"C/0.5 mm Hg) to provide a colorless liquid which solidified on cooling. The compound obtained was identical to AF125 obtained by method (c)

(i)<.>

d) Optical resolution of Afl25

(Preliminary note: due to the possibility that

polarimeters used to determine optical rotation data may not have been reliable, the purity of the optical isomers was assessed using ^-H-NMR, as described below).

To a stirred cold solution of AF125 (15.5 g, 0.085 M) in acetone (150 mL), a solution of dibenzoyl-L-tartaric acid (18.8 g, 0.05 M) was added dropwise. A crystalline material was immediately separated. The precipitated salt was collected and purified by dissolution in hot isopropanol; cooling and adding ether to the cold solution until it was turbid; a repetition of this procedure gave a product with constant optical rotation [cx]j)<02>= 28°(MeOH); m.p. 145°.

In a similar manner, the corresponding salt of the other enantiomer was obtained starting from AF125 (14.74 g, 0.08 M) and dibenzoyl-D-tartaric acid (17.76 g, 0.047 M) of the corresponding tartaric acid. After 3 crystallizations, the product had [α]D<02>= 28°(MeOH); m.p. 145°.

The respective free bases were obtained by neutralizing the salts with a 10% solution of K 2 CO 3 and several extractions with chloroform. The free base derived from the salt of dibenzoyl-D-tartaric acid) had [a]rj<02>= -43°(MeOH), while that derived from the salt of dibenzoyl-L-tartaric acid had [a]j)°<2> = 36° (MeOH).

As indicated above, the optical purity of the enantiomers was shown by 1-H-NMR.<1->H-NMR spectrum of AF125 as a racemate in the presence of optically pure solvent (s)( + )2,2,2 trifluoro-l -(9-anthryl)-ethanol thus shows two optical isomers. The difference in chemical shift (in C5D5) of one of the hydrogens attached to C2 allows determination of the optical purity of AF125. Each separated enantiomer shows only one doublet (half of an AB-type spectrum) for one of these hydrogens on Cg.

Example 2: 2-Etvlspiro (1.3 oxazoline 5.3') qulnuclidine (AF123).

Similar to Example 1(c), the title product was obtained by condensation of 3-aminomethylquinuclidin-3-ol with propanoic acid followed by azeotrophic distillation in xylene. Purification of crude AF123 was achieved by vacuum distillation under reduced pressure.

m/z: M 195, base peak.

1-H NMR (CDCl 3 -TMS): S 4.0(d, 1H)(J=13Hz); 3.55 (d , 1H ) (J=13Hz ); 3,1(d,1H); 2.7-2.9(m); 2.3(q,2H)(J=7.5Hz), CH2CH3); 1.1(t,3H)(J=7.5Hz,CH3 ).

GC (same conditions as for AF125; one peak): retention time, 8.86 min.

EXAMPLE 3: 2-amino-spiro (1.3-oxazoline-5.3')quinucllidine AF125(N)

The amino-oxazoline derivative AF125(N) is an example of an electron-rich oxazoline ring due to the amino nitrogen being conjugated to the double bond, which promotes the negative charge on the imino nitrogen.

3-Aminomethylquinuclidin-3-ol (6 g) and cyanogen bromide (3.5 g) were dissolved in methanol (200 mL). The reaction mixture was stirred at room temperature for 5 hours, and then concentrated under vacuum. The residue was purified on an alumina column

(300 g), using a multisystem solvent as an eluent (1.5:1.0:0.2:0.06 CHC 2 /ether/methanol/NH 4 OH). An almost pure fraction of 450 mg was obtained and was finally purified by trituration with acetone to provide a pure crystalline material. The first chromatographic separation also gave various products including 2 g of crude desired product which was subjected to further separation on a silica column using as eluent 2* NH 3 in methanol. The raw product has a m.p. 135-136°C.

1-H-NMR (CDCl 3 -TMS) : δ 3.83, 3.43, 3.2 (3 doublets, 3H, Hb and Hc, J=0.048Hz); (m, 5H); 1.9 (b, 2H); 1.5 (m, 3).

FAB-MS showed a molecular ion (M+1)<+>182.

EXAMPLE 4: 2-phenyl-spiro(1.3-oxazoline-5.3')quinuclidine AF125(Ph)

A suspension of 3-aminomethylquinuclidin-3-ol (2 g, 0.01 mol) and ethylimidobenzoate hydrochloride (2 g, 0.01 mol) in ethanol (200 mL) was stirred for 24 h at room temperature. After filtration and concentration of the filtrate by evaporation, the crude residue was made basic with NaHCO3 and extracted with dichloromethane. The organic extracts were concentrated by evaporation and the crude residue was separated on an alumina column (30 g, 1.5 cm diameter) using a multi-solvent (1.0:1.0:0.2:0.026 CHCl3/ether /- methanol/32* aq. NH4OH as eluent. The product eluted rapidly (after 40 ml). The solvent was evaporated without heating and a white solid was obtained, mp 65-70°C.

<1>H-NMR(CDCl 3 ): S 7.955 (2H), 7.72 (2H), 4.16 (d, 1H), 3.73 (d, 1H), 3.26 (d, 1H), 2.93 (d, 1H) and (t, 2H), 2.77 (t, 2H), 2.2 (m, 1H), 1.95 (m, 1H), 1.60-1.52 ( m, 3H) ppm.

IR (CHCl3) 2920, 2900,, 2850sh, 1640sh, 1630, 1625sh, 1440, 1337, 1265, 1250, 1070, 1050, 1035, 1005, 965 cm-<1>.

m/z: 242 (60*) [M+] , 198 (30*, 171 (12*), 149 (10*), 139 (50*0, 124 (17*), 122 (20*), 121 ( 20*), 117 (30*), 111 (12*), 105 (33*), 96 (100*), 82 (50*), 69 (42*), 55 (36*).

CHEMICAL AND PHYSICAL PROPERTIES OF THE COMPOUNDS OF FORMULA (I)

Most of these compounds are relatively stable. AF125, for example, is thermally stable and can be distilled at 130° without decomposition. The chemical behavior is similar to that of the known 2-oxazoline derivative. In aqueous solution, the free base shows some degradation after a few hours which gradually decreases with time. The formation of the degradation products can be recorded by<1>H-NMR by inspection of two regions S 3.3-3.5 (appearance of two doublets9) and 1.9-2.0 ppm (appearance of two additional singlets).

The expected decomposition pattern (when, for example, X is 0 and Y is N) involves opening of oxazole lnr none to form both amide and ester as shown in Scheme E, where quinuclidine-derived compounds are given illustratively. The formation of two new singlets at S 1.9-2.0 ppm as expected for R = Me of these two structures is consistent with this assumption.

The same decomposition pattern can be shown for R=Et (AF123). Ring opening is much faster in the presence of dilute hydrochloric acid. It is noted that heating the tartrate and the mandelate salts in tetrahydrofuran also results in partial decomposition. The tendency to decompose such salts complicates the optical separation of compounds such as AF125 and AF123. However, this problem can be solved using the method described for the optical separation of the enantiomers of AF125, or alternatively amino alcohols such as 3-aminomethylquinuclidin-3-ol can be dissolved and the separated enantiomers can then be cyclized using an ester or an imidate as already described. In another alternative, the N-acyl derivatives of (eg) 3-aminomethylquinuclidin-3-ol can be resolved and the separated enantiomers can then be cyclized themselves as shown in Scheme K:

In this cyclization, e.g. p-toluenesulfonic acid or boron trifluoride etherate be used.

EXAMPLE 5: 2-Methyl-spiro(1-.3-oxazoline-5.4')-1'-methylpiperidine

(II; R = R' = methyl - "AF150")

(a) 1-methyl-4-nitromethylpiperidin-4-ol hydrochloride This starting material was prepared using a slight modification of the method of A.S. Cale (U.S. 4,746,655, 1988). A mixture of N-methylpiperidinone (142 g, 1.28 mol) and nitromethane (78.1 g, 1.28 mol) was added to a well-mixed solution of sodium ethoxide (1.28 mol), 20% in ethanol, at maintaining the internal temperature at 5-8°c. A white solid precipitates and stirring is continued for 20 minutes and a further 40 minutes at room temperature. The resulting solution was acidified with 500 mL of 7.2 N BCl in isopropyl alcohol. The hydrochloride and inorganic salts were extracted with CH 3 OH (3x200 mL) and the solvent removed in vacuo to provide the title compound, m.p. 180-182°C (not hygroscopic). m/z: 174 (M<+>of free base, 100*), 157 (M-OH, 20*), 127 (M-H-NO 2 -CH 3 , 40*).

(b) 4-aminomethyl-1-methylplperidin-4-ol hydrochloride

Palladium on charcoal (10*, 4 g) was added portionwise to a solution of 1-methyl-4-nitromethylpiperidin-4-ol (133.5 g) in methanol (1500 ml). The compound was hydrated in a Paar at a pressure of 379.2 KPa at room temperature for 48 hours. The solution was continuously filtered, treated with activated charcoal, the solvent removed and the residue triturated with ethanol (200 ml) to provide the title compound, m.p. 177-179°C.

m/z: 144(M<+>of free base, 15*), 127 (M-0H, 25*).

114 (M-CH 2 NH 2 , 100*).

(c) 2- methyl- spiro(1, 3- oxazoline 5. 4')- 1'- methylpiperidine (AF150)

A solution of KOH (1.43 g, 86*) in methanol (50 mL) was added to a solution of 4-aminomethyl-1-methylpiperidin-4-ol hydrochloride (3.61 g, 0.02 mol) in absolute methanol (50 mL). After stirring for 10 minutes, a solution of ethylace thymidate hydrochloride (2.7 g) in 20 ml of absolute methanol was added and stirring was continued for 30 minutes at room temperature. The solvent was removed and the remaining solid was dissolved in a solution of 2.8 g Na 2 CO 3 in 50 ml water, which was concentrated to dryness in vacuo. The white solid was extracted with 2 x 50 mL chloroform, treated with activated charcoal, dried (NagSO 4 ) and the solvent removed to provide the title product (62.5* yield), mp 45° C. (sublimes at 40° C/0 .05 mm Hg) and gives a single spot on silica TLC eluted with 2* NH 3 i CH 3 OH, Rf=0.4.

m/z: 168 (M<+>of free base, 100* at 7.5 ev).

<1>H-NMR(300 MHz, CDCl3): δ 3.56 (2H, q, J=1.5Hz), 2.53 (4H, m), 2.34 (3H, s), 1.96 (3H, t, J=1.5 Hz), 1.82 (4H, m).

Replacing the K0H used in this example with the equivalent amount of NaOH or Et3N gave similar results.

(d) 2- methyl- spiro( 1. 3- oxazoline 5. 4')- 1'- methylpiperidine Dlbenzoyl- D- tartrate

A hot solution of dibenzoyl-D-tartaric acid (5.4 g, 15 mmol) in 500 mL of toluene was added with stirring to AF150 (5.5 g, 32 mmol) dissolved in 200 mL of dry toluene. The precipitate was set and the supernatant liquid was decanted off. The remaining solid was washed with 3 x 100 mL of dry toluene and dried under reduced pressure to provide 8.4 g (80* yield) of a white somewhat hygroscopic solid.

TLC chloroform/aluminum (Merck Art 5581) Rf=0.4.

m/z: 168 (M<+>)

<1>H-NMR(300 MHz, D2O containing 1.5 mg Na2C03/0.5 ml D2O): S 1.95 (s, 6H, CH3-C), 2.35 (s, 6H, CH3-N ), 3.5 (s, 4H, CH2), 5.7 (s, 2H), 7.5-8.2 (m, 10H, aromatic hydrogens).

EXAMPLE 6: 2-ethyl-spiro(1.3-oxazoline-5.4')-1'-methylpiperidine

(II; R = ethyl, R<*>= methyl)

This compound was prepared similarly to the compound of Example 5 using the equivalent amount of ethyl propionimidate hydrochloride in place of ethyl acetimidate hydrochloride. The product was obtained as a liquid, bp 53°C/0.03 mm Hg, in 60.5* yield).

1-H-NMR (300 MHz, CDCl 3 ): δ 3.52 (2H, 5, J=1.5Hz), 2.47 (4H, m), 2.30 (3H, s), 2.26 [ 2H, quartet (J=7 Hz), triplets (J=1.5Hz)], 1.86 (2H, m), 1.72 (2H, m), 1.18 (3H, t).

EXAMPLE 7; 1- methylpiperlidine-4-spiro-4'-(2'-methyl-1'.3'-oxazoline (II; R = R<*>= methyl - "AF151")

(a) 1-methylpyridine-4-spiro-5*-hvdantoin

A mixture of the solutions of 1-methylpiperidin-4-one (36.44 g, 0.322 mol) in ethanol (150 mL), ammonium carbonate (93.0 g, 0.968 mol) in water (400 mL) and potassium cyanide (25.8 g, 0.396 mol) in water (82 ml), was heated at 60° C. for 2.5 hours and then left at room temperature overnight when 1-methylpiperidine-4-spiro-5<*->hydantoin separated. It was filtered off and washed with small amounts of cold water, ethanol and ether to provide a crystalline powder (27.0 g). Concentration of the filtrate and washings gave a crop No. 2 (20.0 g). The product was crystallized from methanol: m.p. 265-276°C (dec.) IR (KBr) 3170 (NH); 1700 (C=0) cm-<1>

m/z 183(M<+>, 38*); 71 (100*)

^H-NMR (300 MHz, D2O): S 1.8 (2H), 2.06 (six, 2H), 2.49 (S, -CH3), 2.58 (t, 2H), 3.14 (t, 1H), 3.20 (t, 1H).

(b) 4-Amino-1-methylpiperidine-4-carboxylic acid

1-methylpiperidine-4-spiro-5'-hydantoin (9.75 g, 0.0533 mol) and barium hydroxide octahydrate (28.8 g, 0.00913 mol) in water (150 mL) were heated at 160°C in an autoclave for three hours. The contents of four such batches were combined and precipitated barium carbonate was filtered out. The filtrate was neutralized with a solid carbon dioxide and the precipitate was removed by filtration. The filtrate was concentrated to a small volume to

afford 4-amino-1-methylpiperidine-4-carboxylic acid (32.0 g, 95% yield), m.p. 275-280°C (dec.).

IR (KBr) 3300, 1655, 1580 cm-<1>

m/z 158 (M<+>, 90*); 141 (98*, M-OH); 113 (12*, M-CO 2 H); 96 (100*); 71 (52*)

1-H-NMR (300 MHz, C5D5N + D2O): S 1.2 (m, 2H), 1.48 (s, CH3-N-), 1.7 (m, 2H), 1.9 (m , 2H), 2.0 (m, 2H).

(c) 4-amino-4-hydroxymethyl-1-methylpiperidine

Lithium aluminum hydride powder (15.62 g, 0.412 mol) in dry tetrahydrofuran (THF) (600 mL) was heated under reflux for 15 minutes, after which 4-amino-1-methylpiperidine-4-carboxylic acid (31.0 g, 0.196 mol) in the form of a dry powder was added portionwise under nitrogen with effective stirring. After the addition was complete, the reaction mixture was heated under reflux for four hours, cooled to 0°C under nitrogen with effective stirring, worked up by careful slow addition of water (20 mL), 15* aqueous NaOH (20 mL) and fresh water ( 10 ml).

The reaction mixture was filtered and the precipitate extracted with boiling THF (3 x 150 mL). The THF filtrate and extracts were combined and the solvent removed at 25 mm to provide a yellow viscous oil (28.0 g, 98.9* yield).

IR (neat) 3320 (NH); 3200' (br. OH), 1587 (NH2), 1468, 1448 cm-1 m/z 144 (M<+>, 15*); 127 (M-OH); 113 (M-CO 2 H); 96 (100*); 70 (41*).

<1->H-NMR (300 MHz, CDCl3): δ 1.41 (m, 2H), 1.60 (m, 2H), 2.24 (s, CH3-N), 2.29 (m, 2H), 2.48 (m, 2H), 2.50 (br., -NH 2 ), 3.29 (s, -CH 2 OH).

(d) 1-methylpiperidine-4-spiro-4'-(2'-methyl-1'.3'-oxazoline)

(AF151)

A mixture of 4-amino-4-hydroxymethyl-1-methylpiperidine (1.80

g), with acetic acid (20 ml) and xylene (20 ml) was azeotrophically distilled for 28 h. Remaining acetic acid and xylene were

removed at reduced pressure (25 mm Hg) and gave a resid

viscous oil that was basified to pH 11 with an aqueous solution of K2CO3. Extraction with chloroform and evaporation of the extract gave a small amount of residual brown oil (0.27 g). The aqueous solution remaining after the chloroform extraction was evaporated to remove water, det

the remaining solid was extracted with chloroform and the extract dried (NagSO4) and evaporated to provide as a residue a very hygroscopic solid (3.0 g). TLC showed that the latter gave mainly a spot that was more polar than the starting amino alcohol.

A portion of the hygroscopic solid melting at 150-160°C was heated under vacuum and began to distill almost immediately as a colorless oil at 45°C/0.15 mm Hg. This oil, when kept in the freezer, formed crystalline needles which melt at room temperature. The distillate was the acetic acid salt of the title compound.

IR (neat) 1664 (-C=N); 1565 & 1398 (-C02—); 1256 (C-0) cm-<1>m/z 168(M<+>of free base); 109; 70.

<i>H-NMR (300 MHz, CDCl3): S 1.77 (m, 2H), 1.96 (m, 2H), 1.98 (s, CH3-), 2.0 (s, CH3- ), 2.49 (s, CH 3 -N-), 2.91 (m, 4H), 3.95 (s, -CH 2 O-), 9.30 (br.s, -CO 2 <H>).

13C-NMR (300 MHz, CDCl3): S 14.0 (CH3C02-), 22.9 (CH3C=N-), 35.6 (C3and C5), 44.4 (CH3N<+>), 51.1 (C2 and C6), 67.0 (C4), 77.4 (C5'), 164.3 (C-=N), 176.7 (-C02-).

1-H-NMR of free base (300 MHz, CDCl3): δ: 1.64 (m, 2H), 1.84 (m, 2H), 1.98 (s. CH3-), 2.26 (m , 2H), 2.30 (s, CH3-), 2.69 (m, 2H), 3.94 (s, -CH2-).

EXAMPLE 8: 1-methylpyridine-4-spiro-4'-(2'-ethyl-1'.3'-oxazoline

A mixture of 4-amino-4-hydroxymethyl-1-methylpiperidine (3.0 g) with propionic acid (50 ml) and xylene (90 ml) was azeotrophically distilled for 5 hours. The residue (7 ml) was basified to pH II-12 with an aqueous solution of K 2 CO 3 . Extraction with chloroform and evaporation of the extract provided a mixture of non-polar compounds (0.80 g). The aqueous solution remaining after chloroform extraction was evaporated to remove water, the remaining solid was extracted with chloroform and the extract was dried (NagSC^) and evaporated to provide as a residue a hygroscopic solid (3.6 g). TLC showed that the latter gave mainly a spot, which was more polar than the starting amino alcohol (silica gel, solvent 40:58:2 methanol-chloroform-aqueous-ammonium lac).

A portion of the hygroscopic solid (1.5 g) was heated under vacuum and almost immediately began to distill as a viscous colorless oil at 50"C/0.1 mm Hg. The distillate is the propionic acid salt of the title compound. m/z 182 ( M<+>of free base, 14*); 167 (5*), 154 (71*), 125 (9*), 109 (100*), 96 (45*), 81 (30*), 74 ( 57*), 70 (89*), 57 (64*).

<1->H-NMR (300 MHz, CDCl3): S 1.12 (t, J=7.5 Hz, CHCH2-), 1.17 (t, J=7.6 Hz, CH3CH2-), 1 .75 (m, 2H), 2.00 (m, 2H), 2.29 (q, J=7.5, CH3CH2-), 2.30 (q, J=7.6, CH3<C>H2 -), 2.56 (s, CH3N-), 3.02 (m, 2 CH2-), 3.95 (s, -CH2O-), 7.52 (br. -CO2H).

To a stirred solution of the above propionic acid salt (700 mg) in chloroform was added a saturated aqueous solution of K 2 CO 3 until the evolution of CO 2 had ceased. The mixture was then stirred for 0.5 h and the phases were separated. The aqueous phase was extracted with chloroform, combined separated chloroform phase and the extracts were dried (Na 2 SO 4 ) and the solvent was evaporated to provide the title compound in free base form as a residual colorless oil (550 mg) which showed a single spot on

TLC.

%-NMR (300 MHz, CDCl3): S 1.17 (t, J=7.6 Hz, CH3CH2-), 1.61 (m, -CH2-), 1.86 (m, -CH2-), 2.18 (m, -CH2-), 2.29 (q, J=7.6, CH3CH_), 2.30 (s, CH3<N->), 2.71 (m, -CH2-), 3.94 (s, -CH 2 O-). m/z 182(M<+>25%), 167 (9*), 154 (78*), 125 (17*), 109 (100*), 96 (65*), 81 (54*), 70 (96*), 57 (77*).

An alternative route to compounds such as AF150 and AF151 depends on the cyclodehydration of appropriate amides, as shown in the following illustrations:

Dehydrating agents such as P2O5, sulfuric acid, BF3 etherate, CaCl2 and molecular sieves can be used for the above reactions. Corresponding thiazolines instead of oxazolines can be obtained by analogous reactions using P2S5-

EXAMPLE 9: 2-methyl-spro(1.3-thiazoline-5.4')-1'-methylpiperidine

(II; R = R' == methyl - "AF150(S)")

(a) 4-acetamidomethyl-4-hydroxy-1-methylpiperidine 4-aminomethyl-4-hydroxy-1-methylpiperidine (0.83 g, 5.7 mmol) was dissolved in 10 ml of chloroform, and acetic anhydride (0.58 g , 5.7 mmol) was added. The reaction mixture was spontaneously heated to 40-50°C. After 30 minutes the solvent was evaporated and the crude residue was chromatographed on a silica gel column (Merck 7734) using 33:67 2% aqueous ammonia-methanol as eluent.

m/z 186 (M<+>)

<1->H-NMR (300 MHz, CDC13): S 1.60 (multiplet, 4H, H3 and H4), 2.01 (singlet, 3H, CH3-C), 2.29 (singlet, 3H, CH3 -N), 2.38 (multiplet, 2H, H1), 2.55 (multiplet, 2H, H2), 2.98 (multiplet, 1H, NH), 3.26 (doublet, 2H, H5) ppm.

1-H-NMR (300 MHz, D20): S 1.42 (multiplet, 4H, H3 and H4), 1.81 (singlet, 3H, CH3-C), 2.08 (singlet, 3H, CH3-N ), 2.27 (multiplet, 2H, H1), 2.46 (multiplet, 2H, H2), 3.03 (singlet, 2H, H), ppm. The impurity gives a peak of 3.44 ppm.

(b) 2-methyl-spro(1.3-thiazoline-5.4')-1'-methylpiperidine

A mixture of 4-acetamidomethyl-4-hydroxy-1-methylpiperidine (6.5 g, 35 mmol) with phosphorus pentasulfide (10 g, 22 mol) was heated at 220 °C for 30 min, cooled and dissolved in 30 mL of concentrated hydrochloric acid . The acidic solution was transferred to 100 ml of cold concentrated aqueous sodium hydroxide, extracted with 2 x 100 ml of chloroform and combined extracts were dried and evaporated to provide 5 g of a black oily residue which was purified by distillation at 75°C/l mm Hg. to provide 1.8 g of clear liquid.

<1>H-NMR (300 MHz, CDCl3): S 1.8-2.0 (m, 4H), 2.17 (t, 3H, CH3-C), 2.2 (s, 3H, CH3- N), 3.9 (q, 2H, CH2-thiazoline ring)

Biological testing

The compounds according to the invention exhibit pharmacological activity and are therefore useful as pharmaceutical agents, e.g. for therapy. The agonists show particular activity in the tests described below. In the tables reporting the results of such tests, certain compounds may be designated by the following reference numbers:

Test 1; Isolated guinea pig intestinal preparation

The compounds of the invention were tested for their agonistic and antagonistic activities in guinea pig intestine preparations. Table 2 qualitatively summarizes the results obtained with some of the compounds tested where the most potent agonist was AF125. AF125 was found to be a full agonist in the guinea pig intestine preparation and its EC5Q was calculated as 1.3 µM. EPMRAChtil AF125 was approximately 14 compared to values ranging between 50-100 found for AF102B. This compound also showed high affinity towards muscarinic binding sites in the brain. The comparison between AF102B and AF125 showed that AF125 is more active by an order of magnitude than AF102B when it comes to the ability to contact the guinea pig intestine. AF125 therefore induces a much greater contractile response than AF102B at the same concentration. The amino analog of AF125, [AF125(N)] was also active as an agonist, but higher concentrations of this compound were required to contract the intestine. AF125(N) is thus only active at concentrations that are higher by an order of magnitude compared to AF102B. AF125(Ph) is an antagonist (IC50=0.7 uM). In contrast to the amino analogue of AF125, AF123 is found to be an antagonist.

Similar tests showed that the compound of Example 5 (AF150) is a full muscarinic agonist - with an EC5Q=0.8 µM (blocked by atropine 10 µM). Compared to acetylcholine (ACh), AF150 can induce a maximum contraction of the intestine of 130* (ACh=100*). It is surprising that the shape of the curve of AF150 compared to ACh is different and this curve can be analyzed by a two- or even a three-site model. This may indicate that AF150 has different affinities for the subtypes of muscarinic receptors found in this preparation. The compound of Example 7 (AF151) is also a full muscarinic agonist, with an EC5Q=3jjm (blocked by atropine 10jjM).

Test 2: Binding to muscarinic receptors in the brain Binding of agonists to Ml muscarinic receptors and the effect of quanylylimidophosphate (GppNHp). Agonists are known to be coupled to guanine nucleotide binding proteins (G proteins). Many studies have been performed on agonist binding to M2 muscarinic (low-affinity pirenzepine) receptors, but relatively few studies have examined agonist binding to M1 muscarinic (high-affinity pirenzepine) receptors. The primary reason appears to be that most of the agonists (except AF102B for example, US Patent No. 4,855,290) have a higher affinity for the M2 subtype than for M1 muscarinic receptors.

Non-hydrolyzable guanine nucleotides such as GppNHp dissociate the G protein from the receptor and reduce the affinity of the receptor for agonists but not for antagonists. Under special conditions, it is possible to analyze both the affinity and the efficacy of the agonist with M1 muscarinic receptors using low concentrations of <3>H-pirenzepine (<3>H-PZ) below its Kp [Potter et al, Cell. Molec. Neurobiol 8:181-191 (1988); Potter and Ferrendelli, J. Pharmacol. Exp. Ther. 248: 974-978 (1989); Flynn et al., Eur. J. Pharmacol. 172:363-373 (1989)].

Under these conditions, agonists with high efficacy ("full agonists") bind directly to the M1 receptor subtype with two affinities. The high affinity state of the M1 receptor subtype is sensitive to guanine nucleotides and GppNHp, for example, converts the high affinity state to a low affinity state. The ratio of low to high affinities (Kl/Kjj) to the M1 receptor may predict the relative efficacies of antagonists for activation of putative M1 receptor-mediated second messenger systems.

Table 3 summarizes the results obtained for oxotremorine-M and carbachol (two full but non-selective M1 and M2 agonists), for the selective M1 agonist AF102B (US Patent No. 4,855,290), AF150 and AF151. As expected, the quaternary full agonists oxotremorin-M and carbachol show a two-affinity state and the relative efficacy of these compounds is predicted by the Kl/Kh ratio. In contrast, AF102B and AF125 show a mass action curve.

Notes 1 table 3:

Ki=Kg or Kl = IC50/I+C/K]) where C = 4.4nM is the concentration of the radioactive ligand (<3>H-PZ) and Krj=13.9nM is the dissociation constant thereto. Kg, Kl, *H and *L are apparent inhibition constants of the tested ligands for high and low affinity sites. G is GppNHp (guanylyl imidodiphos phat) and was used at 0.1 mM concentration. Blocking of<3>H-PZ from cortical binding sites was performed in Tris/Mn buffer (pH 7.4) for one hour at 25°C and the buffer included 1 mM EDTA and 2 mM Mn+<+>ions. The procedure is similar to that of Potter and Ferrendelli (J. Pharmacol. Exptl. Therap. 248: 974-978, 1989). The binding data were analyzed using the GraphPAD program and were fitted to one or two components. Values are averages from 1-3 experiments performed in triplicate.

It was surprising that AF150 and AF151 show a typical two-affinity state in the presence of <3>H-PZ with a very high Kl/Kh ratio for AF150. GppNHp, on the other hand, shifted the high-affinity state to the low-affinity state. AF150 and AF151 both show high affinity for M1 putative receptors and high efficacy at the same receptor. This can be concluded because<3>H-PZ at the concentrations used (1/3 of Kpj) is only bound to M1 receptors. The data show that both AF150 and AF151 are highly effective agonists that activate M1 receptors and are expected to activate the transduction mechanism involved in secondary messengers coupled to M1 receptors, such as phosphatidyl inositol transfer and Ca<2+>mobilization, for Example. Simultaneous activation of both H and L sites may prove to be necessary functional effects. In this regard, AF150 and AF151 may be particularly suitable for the treatment of disorders with cholinergic hypofunction such as SDAT.

Test 3

The two-affinity state of AF150 is conserved from homogenates of rat cerebral cortex even when the substitution of <3>H-PZ is carried out in a 10M sodium potassium buffer indicating that this is a unique agonist compared to other putative muscarinic agonists (Table 4). . AF150 shows a good selectivity and efficiency for M1 receptors as shown in Tables 4 and 5. In this regard, AF150 may be a good drug for the treatment of disorders with cholinergic hypofunction such as SDAT due to the M1 selectivity and high efficiency of particular therapeutic value in this type of disturbance.

The potency of the compounds according to the invention (and for comparison other cholinergic compounds) to remove from rat brain homogenates, cerebellum or frontal cortex, the following 3H -labelled compounds, i.e. (-)3H-quinuclidinyl benzilate (<3>H-0NB; a non-selective Ml and M2 antagonist) and<3>H-Pirenzepine (<3>H-PZ; a selective M1 antagonist), were investigated. For comparison, oxotremorine (an M2>M1 tertiary agonist), oxotremorine-M and carbachol (mixed M2 and M1/M2 quaternary agonists), AF102B (see US Patent No. 4,855,290) and McN-A-343 (a Ml quaternary agonist) were included. The results are shown in tables 4 and 5.

Removal of <3>H-PZ and <3>H-QNB binding was performed in 10 and 50 mM phosphate buffers. K A = Kg or KL was as expressed in Table 3. The KD values for <3>E-PZ and <3>H-QNB were 13.9 and 0.093 nM (Fisher et al, J. Pharmacol. Exptl. Therap. 1991 , for printing).

Table 5:

Apparent affinity states for Ml (removal of<3>HL-QNB from rat cerebral cortex) and for M2 muscarinic receptors (removal of<3>H-QNB from rat cerebellum)

<*>Competition curves were considerably better adapted to a one-site model (mass effect curve). Note: The K values were generated by a non-linear, least-squares curve fit of the data using GRAPHPAD-2 two- and one-site analysis and statistics, as expressed in Table 1.

Removal of <3>H-QNB binding (KD value for <3>H-QNB of 0.093 nM) was performed in 50 mM phosphate buffer for one hour at 37°C (Fisher et al, J. Pharmacol. Exptl. Therap ., 1991, in press).

Tables 4 and 5 show that AF150 exhibits high selectivity for M1 muscarinic receptors. Where AF125 shows an apparent M2 selectivity, AF150 surprisingly shows a high selectivity for M1 receptors.

Test 4: Modification of muscarinic agonist mixture to cortical membranes by Cu<++> treatment

Transition metal ions and quanine nucleotides exhibit strong effects on the affinity of agonists for muscarinic receptors, which probably result from modification in proportions of different agonist binding states (Gurwitz and Sokolovsky, Biochem. Biophys. Res. Commun. 96: 1296-1301, 1980; Aronstam et al , Mol. Pharmacol. 14: 575-582, 1978). Addition of Cu<++>, which like certain other transition metal ions is known to form stable coordination complexes with protein sulfhydryl residues, has been shown to increase the apparent affinity of muscarinic agonists by increasing the proportion of high affinity sites and eliminating sensitivity to quanine nucleotides (Farrar and Hoss, Trans. Am. Soc. Neurochem. 13: 250, 1982; Gurwitz et al, Biochem. Biophys. Res. Commun. 120: 271-276, 1984).

As shown in Table 6, the Cu<++> treatment did not modify the affinity observed for the classical non-selective muscarinic antagonists atrophine and scopolamine, as well as for the M1-selective antagonist pirenzepine. In contrast, the binding parameters of the classic non-selective agonists, carbachol and oxotremorine, showed that the Cu<++>treatment increased their apparent affinity, i.e. the competition curves were shifted to lower agonist concentrations. Analysis of competitive binding data according to a two-site model is summarized in Table 6, and indicates that the Cu<++-> treatment increased the proportion of high affinity binding sites from 32 to 51* for oxotremorine and from 25 to 44* for carbachol with no significant changes in their two affinity constants Kjj °g KL.

The binding parameters of AF102B were also affected by Cu<++>because, as shown by the data in Table 6, the interaction with untreated membranes represents binding to a single population of sites with Kj = 3.3 jjM, whereas after Cu<+ +> treatment, a high affinity binding state was also observed, representing 26* of the total sites with Kg = 0.02 uM. No change was observed in the low affinity binding constant. It has thus been shown that AF102B reacts with muscarinic receptors in cerebral cortex membrane preparation in a way that is typical for agonists, i.e. the receptor-ligand complex can react with the G-protein(s) (cf. discussion by Gurwitz et al, 1984) .

AF150 also exhibits agonistic binding characteristics in these studies. In untreated membranes it showed many affinity states (0.32 µM and 33.5 µM for high and low affinity sites). The proportion of high affinity sites was increased from 14 to 37* after Cu<++> treatment and this suggests that AF150 may act as an agonist for cerebral cortex muscarinic receptors.

In the above table, values given are mean ± SEM of at least 3 experiments and each experiment is performed in triplicate samples. The analysis is carried out in modified Krebs buffer at pH 7.4 for 2 hours at 25 "C with and without pre-treatment for 30 minutes with Cu<++>(100 jjM) if rat cerebral cortex membranes (Fisher et al, J . Pharmacol. Exptl. Therap., 1991, in press).

Test 5: The Effects of the Test Compounds on the Expression 1 Cell Lines of Muscarinic Receptor Subtypes On the basis of pharmacological data, muscarinic receptors have been divided into three subtypes (M1, M2 and M3). Molecular cloning studies have identified five genetically distinct muscarinic receptor subtypes (m^-ms). Functional expression of these genes has indicated a correlation between genetic and pharmacologically defined subtypes, where Ml=m^, m4 and 1115; M2=m2?and M3=m3 (Buckley et al, Mol. Pharmacol. 35: 469-476, 1989).

Stimulation of m^ and m3 receptors promotes dose-dependent increases in hydrolysis of phosphoinositides (PI). Agonist activation of the m^ receptor also promotes increases in basal and forskolin-stimulated cAMP, whereas the m3 receptor has no effect on intracellular cAMP levels (Buck and Fraser, Biochem. Biophys. Res. Comm. 173: 662-672, 1990). Stimulation of mg and m4 receptors by muscarinic agonists leads to preferential inhibition of adenylate cyclase (AC) activity (Mei et al, Life Sciences, 45: 1831-1851, 1989).

The new compounds AF150, AF151, AF150(S) and AF125 were analyzed for their ability to stimulate PI in CHO cells transfected with m^ and m3 receptors, (Buck and Fraser, loe eit), and in cultured human neuroblastoma cells, line SK -N-SH, which mainly expresses the m3 subtype but not the m^ subtype (Pinkas-Kramarski et al, Neurosci. Lett. 188: 335-340, 1990; Luthin et al Mol. Pharmacol. 34: 327-333, 1988).

These compounds were also analyzed on cultured PC12 cells which mainly express m4 subtype receptors and are linked to the inhibition of the AC activity (Pinkas-Kramarski et al, loe eit).

Stimulation of PI degradation was analyzed using the method of Stein et al (EMBO J. 7: 3031-3035, 1988), and modulation of AC activity was analyzed by the method of Stein et al, loeit and Pinkas-Kramarski et al (FEBS Lett., 239: 174-178, 1988). The results are shown in Table 7 and are compared with carbachol, a full muscarinic agonist (mixed M1/M2 type) and with AF102B.

Notes to table 7:

EC50: effective concentration (jjM) at 50* activation

+++ full agonist compared to carbachol (full agonistic activity was tested for all compounds at

lmM)

+ partial agonist compared to carbachol no stimulation of PI or inhibition of AC

PI phosphoinositide hydrolysis

AC adenylate cyclase activity expressed by change in cAMP concentration

NT not tested

a: * of carbachol action

b: effective concentration (jjM) inducing the effect

c: 22jjM AF102B inhibited the action of 1mM carbachol d: 1mM AF102B inhibited the action of 1mM carbachol

In Table 7, the agonistic effect on m^ receptors is shown by increased PI hydrolysis and AC activity, while stimulation of m4 receptors is reflected by the inhibition of AC activity; The m3 stimulation leads to increased PI hydrolysis. It appears from this table that it is surprising that both AF150 and AF151 are full agonists on the m^ and m4 muscarinic receptor subtypes, but partial agonists on the m3 subtypes. As AF150 and AF151 are full agonists on m^ receptors shown by PI hydrolysis, these compounds are surprisingly different compared to carbachol and this is seen due to their very weak effect on potentiation of AC activity on the same receptors. Carbachol-induced activation of m^-muscarinic receptors thus promotes the increase in basal and forskolin-stimulated cAMP (232-356* and 546-1382*, respectively), but both AF150 and AF151 are equipotently very weak in this test (33*, respectively) and 76*).

In a typical experiment with SK-A-SH neuroblastoma cells, it was found that accumulation of inositol-l-phosphate (IP^) was stimulated 7.46-fold above the basal level by 10 mM carbachol, 5.24-fold by 1 mM oxotremorin-M, and 1.42-fold by 1 mM AF125. In contrast, the M1-selective agonist AF102B (US Patent No. 4,855,290) did not stimulate phosphoinositide hydrolysis in these cells even at 1 mM, whereas at that concentration it could completely block oxotremorine-M-induced signaling. This inhibition was dependent on the oxotremorin-M concentration, for example, 1 mM AF102B completely blocked the phosphoinositide hydrolysis induced by 10 µM oxotremorin-M while inhibiting the signal of 1 mM oxotremorin-M by 34*.

In addition, the combined stimulation of the phosphoinositide hydrolysis by carbachol and certain new compounds was investigated: in the same experiment, the accumulation of IP^ was stimulated 6.11-fold above the basal level by a combination of 10 mM carbachol with 1 mM AF125. The novel compound AF125 thus stimulates phosphoinositide hydrolysis in SK-N-SH human neuroblastoma cells to approximately 20-25* of the stimulation obtained with the non-selective full agonist cabachol, while it only minimally (10-20*) interfered with the action of carbachol. The significance of these results is that AF125 exhibits a unique activity as a muscarinic agonist in this cell line which expresses mainly the m3 muscarinic receptor subtype.

It is concluded that these compounds exhibit a unique activity and selectivity and may therefore be particularly useful for the treatment of SDAT (Braan et al, FEBS let. 230; 90-94, 1988).

Test 6: The effect of the test compounds on synaptosomal acetvlcholln (ACh) release

The effects of AF150 (and for comparison AF125, MeN-A-343 and oxotremorine) on basal and K<+->activated release of<3>H-ACh were studied on synaptosomes prepared from rat cerebral cortex using the methods described in detail by Pittel et al (J. Neurochem., 55: 665-672, 1990). The results are shown in Table 8. It was surprising that the two closely related structures, AF150 and AF125, had different effects on <3>H-ACh release. So that while AF125 (lmM) inhibited the K<+->activated release of ACh, AF150 (lmM) surprisingly potentiated it. These results indicate that AF125 acts on presynaptic receptors such as oxotremorine, a classical M2 agonist. In contrast, the action of AF150 on presynaptic muscarinic receptors causing an increase in K<+->activated ACh release can be considered either an M2 antagonistic or an M1 agonistic action. Hadhazy and Szerb (Brain Res. 123: 311-322, 1977) and Gulya et al (Neurochem. Int. 15: 153-156, 1989) demonstrated that muscarinic antagonists stimulate the K<+->activated ACh release. Suzuki et al (Neurosci. Lett. 84: 209-212, 1988) and Pittel et al, et al. showed that muscarinic antagonists can also stimulate basal ACh release. The latter study suggests that stimulatory muscarinic receptors that modulate ACh release are present in the CNS and may pharmacologically be considered M1 subtype. The existence of muscarinic autoreceptors that increase ACh release was also manifested in the PNS in smooth muscle preparations (Kilbinger and Nafziger, Naunyn Schmiedebergs Arch. Pharmacol. 328: 304-309, 1985).

Notes to Table 8:

The results are from 1-3 experiments unless otherwise stated<*>

NT = not tested

<**>nM

The antagonistic effect of pirenzepine (an M1 antagonist) on the potentiating effects of AF150 on K<+->evoked<3>H-ACh release (as indicated in Table 8), could indicate that AF150 could be considered as an M1- agonist, thereby activating M1 excitatory autoreceptors. Regardless of the mechanism of action of AF150, i.e. whether it can be caused by e.g. (a) M1 post- and pre-synaptic agonistic activity or (b) M1 postsynaptic agonistic and an M2 presynaptic antagonistic action, this compound may be particularly promising for the control of SDAT and related disorders.

Test 7: Electrophysiological assessment of AF150 in rat hippocampal slices

Acetylcholine (ACh) has a number of actions in the mammalian brain. The main effects were observed in hippocampal slices and include: (1) a blockade of sustained outward potassium (K<+>) current activated by depolarization (Irø); (2) a blockade of Ca<++>dependent K<+>current underlying the slow afterhyperpolarization (AHP); (3) a blockade of sustained K<+>current at rest; (4) a transient outward K<+>current; (5) reported effects of ACh on postsynaptic potentials (an increase in spontaneous and decrease in evoked) and on Ca<++>currents.

(Segal, Brain Res. 452: 79-98, 1988; Duttar and Nicoll, J. Neurosci. 8: 4214-4224, 1988).

The results using gallamine (an M2 antagonist) and pirenzepine (PZ, an M1 antagonist) suggest that an M2 receptor can mediate reduction of EPSP and blockade of Irø. In contrast, the depolarization and blockade of the AHP that is insensitive to gallamine and blocked by 0.3 µM PZ is likely to be mediated by M1 receptors (Duttar and Nicoll, et al.).

The effects of microdroplets of AF150, ACh and PZ applied to rat hippocampal cells were recorded intracellularly. The exact concentrations of the drugs at the receptor sites are not known using this method. However, this method has an advantage over the perfusion method because it provides a more correct picture from an electrophysiological point of view (Segal, et al.).

Intracellular responses to AF150 of CA1 neurons in the hippocampal slices were recorded using the method of Segal. Small hyperpolarizing current pulses (current traces not shown) were altered with stimulation of Schaffer commissural afferents producing postsynaptic potentials. AF150 applied topically by microdroplets (100 µM) caused a potent depolarizing response. Hyperpolarizing the cell to control potentials shows a real increase in the input resistance at rest. AF150 slowly blocks the AHP mediated by a Ca<++->dependent K<+->conductance. AF150 causes a small decrease in postsynaptic potentials (PSPs). Pirenzepine (10jM, an M1 muscarinic antagonist) significantly reduces the depolarization caused by AF150. Increases in inward resistance at rest were also prevented by pirenzepine. Blockade of slow AHP at AF150 was significantly reduced in pirenzepine-treated slices. The reduction in PSP by AF150 was unaffected by pirenzepine.

Conclusions

(1) AF150 causes depolarization, increased conduction resistance, and slowly blocks the AHP, and these effects are antagonized by the M1 receptor antagonist pirenzepine, but an effect on PSP that was small compared to ACh was detected and was not affected by pirenzepine. (2) It was surprising that AF150 appeared to be a more specific agonist for M1 receptors than for M2 receptors. (3) Compared to AF102B, it is clear that AF150 is much more effective and causes a depolarization (blocked by pirenzepine) that is not seen in the case of AF102B. Both AF150 and AF102B slowly block the AHP (pirenzepine-sensitive action).

Test 8: Pharmacodynamic profile and general toxicity

PART A

1. Purpose:

The purpose of this study was to establish the primary pharmacodynamic profile and to assess the overall toxicity of AF150 by observing mice after injection at 3 dose levels of AF150.

2. Materials and methods:

a. Animals: mice, male CD strain, 20-30 g

b. Group size: n=5

c. Route of administration: intraperitoneal (i.p.)

d. Dose levels (1.5 and 10 mg/kg)

e. Dosage volume: 10 ml/kg

f. Preparation of the test material: AF-150 was dissolved in salt water solution.

g. AF150 at 3 dose levels was administered i.p. to mice (n=5). Behavioral changes up to two hours after dosing

was recorded in addition to further observations up to 24 hours administration for the occurrence of death. Analgesic effects were assessed using the tail-pinch method 15, 30, 60 and 120 minutes after dosing. Apart from analgesia, the animals were observed for the following effects: appearance of tremors, convulsions, respiratory distress, salivation, diarrhoea, change in pupil diameter, exophthalmos, motor coordination, hypo- or hyperactivity and vocalization.

3. Experimental results

1 mg/kg i.p.: Very slight excretion and diarrhea were observed for about 30 minutes after injection. No pain relief was observed at any time during the observation. No other central autonomic effects were observed.

5 mg/kg i.p.: Reduced motor activity was observed up to 60 minutes after administration and then normal activity was achieved. Partial pain relief was detected 15 minutes after injection. Motor coordination was reduced up to 30 minutes after the injection. Mydriasis was observed 5 minutes after the injection. Some salivation and diarrhea were observed from 10 to 6 minutes after the injection.

10 mg/kg i.p.: Complete hypoactivity was observed from 5 to 75 minutes in 2 animals, and the other 3 regained activity after 120 minutes. Certain tremors were observed 10 minutes after injection for a short period. Motor coordination was markedly affected during the 2-hour observation. Pain relief was complete 15 and 30 minutes after injection and no pain relief was observed after > 60 minutes. Moderate salivation, diarrhoea, lacrimation and mydriasis were observed 5 minutes after injection and became severe 10 minutes after injection, but the degree of severity was reduced and all signs were gone 90 minutes after injection.

PART B

White hanimis from the CD-1 strain (Charles River, UK) within the body weight range 18-26 g were used in this study carried out on AF123, AF125 and AF125(N).

AF125

The dose range of AF125 injected i.p. into mice at dose levels ranging from 50 to 200 mg/kg showed that the toxic-lethal dose falls within the range of 100-200 mg/kg. These original experiments clearly showed that the main signs of reactions to treatment consisted mainly of signs characteristic of potential cholinomimetic activity of the test material, such as tremors, salivation, lacrimation, diarrhea, analgesia, hypothermia and vasodilation. The survivors showed initial signs of healing 2 to 3 hours after injection. Results after p.os and i.v injection of AF125 at non-lethal doses are shown in the following table 9.

Tree=Trees

Con = Seizures, mostly of the clonic-tonic type Res = Respiratory disorder, mostly hyperpnoea and tachypnoea

Sal = saliva secretion: sli = slight; mod = moderate; sev = severe

Lac = Lacrimation (sli; mod; sev)M Dia = Diarrhea (sli; mod; sev)

Ana = Analgesia, indicated by a lack of response to tail pinching

Myd = Mydriasis, (sli; mod; sev)

Mot = Changed spontaneous motor activity, either increased (Inc) or decreased (dec)

Arrow = Arrow erection (sli; mod,; sev)

Hyp = Hypothermia (sli; mod; sev)

Middle Lethal Dose (LD50) of AF125

The following are the LD5Q(95* tllllt limits) values obtained;

I. Intravenous injection 68.9 mg/kg (66.6-71.2)

II. Oral administration 134.4 mg/kg (118.4-152.5).

In light of the primary screening results obtained in 1 mouse, AF125 has primary cholnomimetic activity. It is also worth noting with regard to the duration of activity, healing began approximately 1 to 2 hours after treatment depending on the dose administered. Especially under the conditions of oral dosing, the comparative onset of healing appeared to be relatively rapid. Close relationships between the two respective LD5Q values, as well as comparative "active" dose levels after an i.v. or p.os treatment indicates that AF125 is fairly quickly absorbed by the enteric route.

AF125(N). AF123

The general pharmacological actions of these compounds are shown in Table 10.

* Numerical values of the data presented refer to the number of animals affected based on the total number of animals treated. Regarding other abbreviations see the details below and in addition:

P/H = Piloerection/hypothermia (sli; mod; sev)

Test 9: The effects of the test compounds in scopolamine-treated rats 1 an example of passive avoidance (PA).

Naïve male Sprague-Dawley rats, three months old (obtained from Charles River Breeding, U.K.) were used (weight 200-300 g). The passive avoidance (PA) test is performed according to Fisher et al, Neurosci. Easy. 102: 325 (1989), except that in these rats amnesia was induced by scopolamine.

Eight groups of 17-20 naive rats were used. Each group was divided into two subgroups of 7-10 rats each: subgroup 1 was injected with scopolamine HBr (0.5 mg/kg in

saline, s.c, 15 min before shock) and subgroup 2 was injected with saline (1 ml/kg, s.c, 15 min before the shock). Seven of the groups were treated within one minute of the shock with one of the following doses of the test compound: 0.1, 0.5, 1, 3, 5, 8, 10 mg/kg (in saline, i.p.) and one group received saline (1 ml/kg, i.p.).

AF125

Initial latency and retention latency measurements were analyzed by a two-way ANOVA (pre-shock scopolamine treatment/post-shock AF125 treatment). Tables 11 and 12 present the mean ±S.E.M. of initial latencies and retention latencies, respectively.

CONCLUSIONS

The reverse screening experiment using scopolamine as a preshock treatment and AF125 as a postshock treatment provided a significant dose response curve. The doses of 3, 5, 8 and 10 mg/kg had a visible advantage compared to all other doses. Rats treated with the above doses exhibited the longest retention latencies compared to rats treated with saline. The lower doses exhibited short retention latencies that did not indicate any improvement in cognitive functions. No significant differences were found in the initial latencies between all the groups tested.

The effect of the post-shock AF125 treatment was found to be significant (F=104.77, df=l/126, p<0.001), 10 mg/kg (p<0.05), significantly increased the retention latency of scopolamine-treated rats. In addition, AF125 (5 mg/kg) significantly (p<0.05) reduced the retention latency of pre-shock saline-treated rats. A significant difference was found between the retention latency of rats treated with pre-shock scopolamine/post-shock saline and pre-shock saline/post-shock saline treated rats (p<0.001). All pre-shock saline-treated rats that received AF125 post-shock at the doses: 3, 5, 8 and 10 mg/kg had side effects. The group receiving 3 mg/kg had some diarrhoea, the group treated with 5 mg/kg had stronger diarrhoea, and the groups treated with 8 and 10 mg/kg had severe diarrhea with lacrimation. It is important to note that rats treated with pre-shock scopolamine did not show any side effects after AF125 treatment.

Compared to AF120B (US Patent No. 4,855,290), AF125 exhibited a broader dose-response curve; AF102B had a significant reinforcing effect on the retention behavior using the doses of 3 and 5 mg/kg, while the reinforcing effect of AF125 continued in a larger range from 3 to 10 mg/kg. All these results indicate that the beneficial action of AF125 in this model is caused by its potent central agonistic action on muscarinic receptors involved in cognitive functions.

AF125(N)

Individuals

Naïve male Sprague-Dawley rats three months old (obtained from Charles-River Breeding.U.K.) were used (weight 200-300 g).

Behavioral test.

Eight groups each of 18-20 naive rats were used. Each group was divided into two subgroups each with 9-10 rats: subgroup 1 was injected with scopolamine HBr (0.5 mg/kg in saline, s.c, 15 min before shock) and subgroup 2 was injected with saline (1 ml/ kg, s.c, 15 min before the shock). Seven of the groups were treated within one minute of the shock with one of 10235 in the following AF125(N) doses: 0.1, 0.5, 1, 3, 5, 8, 10 mg/kg (in saline, i.p.) and one group received saline (1 ml/kg, i.p.).

The baseline measures of baseline latency and retention latency were analyzed by a two-way ANOVA (pre-shock scopolamine treatment/post-shock AF125(N) treatment). Tables 13 and 14 present the mean ±S.E.M. of initial latencies and retention latencies, respectively.

A significant difference (F(7/140)=2.37, p<0.05) in the initial latency was found between the groups treated with AF125(N) (Table 13). An interaction between AF125(N) treatments and the pre-shock treatment (saline vs scopolamine) was also found (F(7/140)=22.43, p<0.05). More specifically, a Scheffe test showed that the initial latency for pre-shock scopolamine-post-shock AF125(N) 0.5 mg/kg treated rats (62.9±17.4 sec) was significantly (p<0.025) longer than the initial latency for the control group (preshock-scopolamine-post-shock saline, 39.1±17.6 sec). The group treated with preshock-saline-postshock AF125(N) 5 mg/kg also had a significantly longer initial latency compared to the control group: Preshock saline-postshock saline-treated group (65.1±18.5 vs 26.7±4, 7 sec p<0.001). The effect of the post-shock AF125(N) treatment was found to be significant (F(7/140=3.51, p<0.05) and Table 14). A Scheffe test specifically showed that the dose of 0.5 mg/kg AF125(N) significantly increased the retention latency of scopolamine-treated rats compared to rats treated with saline (332.7±80.9 vs 141.1±5.5 sec. , p<0.005).

In addition, AF125(N) (5 mg/kg, i.p) significantly reduced the retention latency of pre-shock saline-treated rats compared to the control group (290.3±52.1 vs 483.6±64.9 sec, p<0.005). Finally, a significant difference was found between the retention latency of rats treated with pre-shock scopolamine/post-shock saline-treated rats and rats treated with pre-shock saline/post-shock saline (141.1±55.5 vs 483.6±64 .9, p<0.001). The treatment with AF125(N) did not cause obvious side effects in any of the doses when administered either post-scopolamine or post-saline. The reverse screening experiment using scopolamine as a pre-shock treatment and AF125(N) as a post-shock treatment provided only one positive significant response after one dose: 0.5 mg/kg, i.p. Rats treated with this dose exhibited longer retention latencies compared to rats treated with saline.

Test 10: Effects of AF150 in AF64A-treated rats in a passive avoidance (PA) paradigm.

This animal model mimics to some extent the cholinergic hypofunction in SDAT (Fisher and Hanin, Ann. Rev. Pharmacol. Toxicol. 26: 161-181, 1986). The procedure described in Fisher et al (J. Pharmacol. Exptl. Therap., 1991 (in press)) and in Fisher et al (Neurosci. Lett. 102: 325-331, 1989) was used here. The results are shown in Table 15.

As shown in the table, AF150 was beneficial in this animal model at all three doses tested, by i.p. injection. In addition, AF150 (in free base form) significantly improved the retention latency of A64A-treated rats when administered orally at 0.2 and 1.0 mg/kg (not shown). The latest finding indicates that the compound is well absorbed and is effective when administered orally at very low doses. The low effective doses in the PA test are free of obvious side effects. AF150 thus has a wide margin of safety as shown in learning and memory tests such as PA.

No significant differences were found among the different groups of animals tested in the initial latency test and the initial latency ranged from 20 and 31 seconds. In all experiments using AF150, the compound was dissolved as free base in 10 mM phosphate buffer at pH 7.35.

Notes to table 15

Significant interaction reaches F(2,54)=6.33, p<0.005. When analyzing the previous data according to Scheffe's main contrasts, the following significant results were found:

(p<0.005) AF150 (0.1 mg/kg) vs. AF64A buffer

(p<0.025) A64A-AF150(0.2 mg/kg) vs. AF64A buffer

(p<0.001) A64A-AF150(1.0 mg/kg) vs. AF64A buffer

(p<0.05) A64A-AF150(1.0 mg/kg) vs. AF64A-AF150(0.2 mg/kg)

(p<0.05) Saline buffer vs. AF64A buffer (p<0.005) saline buffer vs. AF64A-AF150(1.0 mg/kg)

(p<0.025) saline buffer vs. saline AF150(1.0 mg/kg)

Test 11: Effects of AF125 and AF150 in AF64A-treated rats in an example of 8-arm radial paw wringing (RAM)

This animal model mimics the cholinergic hypofunction and cognitive dysfunction in SDAT (Fisher and Hanin, loe eit; Fisher et al, Neurosci. Lett. 102: 325-331, 1989); the method described in the latter publication was used here.

PART A - AF125

The potential effect of AF125 in reversing amnesia was assessed on the performance of AF64A-injected mature rats in the RAM.

Procedures

a) Individuals

Forty-eight mature male rats (11-12 months) were used in this

the study. They were injected icv with AF64A (3 nmol/2 ul/side) 8 months earlier at three months of age. One week before the behavioral testing, the rats were transferred to individual cages and they were not fed until they reached 90* of their weight with free food access (the rats had access to water). The room was illuminated 12 hours per day (6:00 a.m. to 6:00 p.m.) and behavioral testing sessions were conducted during the mornings. After reaching 90* of the free food weight, the rats received approximately four food pellets per day (Altromin, 15 g) to further reduce the rats' weight. Two days before the behavioral testing, the rats were fed precision pellets (Bioserv Inc.) which were later used for reinforcement in the maze.

b) Apparatus

Behavioral tests were performed in an elevated (70 cm) 8-arm

radial labyrinth made of PVC. The arms (75 cm long and 10 cm wide) went out from an octagonal central arena (40 cm width). At the end of each arm was a self-feeder (45 mg pellet dispenser model 8000 (Lafayette Instrument Company).

c) Behavioral testing

1) Pre-training

Before the actual test began, the rats were familiarized with the RAM. The pellets were distributed throughout the maze area. The rats were placed in the middle, one at a time, always placed in the same direction, and they were allowed to run from arm to arm until they had visited all the arms or until 10 minutes had elapsed. The preliminary training lasted two weeks (5 days per week).

2) Training

Due to the size of the batch (48 rats), the experiment was conducted in two phases. Twelve AF64A-injected rats and 12 saline-injected rats participated in each phase. The rats were randomly divided into the different phases. The rats in each phase were further divided into two subgroups, drug-treated and controls. On each training day, each rat was placed in the center 30 min after injection of either AF125 (0.5 mg/kg solution in saline, i.p.) or saline (1 mg/kg, i.p.). The rats could run from arm to arm until eight pellets were collected or until 15 minutes had passed. For each rat, correct choice, number of errors and total time to the five training days were grouped into a block. Correct choices, number of errors and total time were analyzed using a two-way ANOVA (2x2) (Injection-AF64A/saline and Treatment-AF125 0.5 mg/kg/saline).

Correct choices: A significant difference was found in the average number of correct choices from the first eight entrances between the performance of AF64A-injected rats (6.0±0.-24) and that of saline-injected rats (6.8±0, 15) (F(l/36)=9.29, p<0.005). In addition, a significant interaction was found between injection and treatment (F(l/36)=6.35, p<0.025). Scheffe's tests showed that AF125 improved the performance of AF64A-injected rats (6.53±0.36) compared to the performance of saline-treated rats (5.49±0.26), (p<0.025). No significant difference was found between the performance of AF64A-injected rats treated with AF125 and that of saline-treated rats injected with saline.

Number of errors: AF64A-injected rats made significantly more errors (7.69±1.1) than saline-injected rats (3.5±0.59) (F=(l/36)=10.11, p<0.001 ). AF125 treatment had no effect on this parameter.

Total time: No significant difference was found between any of the groups in measuring the total time.

Body weight: Comparison of the mean weight of the rats before the behavioral testing using a t-test showed an overall significant difference (t=(38 )=4.05, p<0.001) between AF64A-injected rats (489.5 g) and saline-injected rats (579 g). The effect of AF125 on body weights was not tested because the rats were deprived of food.

Side effects: The dose of 0.5 mg/kg used in this study caused less side effects than the higher doses (1 and 3 mg/kg) used in the MWM experiment. Only two saline-injected rats treated with AF125 had diarrhea. No other side effects were found.

CONCLUSIONS

1. AF64A-injected rats showed a significant reduction in performance as expressed in both parameters: the number of correct choices of the first eight entrances and the number of errors.

2. AF125 (0.5 mg/kg, i.p.) significantly improved the performance of AF64A-injected rats. This result was only illustrated by the parameter for correct from the first eight entries.

3. Side effects of the test compound AF125 were minimal probably due to the low dose of 0.5 mg/kg compared to the higher doses (1 and 3 mg/kg) used in the MWM experiment.

PART B - AF150

Saline and A64A-pretreated rats (3 nmol/2 µl/side, icv) were used in these experiments, 2.5 months post-injury. The experiment was conducted over a three-week period. During the first week, the rats were trained to find food pellets in all 8 arms of the maze. After this was learned, the rats were trained using delay procedures. During a pre-delay session, four of the arms of the maze were closed and the rats collected the food pellets from the remaining four open arms. During this period, the memory errors were reduced. After a two-hour delay, the rats were returned to the maze. All the arms of the maze were now open, but this only applied to 4 of them. Those that were closed during the pre-delay session were provided with bait. The rats had to collect the food pellets without entering any previously visited arms during either the pre-delay or the post-delay session. After achieving a baseline level, the rats were tested for their performance during their third experimental week in which either 10 mM phosphate buffer (pH 7.3), or AF150 (0.07 or 1.0 mg/kg free base dissolved in 10 mM phosphate buffer, pH 7.3), was administered i.p., immediately after the pre-delay session, once per day for 5 days. The results were listed according to the week (II or III) and are shown in table 16.

Current memory traps increased during the pre-delay session in the group of AF64A + buffer-treated rats; and it appears that as the learning process progresses the rats become more confused perhaps because a previous experience does not facilitate but interferes with the next one. AF150 at tested doses (0.07 or 1.0 mg/kg, i.p) was significantly beneficial in consolidating the working memory (only in the pre-delay session) of AF64A-treated rats; and the lower dose of 0.07 mg/kg, I.p. is somewhat better than the higher tested dose. The beneficial effect of AF150 in this experiment is prevention of impairment of AF64A-treated rats rather than improvement. AF150 had no ameliorative effect on current memory errors during the post-delay session despite the fact that errors also increased during that period. The reason for this effect may be: (a) a pre-delay session is easier than the post-delay session and therefore requires less cognitive ability and is easier to improve; (b) AF150 was administered after the predelay session and thus improves the consolidation of the memory traces of the first four entrances. It is possible that this post-administration of the drug had no effect on the post-delay session, ie on the learning of the other four entries, which may be another cognitive process.

These results, together with the results of AF150 in the PA test indicate that this compound is a very potent cognitive activator, surprisingly more potent than AF102B. AF150 may therefore be particularly promising for controlling SDAT.

Test 12: Effects of AF151 in AF64A-treated rats in an example Morris water maze (MWM).

This animal model that mimics the cholinergic hypofunction and cognitive impairments in SDAT was used as described in Fisher et al (J. Pharmacol. Exptl. Therap, 1991, in press).

The rats were injected with AF64A (3 nmol/2 µl/side, icv) or saline (2 µl, icv), in the presence of phosphate buffer or AF151. The results with regard to the moved path length (cm) of the rats are shown in table 17.

AF151 showed at all three dose rates a significant dose-dependent beneficial effect in restoring cognitive dysfunction in A64A-treated rats. AF151 may thus be a promising drug for the treatment of SDAT because it is effective at very low doses without having visible side effects.

The statistical significance of the data recorded in Table 17 is as follows.

AF151 (1.0 mg/kg, i.p.) vs AF64A + phosphate buffer: p < 0.01 AF151 (0.3 mg/kg, i.p.) vs AF64A + phosphate buffer: p < 0.05 AF151 (0.07 mg/kg , i.p.) vs AF64A + phosphate buffer: p < 0.07.

Notes to table 17:

(Cf. Fisher et al, J. Pharmacol. Exptl. Therap., 1991, in press). Training continued for five consecutive days and each rat received four trials on each day and results are expressed as blocks (eg four trials/day). During trials 1-16 (days 1-4, training stage) the platform was located in the middle of the north-west quadrant of the pool. During trials #17, on the fifth day, the platform was completely removed from the pool (a transfer test). In this experiment, the rat was placed in water for a limited period (60 sec) and the spatial area was measured. During trials 18–21 on the fifth day, the platform was removed to the center of the south-east quadrant (a reversal (opposite) test). Four exit locations were used: north, south, east or west around the perimeter of the pool. The sequence of locations was semi-randomly changed each day. AF151 and phosphate buffer were administered once daily for five days, 30 minutes before testing. The experiment was performed using a tracking system consisting of an image analyzer (cis-2) connected to a microcomputer (8 MZHz - IBM AT, Galai Laborato-ries, Ltd.)

The representative structures such as AF150 and AF150(S), relative to reference cholinergic compounds (eg, acetylcholine, carbachol, bethanechol and nicotine; Mount et al, J. Neurochem. 63, 2065-2073, 1994), show surprising neurotrophic effects even without the presence of

NGF.

The compounds of the present invention show unexpected activity by being synergistic with NGF, basic fibroblast growth factor and K252a (an alkaloid that inhibits the neurotrophic effects of NGF) in increasing neurite outgrowth and enhancing the secretion of the amyloid precursor protein(s) (APP). This synergistic effect was shown in cell cultures transfected with ml muscarinic receptors. It is important to mention that incorrect metabolism of APP can induce Alzheimer's disease (AD), ml agonists such as AF150(S), AF151(S), AF150 and AF151 can, by stimulating ml muscarinic receptors, increase the cleavage of APP in its middle (3 -amyloid region. This cleavage produces secreted, non-amyloidogenic APP and prevents the formation of the AP peptide. This beneficial effect of AF150(S) was also shown in neuronal primary cell cultures by an enhanced secretion of APP.

Chronic administration in apolipoprotein of AF150(S) surprisingly restored to normal missing mice recognition dysfunctions, brain cholinergic hypofunction as shown by brain biochemical parameters such as: acetylcholinesterase, choline acetyltransferase and ml muscarinic receptor binding and overphosphorylated tau protein in the area of tau (not in C - and N-termini, but in the microtubule binding domain of tau. This last finding is surprising because it indicates that AF150(S) promotes the binding of tau to tubulin (A. Fisher, D. Michelson, submitted to J. Neuroscience ).

The compounds according to the invention

Below follows detailed and more specific data.

TOXICOLOGICAL/PHARMACOLOGICAL SUMMARY OF AF150(S)

Pharmacological aspects of AF150(S) in vitro:

AF150(S): Pharmacology; pharmacokinetics & toxicology

Claims (6)

1. Analogous process for the preparation of therapeutically active compounds of general formulas (I) and (II) including enantiomers, racemates and acid addition and quaternary salts thereof, wherein the formula (I) 0 is ( CE2 ) m or C(CH3 ) 2 attached to one of the 1',4' or 1',5' positions in the six-membered ring, where m is 1, 2 or 3, and in formula (II) Q is two hydrogen atoms, (CH2)m or C(CH3)2 wherein m is 1, 2 or 3 and n and p are each independently 0, 1,2 or 3, with the the assumption that n + p = 1-3, r<0>is hydrogen, methyl or hydroxyl, the part indicates any of the following: segment with a 4-spiro substituted 1,3-oxazoline, segment of a 5-spiro substituted 1,3-oxazoline, segment of a 4-spiro substituted 1,3-thiazoline or segment of a 5-spiro substituted 1,3-thiazoline; R is selected from hydrogen, NH 2 , NH-C-j-alkyl, H{C 1- b -alk<y>1) 2 . C<-j>^-alkyl, <C>2-6-alkenyl, C2-6_alkynyl, C3-7-cycloalkyl, C^-^-alkyl substituted with 1-6 halogen atoms, hydroxy-<C>1-6-alkyl, C^^-alkoxy, <C>1_6-alkylthio, C1_f}-Alkoxy-C-j^-alkyl, carboxy-C-j^-alkyl, (Cj^-a<l>coco<y>)-carbonyl-Ci-j-alkyl, amino-C-j ^-alkyl, mono-(Cj,^-<a>alkyl)-amino-C-j^-alkyl), di-(C^_£,-alkyl iamino-C^,^-alkyl, 2-oxo-pyrrolidine- 1-ylmethyl, aryl, diarylmethylol and C1-4-alkyl substituted with one or two aryl groups, R' is independently selected from the group from which R is selected and C1-6-alkanoyl and arylcarbonyl; and aryl denotes unsubstituted phenyl or phenyl substituted with 1-3 substituents selected from halogen, C^_^-alkyl, C1_fc-alkoxy and CF3, subjected to the conditions (i), (ii), (iii), (iv) and (v), i.e. : (i) in formula II, when Q is two hydrogen atoms, n=p=1, R° is hydrogen, R is NH2 and R' is methyl, then the part different from the segment of a 5-spiro substituted 1-3-oxazoline, (ii) in formula II, when Q is two hydrogen atoms, n=p=1, Ro is hydrogen and R is phenyl, R' is not tertiary butyl, ( iii) in formula II, when Q is two hydrogen atoms, n=p=1, R° is hydrogen, R is p-chlorophenyl and R' is tertbutyl then the moiety is neither segment of a 4-spiro substituted 1,3-oxazoline or segment of a 5-spiro substituted 1,3-oxazoline, (iv) 1 formula II, when 0 is two hydrogen atoms, n = p = 1, R° is hydrogen, R is 3,5-dichlorophenyl and R' is tertiary butyl, then the moiety is different from the segment of a 5-spiro substituted 1,3-oxazoline, and (v) in formula I, Q is not CHg in the 1',4'-position, characterized in that to prepare a compound of structural formula I becomes a compound of general formula wherein R° and Q are as defined above, subjected to oxazoline or thiazoline cyclization reaction at the carbonyl group or epoxide group of general formula (Ia) or (Ib) to produce a compound of general formula (I) wherein X, R and Y are as defined above; and wherein, to produce a compound having the structural formula II will a compound having the general formula wherein R°, R^, Q, n and p are as defined above, be subjected to the oxazoline or thiazoline cyclization reaction at the carbonyl group or the epoxide group of general formula (Ila) or (Ilb) to prepare a compound of formula (II) wherein X , R and Y are as defined above. 2. Analogous method according to claim 1, characterized in that the compound of general formula (Ia) or (Ib) or of general formula (Ila) or (Ilb) is initially converted into a 3-aminomethyl compound or 3-hydroxymethyl compound of general formula before the formation of an oxaline compound is achieved. 3. Analogous method According to claim 2, characterized in that conversion from a compound of general formula (Ib) or (Ilb) is achieved by reaction with alkali metal azide followed by contacting with wet, active Raney nickel. 4. Analogy method according to claim 2 or 3, characterized in that when part denotes the segment of a 4-spiro substituted 1,3-oxazoline or segment of a 5-spiro substituted 1,3-oxazoline, the 3-aminomethyl compound or the 3-hydroxymethyl compound is condensed with a carboxylic acid RCOOH or an amidate of formula RC(:NH)-0-alkyl, in which R is as defined above in claim 1 or with cyanogen bromide when R = NH2 and when part indicates the segment of a 5-spiro substituted 1,3-oxazoline or thiazoline, or the segment of a 4- spiro substituted 1,3-oxazoline or thiazoline, the 3-aminomethyl compound or 3-hydroxymethyl compound is condensed with an acid anhydride (RCOjgOGg the resulting reaction product is reacted with P2X5 , where X is 0 or S. 5 . Analogy method according to any one of the preceding claims, characterized in that each of the symbols in the formulas (I) and (II) have the same meanings as defined in claim 1, in that part indicates the segment of a 4-spiro substituted 1,3-oxazoline or the segment of a 5-spiro substituted 1,3-oxazoline, and R is as defined in claim 1 with the exception that it cannot be C 1-3 -alkyl or C 1-3 -alkyl. 6. Analogous process according to claim 5, characterized in that it is carried out to produce a compound selected from the group consisting of: 2-aminospiro (1,3-oxazoline-5,3')quinuclidine, 2-methylspiro (1,3-oxazoline-5, 3')quinuclidine, 2-ethylspiro(1,3-oxazoline-5,3')quinuclidine, 2-phenylspiro(1,3-oxazoline-5,3')quinuclidine, 1-methylpiperidine-4-spiro-4'- (2'-methyl-1',3'-oxazoline), 1- methylpiperidine-4-spiro-4'-(2'-ethyl-1',3*-oxazoline), 2- methylspiro(1,3-thiazoline -5,4')-1'-methylpiperidine, 2-methylspiro(1,3-oxazoline-5,4')-1'-methylpiperidine, including enantiomers, racemates and acid addition and quaternary salts thereof.